Table of Content

01 December 2014, Volume 47 Issue 23

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES

The Maximum Threshold Distances of Rice Gene Flow and Its Temporal and Spatial Distribution in the Hainan Crop Winter-Season Multiplication Region of China

HU Ning, YAO Ke-min, YUAN Qian-hua, JIA Shi-rong, HE Mei-dan, JIANG Xiao-dong, XU Li-xin, HU Ji-chao, PEI Xin-wu

Scientia Agricultura Sinica. 2014, 47(23):  4551-4562.  doi:10.3864/j.issn.0578-1752.2014.23.001

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【Objective】The area of three counties of Lingshui, Sanya and Ledong is a major base or center for Hainan crop winter-season multiplication (HCWM) of different types of rice, including genetically modified (GM) rice. The objective of this study is to calculate the maximum threshold distances of gene flow (MTDs) in the region and to draw a small-scale and even more precise picture of MTDs spatial distribution, which may serve as a reference for setting proper isolation distances in plantation of different types of traditional and GM rice. 【Method】 By adopting an established rice gene flow model and the meteorological data recorded at 52 automated meteorological stations in 27 towns as a model input, the MTDs of rice cultivars and male sterile (ms) lines at a town level were calculated. On the basis of these data, the patterns of spatial distribution of MTDs in the region and its key determining factors were analyzed. According to the national standards of rice seed purity issued by Ministry of Agriculture (MOA), China, 1% or 0.1% was used as a threshold value. 【Result】 Results indicated that in the HCWM region, the MTD_1% to ms lines ranged from 53 m to 195 m with a mean of (110±31) m. The MTD_0.1% to ms lines ranged from 75 m to 271 m with an average value of (169±44) m. In contrast, the MTD_1% to common rice cultivars was less than 1 m in all locations, and the MTD_0.1% was 0.6 m to 5.8 m with a mean of (3.4±1.1) m. These data show that there is a 50-fold difference between MTD_0.1% to ms lines and cultivated rice. It was found that there were two centers and 4 spots with high MTDs, and 3 centers and 5 spots with low MTDs in the region. The three counties of Lingshui, Sanya and Ledong are situated in a tropical zone, along the South China Sea coast with a protective screen provided by the Five-finger Mountains (FFM) at north. In the winter season, the prevailing wind direction in the region is north-east monsoon, while in the spring and early summer, it becomes south-east monsoon from the South Pacific Ocean and south-west monsoon from the Indian Ocean. Higher wind speed usually occurs in the plain region along sea coast. Wind direction and speed in the hilly region between the plain region and the FFM is also influenced by the height and trend of hills. Wind speed near the south slope of the FFM is greatly reduced by its protective effect. 【Conclusion】 The topography and the atmospheric circulation that affect wind speed and direction, to a great extent, determine the basic distribution patterns of MTDs in the region: the high MTDs mainly exist at the east and west wing of the region as well as in the south coast plain region; while the low MTDs typically distributed in the south slope region of the FFM that provides an effective protection.

Genome-Wide Identification and Analysis of Dof Transcription Factor Family in Maize

GE Min, Lü Yuan-da, LI Tan, ZHANG Ti-fu, ZHANG Xiao-lin, ZHAO Han

Scientia Agricultura Sinica. 2014, 47(23):  4563-4572.  doi:10.3864/j.issn.0578-1752.2014.23.002

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【Objective】The study was carried out for genome-wide identification and analysis of the maize Dof (DNA binding with one finger) gene family. 【Method】Based on maize V3 version, a genome-wide overview of Dof gene family in maize, including the gene structures, phylogeny, and chromosome distribution, and mRNA expression atlas at 18 different development stages were made. A survey of the transcriptomes of V3 stage leaf tissues collected under nitrogen sufficient and limiting conditions was conducted using RNAseq. 【Result】The analyses revealed 46 Dof genes harbored in maize genome, named ZmV3Dof1- ZmV3Dof46. According to the phylogenetic relationships and sequence similarity, the gene family was divided into 8 subgroups, which ranged in size from 3 to 8 Dof genes. Genome chromosomal location analyses revealed that maize Dofs were distributed on almost all chromosomes, except for chromosome 9. In total, 12 ZmV3Dofs were presented on chromosome 1, 8 and 7 on chromosomes 5 and 3, 4 on chromosome 8, 3 on each of chromosomes 2, 4, 6, 7 and 10. Relatively high densities of Dofs were observed in some chromosomal regions, including the bottom of chromosomes 1, the top and bottom of chromosomes 5. Dof gene expression profiling analysis showed that some of genes have played diverse roles in the process of plant development, indicating the diversity of their function. DNA phylogenetic relationships showed the partial consistency with RNA profiles. There are 35 Dof genes showed different expressions in these two nitrogen treatments, and thirteen out of them showed greater differences. There are 21 Dof genes exhibited a significant response to low nitrogen supply, and eight out of them were induced by low nitrogen condition, suggesting Dof genes may play an important role in the regulation of nitrogen metabolism. 【Conclusion】In this study, the Dof gene family in maize known to date was identified. These findings indicate that members of this gene family may be involved in different plant biological processes, some of which probably are involved in plant nitrogen assimilation and metabolism. The preliminary genomic analysis will provide a foundation for future functional dissection of Dof family gene.

Cloning and Function Analysis of a Salt-Stress-Induced Receptor Like Protein Kinase Gene MsSIK1 from Alfalfa

GUO Peng, XING Xin, ZHANG Wan-jun, JIANG Jian

Scientia Agricultura Sinica. 2014, 47(23):  4573-4581.  doi:10.3864/j.issn.0578-1752.2014.23.003

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【Objective】The aim of this study is to clone stress-induced protein kinase gene 1 (SIK1) gene, to analyze its molecular mechanisms and promote their applications in breeding. 【Method】 The total RNA from the leaves of alfalfa was used as the template to design the degenerate primers based on homology cloning strategy, and then the full-length ORF sequence of MsSIK1 was obtained through a combined reverse transcription-PCR (RT-PCR). Sequence analysis was performed by using homology comparision, the SMART website (http://smart.embl-heidelberg.de/) was used to simulate the protein structure. The subcellular localization transient expression vector was constructed and transformed into onion epidermal cell by particle gun. MsSIK1 and GFP were expressed in a fusion, which could be used to analyze the subcellular localization by the fluorescence signal. Real time-PCR was used to investigate the expression pattern under NaCl, ABA and drought stress, transgenic Arabidopsis plants was obtained by Agrobacterium. Expression identification of transgenic plants was performed by RT-PCR, after the transgenic plants were obtained, T₃-2, T₃-6, and T₃-10 transgenic lines were used for character identification of transgenic Arabidopsis under salt stress at seedling stage. Chlorophyll content and MDA content were investigated for functional verification of salt resistance between the wild type and T₃-2 ,T₃-6, T₃-10 transgenic lines.【Result】The results indicated that ORF of MsSIK1 was 2 478 bp and contained a single open reading frame of 825 amino acid residues. The protein C terminal has high homology with a variety of plant kinase, the simulation of protein structure indicated that the protein encodes a putative leucine-rich repeat receptor-like kinase with various domains. In the N-terminal region, a putative extracellular domain containing 10 amino acid leucine-rich repeats (LRR) was identified, and a transmembrane domain was identified. In the C-terminal cytoplasmic region, a serine/threonine protein kinase domain was predicted. The subcellular localization result suggested that MsSIK1 is located in the plasma membrane of onion epidermal cell. Real time-PCR indicated that the mRNA accumulation of MsSIK1 was induced by salt stress, abscisic acid (ABA) and drought. In the salt treatment, expression was induced quickly and the level of the product peaked at 4 h (about 7 times as to the control). In the drought treatment, MsSIK1 transcription was not induced immediately, but reached its maximum at 2 h (about 6 times compared with the control). In the ABA treatment, MsSIK1 transcripts accumulated rapidly and peaked after 3 h (about 6.8 times compared with the control). The RT-PCR identification of transgenic plants indicated that there were 6 lines with obviously band in T₁, and highest expression in T₁-10, but not detected in the wild type, therefore, it indicated that the foreign gene had been integrated into the chromosome of Arabidopsis thaliana and inherited in the offspring. Compared with the wild type plants, T₃-2, T₃-6, and T₃-10 transgenic lines grew well at seedling stage in salt treatment, indicating that MsSIK1 improved the salt tolerance in Arabidopsis thaliana. Additionally, compared with the control, the content of chlorophyll in transgenic Arabidopsis decreased less in NaCl treatment, among them, the chlorophyll content of wild type decreased by 77%, T₃-3 decreased by 53%, T₃-6 decreased by 44%, T₃-10 decreased by 35%; equally, the content of chlorophyll in transgenic Arabidopsis accumulated less in NaCl treatment, among them, the content of MDA in wild type was 1.3 times as that in T₃-10 line. 【Conclusion】MsSIK1 as a receptor like protein kinase was induced by a variety of stress and could increase salt tolerance by overexpression in A. thaliana.

Selection of Late Rice Cultivars of Japonica Rice Switched from Indica Rice in Double Cropping Rice Area

HUA Jin, ZHOU Nian-bing, ZHANG Jun, ZHANG Hong-cheng, HUO Zhong-yang, ZHOU Pei-jian, CHENG Fei-hu, LI Guo-ye, HUANG Da-shan, CHEN Zhong-ping, CHEN Guo-liang, DAI Qi-gen, XU Ke, WEI Hai-yan, GAO Hui, G

Scientia Agricultura Sinica. 2014, 47(23):  4582-4594.  doi:10.3864/j.issn.0578-1752.2014.23.004

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【Objective】Under the conditions of late rice of double cropping systems, three different types of rice cultivars were used to investigate the differences and interrelations of yield, growth and utilization of temperature and illumination. The result will provide a scientific basis and technical support for the selection of suitable varieties in double cropping systems.【Method】A field experiment was conducted with japonica hybrid rice (including partial japonica of indica-japonica hybrid rice, partial indica of indica-japonica hybrid rice and pure japonica hybrid rice), japonica conventional rice and indica hybrid rice as materials in double-cropping rice areas (Shanggao of Jiangxi) in the middle reaches of the Yangtze River in 2010-2013. Differences and interrelation of yield, growth and utilization of temperature and illumination of rice cultivars with different types under the late rice of double cropping systems were studied systematically using cultivation with wet nursery and big seedlings transplanting.【Result】The yields of different types of rice cultivars under the late rice of double cropping systems all demonstrated japonica hybrid rice＞japonica conventional rice＞indica hybrid rice significantly or very significantly. Compared with indica hybrid rice, the yields of japonica hybrid rice and japonica conventional rice were increased by 19.20%-28.95% and 9.21%-12.17%, respectively. It was found that building enough total spikelets through adequate panicles and the large spikelets per panicle, and keeping a high seed-setting rate and 1000-grain weight was a basic way to improve yield structure comprehensively and obtain higher yield for japonica rice. Compared with indica hybrid rice, the growth periods of japonica hybrid rice and japonica conventional rice were delayed, and their total growth periods were significantly extended by 17.1 days and 9.5 days, respectively. And the utilization efficiency of temperature and illumination of various types of rice cultivars at different developmental stages showed that japonica hybrid rice＞japonica conventional rice＞indica hybrid rice, and the whole growth period trended similarly among which japonica hybrid rice were 3 713.3℃,924.9 h, 90.9% and 93.5% respectively.【Conclusion】The yield, growth and utilization of temperature and illumination among different types of rice cultivars under the late rice of double cropping systems had big differences. The suitable japonica rice varieties could make good use of longer growth yield with high utilization of temperature and illumination and increase the grain yield. According to the requirements of national japonica rice production development planning, considering the stubbles feature of double-cropping rice and heat conditions as well as the requirement of mechanized and simplified cultivation, a batch of late japonica rice cultivars of high yielding which were suitable for cultivation in the local were selected including Yongyou 538, Yongyou 8, Yongyou 2640, Yongyou 1538, Yongyou 1540, Changjiang 19, Changjiang 21, Xiaoyechishu and Zhendao 5108, in order to provide reference for rice production in large area.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY

Regulatory Effects of Exogenous Nitric Oxide on Morphology of Cotton Seedlings Under Nitrogen Stress

CHEN Jing, LIU Lian-tao, SUN Hong-chun, ZHANG Yong-jiang, WANG Zhan-biao, LI Cun-dong

Scientia Agricultura Sinica. 2014, 47(23):  4595-4605.  doi:10.3864/j.issn.0578-1752.2014.23.005

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【Objective】In order to explore the effects of exogenous nitric oxide on cotton seedlings under nitrogen stress, the morphology characteristics of leaves and roots and cotton growth were analyzed, which would provide a theoretical basis for regulation of exogenous nitric oxide in cotton.【Method】The hydroponic experiment was conducted in a growth chamber. Nongdamian 8 was selected as the test material in this study. The experiment consisted of seven different treatments. The control group (CK) was growing under Hoagland's solution. Six treatment groups were grown under different SNP concentrations with the basis of Hoagland's solution without nitrogen. Concentrations of SNP, respectively, were 0 μmol·L^-1(T0), 50 μmol·L^-1(T1), 100 μmol·L^-1(T2), 200 μmol·L^-1(T3), 500 μmol·L^-1 (T4), and 1 000 μmol·L^-1(T5). The leaf area, root morphology characteristics, water consumption and dry weight of cotton seedlings under nitrogen stress were measured. 【Result】The growth of cotton seedlings above-ground and under-ground were inhibited by nitrogen stress. The increase of leaf number and leaf area was inhibited. The root length, root surface area and root volume of fine root (0.05-0.20 mm) and middle root (0.2-0.45 mm) decreased and the water consumption and dry weight also decreased. Different concentrations of exogenous nitric oxide had different effects on nitrogen-stressed cotton seedlings’ above-ground and under-ground growth. The damage of nitrogen stress was alleviated by low concentration exogenous nitric oxide (SNP concentration: 50-100 μmol·L^-1). The upper and down leaf area significant increased by low concentration exogenous nitric oxide. Fine root and middle root growth were affected significantly, whose root length, root surface area and root volume were increased by low concentration exogenous nitric oxide. Water consumption and dry weight also increased by low concentration exogenous nitric oxide. The alleviation effect reduced with the further increase of SNP concentration when the concentration was more than 100 μmol·L^-1. The leaf number, upper and down leaf area, root surface area and root volume of fine root and middle root, water consumption and dry weight all decreased. It was concluded that different concentrations of exogenous nitric oxide could ameliorate the negative effects of nitrogen stress by improving the growth of shoot and root. SNP at 100 μmol·L^-1 was the best in alleviation the nitrogen stress on cotton seedlings, while the high concentration of SNP enhanced the damage of nitrogen stress. 【Conclusion】 Nitrogen stress decreased the growth of cotton seedlings. Nitrogen deficiency was mitigated, the growth of above-ground and under-ground was increased, the tolerance to nitrogen stress was also increased by lower concentration of NO (SNP concentration: 50-100 μmol·L^-1). The 100 μmol·L^-1 SNP has the best mitigative effect on nitrogen stress.

Effect of Diammonium Phosphate Levels and Planting Pattern on Nutrient Uptake in Super-High-Yield Soybean Cultivars

MA Zhao-hui, CHE Ren-jun, WANG Hai-ying, ZHANG Hui-jun, XIE Fu-ti

Scientia Agricultura Sinica. 2014, 47(23):  4606-4617.  doi:10.3864/j.issn.0578-1752.2014.23.006

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【Objective】Nutrient uptake of soybean is closely related to the yield, while the response of soybean cultivars to nutrient is not the same. A pot experiment was conducted to find out the nutrient uptakes of nitrogen (N), phosphorus (P), potassium (K) of super-high-yield soybean cultivars and the common cultivar under the treatments of different fertilizer rates and planting patterns.【Method】Two super-high-yield soybean cultivars Liaodou 14, Zhonghuang 35, and one common cultivar Liaodou 11 were used in the pot experiment to investigate the effects of different diammonium phosphate(DAP) levels and planting patterns on soybean N, P, K accumulation and distribution in 2012 and 2013. The experiment was laid out in a randomized block experiment design with five replications. DAP was applied at seedling stage. The treatments consisted of three levels of DAP (0, 100 and 200 mg·kg^-1 dry soil) and two planting patterns (single seeding and mixed seeding). At the stages of R2, R6 and R8, five plants were selected randomly for measurements. Seeds and aboveground vegetative samples were collected from all the treatments. The plant samples were dried in the oven at 105℃ for 30 min and 80℃ at least for 72 h to achieve constant weight. The plant samples were weighed, grounded and digested with H₂SO₄-H₂O₂ acid mixture method. Nitrogen content was estimated by Kjeldah(KN520) method. Phosphorus and potassium were determinated by molybdenum stibium anti(UV-2450) and flame photometer (PEAA800), respectively. N, P, K accumulation was calculated by multiplying dry mass with N, P, K percentage contents. The nutrient use efficiency was calculated as grain yield divided by the nutrient amounts of grains. The nutrient harvest index was calculated by dividing the accumulative nutrient amounts of grains with total nutrient amounts produced by the plants. 【Result】The results showed that the uptake, transportation and distribution of N, P, K varied with different cultivars and planting patterns. Super-high-yield cultivars accumulated more nutrients and had a higher nutrient utilization efficiency as well as N, P harvest index than those of common cultivar. The accumulative amounts of N, P, K showed an increased tendency with the increment of DAP levels, especially in the super-high-yield soybean cultivar. N contents increasing range of stem and leaf of super-high-yield cultivar were 66.9% and 30.5% higher than those of common cultivar, respectively, at flowering stage under 200 mg·kg^-1 fertilization level, and the difference range of P contents reached 28.1%. At seed filling stage, the P contents difference of pod wall and seed was increased with fertilization level enhanced, and that the difference range of K contents between varieties reached 18.6% at maturity stage under high fertilization level. The capacity of nutrient uptake and its use efficiency of super-high-yield soybean cultivars were significantly higher than those of common cultivar in fertilization and mixed seeding treatments. N utilization efficiency of super-high-yield cultivar and common cultivar were declined, while P, K harvest index increased in mixed seeding treatment with fertilization level enhanced. Mixed seeding treatment increased the difference of nutrient utilization efficiency and nutrient harvest index between super-high-yield cultivar and common cultivar, which was more obvious at medium (100 mg·kg^-1) and high (200 mg·kg^-1) fertilization levels. N utilization efficiency and P utilization efficiency of super-high-yield cultivar were 13.6% and 14.2%, 2.1% and 10.4% higher than those of common cultivar, respectively, in mixed seeding treatment at medium and high fertilization levels. In addition, the difference range of P utilization efficiency between varieties was increased from 4.9% to 10.8% in mixed seeding treatment. 【Conclusion】In the case of existing interspecies competition between super-high-yield cultivar and common cultivar, the former had a stronger capacity of nutrient uptake and higher transferring efficiency of nutrients under abundant fertilizing amount.

PLANT PROTECTION

Stem Rust Resistance Evaluation and Ug99-Resistance Gene Detection of 139 Wheat Cultivars

WU Xian-xin, LI Tian-ya, CHEN Si, WANG Guan-qin, CAO Yuan-yin, MA Shi-liang, LI Ming-ju

Scientia Agricultura Sinica. 2014, 47(23):  4618-4626.  doi:10.3864/j.issn.0578-1752.2014.23.007

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【Objective】 Wheat growing region in Yunnan Province of Southwestern China plays a pivotal role in spread and epidemic of wheat stem rust disease in China. Thus, once Ug99 and its lineage mutants invade in China, it is highly possible that the pathogen will preferably colonize, repeatedly reproduce and disseminate to entire country. The objective of this study is to clarify the resistant levels of the main wheat cultivars (lines) in Yunnan Province and the Ug99-resistant materials of CIMMYT to wheat stem rust races in China and those Ug99-resistant genes contained in these wheat materials, and to provide guidance for the layout of the resistant varieties.【Method】From Oct., 2013 to Mar., 2014, all of the 119 wheat varieties (lines) from Yunnan Province and 20 of Ug99 resistant materials including the susceptible control Little Club (LC), were sown in the 10 cm diameter clay pots, when the primary leaves were fully expanded or at about 7-day-old seedlings, they were inoculated using talc-urediospore powder mixture of the common races 21C3HTTTM, 34MRGQM and a new race 34C3RTGQM which was obtained from an aecia sample on barberry, maintained humidity overnight in humid chamber, and then transferred to greenhouse for incubation at 15-26℃ (night/day). About two weeks later, when the pustules on LC could be scored into type ‘4’, the infection types (ITs) on test cultivars (lines) were investigated and recorded. The IT standard was determined in accordance with ‘0-4’ (0-2⁺ being resistant and 3^--4 being susceptible). The IT data were used to appraise the resistance of the tested materials. In addition, the primary leaf tissue of each healthy test cultivar (line) was taken to extract genomic DNA, amplified with the reported specific primer pair of Ug99 resistant genes (Sr22, Sr25, Sr26 and Sr28)and detected through 2.0% agarose gel electrophoresis as well as polyacrylamide gel electrophoresis for the resistance gene marker (s) to figure out those genes carried in tested cultivars (lines).【Result】Of the 119 tested varieties (lines), 42 materials were resistant (accounting for 35.3%), with 6 materials being immune or nearly immune (5%), 36 materials were highly resistant or moderately resistant (30.3%) and the rest (77 materials) were susceptible with 42 materials being highly susceptible (35.2%) and 35 materials moderately susceptible (29.4%). Of the 20 CIMMYT’s materials, two were highly susceptible (10%) and two moderately susceptible (10%). The result of molecular marker detection indicated that two CIMMYT’s materials contained Sr25, one contained Sr26 and one contained Sr28.Interestingly, 12 of Yunnan materials were met with the same one gene, ie. Sr28.【Conclusion】To the races tested, the resistant level of wheat varieties (lines) in Yunnan is considerably low and a small section of CIMMYT’s materials show some degrees of susceptibility, too. However, the 12-Sr28-carrying materials are Ug99 resistant resources rarely reported in China.

Molecular Characterization and Analysis of Suppressing RNA Silence of P0 Protein Encoded by Sugarcane yellow leaf virus

LIN Yi-hua, XIAO Sheng-hua, LIU Ying-hang, CHEN Jian-sheng, FU Hua-ying, CHEN Ru-kai, GAO San-ji

Scientia Agricultura Sinica. 2014, 47(23):  4627-4636.  doi:10.3864/j.issn.0578-1752.2014.23.008

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【Objective】Yellow leaf (YL) disease of sugarcane is one of viral diseases in major sugarcane-producing regions in China and worldwide, causing a serious threat to sugarcane industry. Sugarcane yellow leaf virus (SCYLV), a member of the genus Polerovirus and family Luteoviridae, is the causal agent of YL. The aim of this study is to characterize the conserved regions of P0 protein encoded by SCYLV, and to test its functionality as a RNA silencing suppressor in natural host sugarcane (Saccharum spp. hybrids). The findings will contribute to further investigate the molecular mechanism of SCYLV pathogenesis at RNA silencing level. 【Method】P0 and its two deletion mutants, P0^?2-15(15-aa deletion in N terminal) and P0^?155-256(102-aa deletion in C terminal) from SCYLV CHN-FJ4 isolate were obtained by RT-PCR and cloned into an expression vector under control of the maize ubiquitin 1 promoter and Agrobacterium nopaline synthase terminator. Each of P0 or its deletion mutant constructs were co-bombarded with the reporter gene coding for enhance yellow fluorescent protein (EYFP) into sugarcane young leaf segments, and transient EYFP expression was quantified using ImageJ software. The Tomato bushy stunt virus-encoded P19 was included as a model suppressor.【Result】Phylogenetic analysis revealed that the CHN-FJ4 isolate clustered with isolates of the BRA genotype and shared 96.1%-98.4% aa identified with them. MEME software online revealed that the P0 proteins contained three significant conserved regions positioned at 1-60, 76-125, and 161-210 aa residues. Besides, eight positive sites were detected using five different approaches under the datamonkey web-server. P0, P0^?2-15 and P0^?155-256 were co-bombarded with EYFP into sugarcane young leaf segments, respectively. In young leaf segments co-expressing a suppressor P0 or P19, EYFP foci account and EYFP expression level were increased at 48-120 h post-DNA introduction compared with that in the absence of a suppressor. At 120 h post-bombardment, EYFP foci account and of EYFP expression levelincreased by more than 1.6-fold and 4.0-fold, respectively. No significant differences (P>0.05) in EYFP expression were found in presence of P0 or P19. However, EYFP expression was not increased by P0^?2-15 or P0^?155-256. The reason would be 15-aa deletion in N-terminal or 102-aa deletion in C-terminal of P0 protein impaired and even disabled the function of RNA silencing suppressor.【Conclusion】The P0 RNA silencing suppressor proved to be efficient in enhancing reporter gene expression in transient assay using sugarcane young leaf segments. The conserved regions of P0, 15-aa in N terminal and 102-aa in C terminal, are necessary for the RNA silencing suppressor activity of P0.

Antennal Ultrastructure of Aphidius gifuensis and the Effect of Cold Storage on Antennae

SUN Zhi-juan, CHEN Dan, FAN Xiu-juan, LIU Li, CHENG Yun-ji, ZHANG Chang-hua, REN Guang-wei, LIU Xiang-dong

Scientia Agricultura Sinica. 2014, 47(23):  4637-4647.  doi:10.3864/j.issn.0578-1752.2014.23.009

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【Objective】 The objectives of this study are to describe the external morphology of the antennae and antennal sensilla of Aphidius gifuensis, examine the effects of cold storage on the antennal sensilla, and to provide the basis for effective use of A. gifuensis . 【Method】 Scanning electron microscopy was used to observe the morphological characteristics of the antennae and sensilla of A. gifuensis adults that emerged under normal conditions, and to compare the sensilla of males and females. Mature larvae of A. gifuensis were stored at 4℃ for 7, 14, 21, and 28 days, and then following cold storage, samples were prepared by ethanol dehydration. Scanning electron microscopy was used to study the effects of cold storage on the external form of the antennae and antennal sensilla. 【Result】 The antennae of males and females were both filiform and had similar morphologies. The average length of the male antenna (19 segments) was slightly longer than that of the female (17 segments). Six types of antennal sensilla were identified, namely, trichodea, chaetica, placodea, Böhm’s bristles, coeleoconica, and basiconica sensilla. Sensilla trichodea type I, which were the most widely distributed and most numerous of the antennal sensilla, were found mainly on the flagella. Sensilla placodea were also distributed on the flagella, occurring from the first flagellum in males and from the second flagellum in females. Sensilla coeleoconica type II were found only on the antennae of adult males. There were differences in the number of different sensilla on male and female antennae. The number of sensilla chaetica type I in females was greater than that in males, whereas the converse was true for the number of sensilla chaetica type II and sensilla placodea. Abnormal placodea and coeleoconica were observed when larvae were stored at 4℃ for 14, 21, and 28 days, with the low temperature influencing the segmentation of the last two antennal subsegments. Fifty-four percent of male antennae showed abnormal segmentation, with antennae comprising 18 segments instead of the normal 19. Similarly, 69% of female antennae showed abnormal segmentation, bearing 16 segments instead of the typical 17. 【Conclusion】The antennae of adult male and female A. gifuensis are filiform. Six types of antennal sensilla were found on the antennae. The types of antennal sensilla in both males and females were similar, but the length of the male antenna was longer than that of the female. Cold storage had a significant effect on antennal segmentation and the form of some sensilla. In order to reduce the damage to A. gifuensis caused by cold storage,preserving A. gifuensis prior to the pupal stageshould be avoided and the storage time should be shortened.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Soil Organic Carbon in Agricultural Soils in China Under Global Climate Change

ZHANG Xu-bo, SUN Nan, XU Ming-gang, ZHANG Wen-ju, LI Jian-wei

Scientia Agricultura Sinica. 2014, 47(23):  4648-4657.  doi:10.3864/j.issn.0578-1752.2014.23.010

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Soil organic carbon (SOC) in agricultural soils plays an important role for ensuring food security and mitigating global climate change. Thus, the past and potential effects of climate change on the SOC in agricultural soils have been widely noticed by global society. The net primary productivity (NPP), carbon input and SOC decomposition rate could be altered by changes of temperature, precipitation and CO₂ concentration which due to the global climate change. Furthermore, carbon cycling could also be changed by the changes of land use and planting systems under climate change. The published data were used to illustrate the effects of global climate change on SOC in upland in China. It is estimated that the increase of temperature and precipitation ranged from 3.9 to 6.0℃ and from 9% to 11% in China until 2050s, respectively, and carbon input in agricultural system will be decreased by 2.3%-10% (average of wheat, maize and rice cropping systems). However, if consider the combined effects of increase of CO₂ concentration with global warming, carbon input will be increased by 13%-22% until 2050s compared with that before the period of 1990s. Model simulation results showed that until 2020s, 2050s and 2080s, SOC will be decreased by 2.7, 6.0 and 7.8 tC·hm^-2 under CO₂ emission scenario, and be decreased by 2.9, 6.8 and 8.2 tC·hm^-2 under high CO₂ emission scenario, respectively, which is ca. 4.5%, 10.5% and 12.7% of the SOC pool in 1980s. Furthermore, SOC pool will be decreased approximately by 10% at the end of the 21st century. If C inputs in agricultural systems are promoted with a 1% annual increase rate, there is a 100% increase in carbon input in agricultural soils by the end of the 21st century in China. An obvious uncertainty still remain whether the climate change will exert huge influence on soil carbon pool. The consensus that the positive and negative of soil carbon sequestration counteract to become carbon sink or source has not been reached. Thus, in the meanwhile of mitigating climate change and increasing soil carbon sequestration, there is a special need for predicting the carbon change in the future and quantifying the interaction between climate change and the feedback from organic carbon in agricultural soils.

The Relationship Between Temperate Meadow Steppe Soil’s Biological Properties and Aboveground Vegetation Under Different Grazing Intensities

TAN Hong-yan, YAN Rui-rui, YAN Yu-chun, CHEN Bao-rui, XIN Xiao-ping

Scientia Agricultura Sinica. 2014, 47(23):  4658-4667.  doi:10.3864/j.issn.0578-1752.2014.23.011

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【Objective】The number of the soil microorganisms and soil enzyme are sensitive indexes for soil quality and ecosystem functional changes, it’s an important biological indicator for evaluating soil ecosystem degradation under interference conditions. This study focused on relationship between soil’s biological properties and aboveground vegetation under different grazing intensities, which explained the soil and plant degradation process and mechanism under grazing, thus giving a theoretical basis for ecological restoration of degraded grassland.【Method】 Based on the cattle grazing control experiment carried out in the temperate meadow steppe in the Hulunbeir Grassland, the number of soil microorganisms and soil enzymes activity changes under four different grazing intensities (control area G_0.00: 0.00 Au·hm^-2, light grazing G_0.23: 0.23 Au·hm^-2, moderate grazing G_0.46: 0.46 Au·hm^-2 and heavy grazing G_0.92: 0.92 Au·hm^-2) were monitored, and the relationship between soil’s biological properties and aboveground vegetation indexes was analyzed. 【Result】The maximum number of ammonifying bacteria, oligotrophic azotobacter microorganisms, oligotrophic cellulolytic microorganisms appeared in the treatment of light grazing G_0.23; the maximum number of aerobe azotobacter, oligotrophic azotobacter appeared in the treatment of moderate grazing G_0.46. Except for catalase, the activities of alkaline phosphatase, urease and invertase decreased significantly as grazing intensity increased (P＜0.05) . The tendency of vegetation coverage, biomass and vegetation diversity indexes obviously decreased with the increase of grazing gradient. The soil bacterial physiological functional groups (except Nitrifying bacteria) were positively correlated with the species richness of aboveground vegetation, vegetation coverage, community diversity indexes and biomass; the soil alkaline phosphatase and urease activities showed a significant positive correlation with vegetation coverage, community diversity indexes and biomass(P＜0.05).【Conclusion】The temperate meadow steppe soil’s biological properties showed different degrees of change under different grazing intensities, soil enzyme activities represented the current state of soil ecological system better than the microorganism community, and it had a better correlation with aboveground vegetation indexes.

Effects of the Potting Media Produced by Activated Sludge Vermicompost Stacked and Added Vermiculite on Eggplant Seedlings

ZHAO Hai-tao, LI Tian-peng, YAO Xu, ZHAO Lei-ming, HONG Qi-dong, YAN Zhi-jun, SHAN Yu-hua, FENG Ke

Scientia Agricultura Sinica. 2014, 47(23):  4668-4679.  doi:10.3864/j.issn.0578-1752.2014.23.012

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【Objective】 Sludge vermicompost contains large amounts of nutrients and bioactive substances, and it has the potential to be exploited as potting media. This paper aims to investigate the effects of the potting media produced by activated sludge vermicompost stacked and added vermiculite on eggplant seedlings development. The changes of the characteristics of the available NPK, pH and the electrical conductivity (EC) of the potting media were also studied. 【Method】 In this experiment, potting media were prepared by adding different amount of vermiculite (volume ratio: vermicompost﹕vermiculite =1﹕0, 2﹕1, 4﹕1, 8﹕1) to fresh sludge vermicompost and stacked sludge vermicompost (stacked for 180 days), and commercial peat potting medium was used as a control. Eggplant seedlings were cultivated in a greenhouse. 【Result】 The results showed that sludge vermicompost-based nursery media were better for eggplant seedling development compared with the traditional peat-based nursery media. Both fresh and stacked sludge vermicompost could significantly increase the evaluation indexes of eggplant seedlings, such as the leaf expanded ness, root surface area, root diameter, root volume, shoot dry weight, total dry weight, root/shoot ratio and the strong seedling index.The chlorophyll of eggplant seedlings increased significantly for growing in fresh vermicompost media, and the root length increased significantly for growing in stacked vermicompost media. The impact of fresh sludge vermicompost added with different amounts of vermiculite on eggplant shoot growth was less than stacked sludge vermicompost, while stacked vermicompost on root growth was more than fresh sludge vermicompost. To sludge vermicompost, stacking resulted the decrease in the available nitrogen, available phosphorus and the pH, while increased the EC. To the fresh and stacked sludge vermicompost, adding vermiculite decreased the available nitrogen, available phosphorus, available potassium, and the EC while increased the pH, and the change scope elevated with the added vermiculite increased. After nursery, the contents of the available NPK in the sludge vermicompost media were significantly higher than that in commercially potting media, but no significant difference in pH changes. It was more conducive to the development of eggplant seedlings that the potting media made from the fresh sludge and vermiculite in the volume ratio of 2﹕1, and stacking sludge vermicompost and vermiculite in the volume ratio of 8﹕1. 【Conclusion】 In short, the sludge vermicompost could be used as raw material for potting media for eggplant seedling nursery. The important reasons for the sludge vermicompost promoted the eggplant seedling growth were continuing supply of a large amount of available mineral nutritious. Although the process of stacking sludge vermicompost reduced the available nitrogen, the available phosphorus, and increased the EC, in the eggplant nursery, from the points of the convenient production, costs reduction, ensuring the quality of seedlings, and improving the vermicompost media use security, the recommended potting medium should be made by stacked sludge vermicompost and vermiculite in accordance with the 8﹕1 of volume ratio.

HORTICULTURE

Three-Dimensional Simulation of Canopy Structure and Light Interception for Tall Spindle Shape of Spur ‘Fuji’ Apple with Dwarf Interstock

YANG Wei-wei, CHEN Xi-long, LIU Hang-kong, ZHANG Man-rang, ZHANG Dong, HAN Ming-yu

Scientia Agricultura Sinica. 2014, 47(23):  4680-4694.  doi:10.3864/j.issn.0578-1752.2014.23.013

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【Objective】The present study was conducted to construct three-dimensional(3D) plant virtual model of canopy structure of apple, to provide precise and quantitative method for evaluating canopy structure and light interception of fruit tree and provide data for growers to make proper training and pruning in high density planting systems. 【Method】 Two 14-year-old tall spindle shape of ‘Fuji’ apple with dwarf insterstock were chosen as materials to construct 3D virtual plant model based on 3D digitizing for these trees and measuring morphological indexes of leaves and shoots in a global range of trees. The 3D distribution of leaf area, light interception according to shoot types and fruit yield were studied quantitatively.【Result】The allometric relationship among indexes of shoot and leaf morphology was established. There existed significant linear correlations between shoot length and shoot leaf number, shoot leaf area, respectively, leaf length and petiole length, leaf width, respectively, and the square of leaf length and leaf area. The three-dimensional plant virtual model of apple was constructed by combining 3D digitizing and the allometric relationship between shoot and leaf morphology indexes. For leaf number of shoot and leaf area of shoot between simulated and measured, the coefficient of determination (R²) was 0.92 and 0.95, respectively, root mean square error (RMSE) was 1.18 and 31.5 cm², and relative prediction error (RE) was 7.15% and 5.86%, respectively. The precision of 3D virtual plant model can meet the requirements for analyzing canopy light interception and structure. The model can be used to quantitatively simulate leaf area, volume, projected leaf area, STAR, overcrowded ratio, relative variance of leaf area density (ξ) and diurnal variation of STAR at shoot and tree scale. For tall spindle shape of ‘Fuji’ with dwarf interstock, the shoots mainly distributed in 0.5-2.5 m height and 20-80 cm to trunk in horizontal distance within canopy, 74.88% leaf area was in this zone. The volume and ratio of overcrowded of canopy were 4.47 m³ and 44.62%, respectively. For vegetative short shoot, vegetative long shoot and bourse shoot, the relative volume to whole canopy volume was 69.73%, 43.50% and 41.26%, respectively, the ratio of overcrowded was 60.77%, 54.12% and 83.15%, respectively, the mean STAR value was 0.10, 0.23 and 0.14, respectively, the STAR value increased gradually from bottom to top and also increased from inner to outer of the canopy regardless of shoot types. Yield of fruits mainly distributed in 0.5-2.0 m height and 20-60 cm to trunk in horizontal distance within canopy and was about 4.1×10³ kg per 667m². Diurnal variation of STAR for the whole canopy, vegetative short shoot and bourse shoot was bimodal curve and for vegetative long shoot was unimodal curve. STAR values, no matter which shoot types, had a significant negative correlation with bourse shoot number and had a significant positive correlation with soluble solids, fruit weight and diameter, respectively.【Conclusion】Three-dimensional virtual plant model can be used to evaluate the tree structure and light interception precisely and quantitatively. STAR also can be used to evaluate canopy light interception efficiency quantitatively.

The Influence of Changes of Endogenous Hormones in Shoot on the Grapes Flower Bud Differentiation in Greenhouse

WANG Hai-bo, ZHAO Jun-quan, WANG Xiao-di, SHI Xiang-bin, WANG Bao-liang, ZHENG Xiao-cui, LIU Feng-zhi

Scientia Agricultura Sinica. 2014, 47(23):  4695-4705.  doi:10.3864/j.issn.0578-1752.2014.23.014

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【Objective】 The objective of this experiment is to study the changes of endogenous hormones in grape shoots during the flower bud differentiation process and analyze its functions on grape flower bud formation and to provide a theoretical basis for solving the problem of “alternate bearing” in grape facilities and promoting early cultivation. 【Method】 Four-year-old beta grafted ‘Jingmi’ (V. vinifera CV. Jingmi, a weak light resistant variety, sustainable productivity can be obtained under early maturing cultivation in sunlight greenhouse without taking any measures) and ‘Summer Black’ (V.vinifera-V. labrusca CV. Summer Black, a weak light unresistant variety, sustainable productivity can be obtained under early-maturing cultivation in sunlight greenhouse needs renewal pruning) were used as materials, the experimental treatments of early maturing promoting cultivation in greenhouse (‘Jingmi’and ‘Summer Black’) and the opening field cultivation (‘Summer Black’) were designed, the process of flower bud differentiation in sampling period and the differentiation ratio were mapped and the flower bud differentiation process was observed by means of paraffin sectioning, through collecting the grape shoots which its roughness of the first node was greater than 0.5 cm and choosing the main winter bud of 2-3 nodes as the research material. At the same time, the contents of GAs, ZRs, ABA and IAA and the ratio of endogenous hormone levels of shoots base 2-3 nodes period were determined by using enzyme-linked immunoassay. 【Result】 The changes of endogenous hormones content in shoot were observed as follows: They were different from ‘Summer black’ with the poorest flowering in greenhouse, endogenous ZRs contents in shoots of ‘Jingmi’ with good flowering in greenhouse and ‘Summer black’ in open field showed a steadily rising trend since undifferentiation stage of embryo shoot growing point (5-7 leaves separated) to Bipartite stage of embryo shoot growing point (early flowering period). Since dome or flat differentiation stage of embryo shoot growing point (inflorescence separated) to anlagen differentiation stages (fruit-setting), endogenous ABA content increased rapidly. Since undifferentiation stage of embryo shoot growing point (5-7 leaves separated) to anlagen differentiation stages (berry enlarging begin), endogenous GAs content showed a trend of down-upward-down. Endogenous IAA content was higher in the whole flower bud differentiation process. The changes of endogenous hormones ratio in shoot were as follows: They were different from ‘Summer black’ with poorest flowering in greenhouse, endogenous ZRs/GAs ratio in shoots of ‘Jingmi’ with good flowering in greenhouse and ‘Summer black’ in open field increased significantly from inflorescence separating stage (from dome or flat stage of embryo shoot growing point to bipartite stage) and berry enlarging stage (from anlagen appearance to formation of the main axis of the inflorescence primordium and second branching of inflorescence primordium). The ratio of endogenous ZRs/IAA rose slightly since inflorescence separating stage (from dome or flat stage of embryo shoot growing point to bipartite stage), then held steady at lower levels from dome or flat stage of growing point to second branching differentiation stage of inflorescence primordium. Endogenous ABA/GAs ratio in shoots increased significantly at the late fruit enlarging stage (from anlagen appearance to formation of second branching of inflorescence primordium). Endogenous ABA/IAA ratio in shoots maintained at a lower level after fruit enlarging stage (from anlagen appearance to differentiation stage of the main axis of the inflorescence primordium). 【Conclusion】 The time before berry enlarging stage was the key moment of differentiation and regulation of flower bud. Endogenous GAs content in shoots showed less changes before berry enlarging stage, ZRs content before flowering beginning and ABA content from inflorescence separating to fruit setting rapidly declined, endogenous IAA content in shoots was low in the flower bud differentiation process. They could be the main reasons why grape in greenhouse could not form good quality flower buds. Endogenous hormones regulate the grape flower formation by balancing and cross-interacting at the specific period. Endogenous ZRs/IAA ratio in shoots maintained lower and stable after inflorescence separating stage was in favor of the formation of flower bud. The ratio of endogenous ZRs/GAs increased significantly at inflorescence separating stage and berry enlarging stage. The higher ABA/GAs ratio and the lower and stable ratio of ABA/IAA promoted the formation of anlagen and the main axis and second branching of inflorescence primordium after berry enlarging stage.

STORAGE·FRESH-KEEPING·PROCESSING

Simultaneous Determination of Main Flavonoids and Phenolic Acids in Citrus Fruit by Ultra Performance Liquid Chromatography

ZHENG Jie, ZHAO Qi-yang, ZHANG Yao-hai, JIAO Bi-ning

Scientia Agricultura Sinica. 2014, 47(23):  4706-4717.  doi:10.3864/j.issn.0578-1752.2014.23.015

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【Objective】The fast method based on ultra performance liquid chromatograph (UPLC) for the analysis of main phenolic acids and flavonoids in citrus was developed in order to provide technological supports for exploitation of phenolic substance of citrus.【Method】 First, the instrument condition optimized the detection wavelength selection based on full-scan (190-400 nm), all material have maximum absorption spectrum at around 283 nm. The optimization of column temperature, mobile phase and flow rate refer to the relevant literature. In order to make the baseline separation using a gradient elution; to optimize the citrus sample processing method, the extracting agent type, extraction times and time comparing by single-factor comparison test. Then citrus samples were extracted with ethyl acetate for 30 min, evaporated and concentrated. The residue was redissolved in methanol for UPLC analysis, peel samples were extracted for 3 times and pulp for 4 times. 【Result】 The chromatographic separation was carried on a ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm) in gradient elution with a mobile phase of acetic acid aqueous solution (0.3%, w/w) (A) and methanol (B) as follows: 95%-80% (0-3 min)A, 80%-80% (3-8 min)A, 80%-70% (8-12 min)A, 70%-20% (12-17 min)A and 20%-95% (17-20 min)A. The column temperature was 35℃, the injection volume was 3 μL and the flow rate was 0.3 mL·min^-1. DAD spectra was recorded and 283 nm was the detection wavelength of phenolic compounds. A total of 19 analytes were separated to the baseline within 18 min with the correlation coefficients higher than 0.999 and linear range from 0.01 to 500 mg·L^-1. The precision, reproducibility and stability were satisfactory with the relative standard deviations (RSDs) less than 5%. Peel samples were extracted for 3 times and pulp for 4 times. Recoveries achieved from the spiked peel and pulp at two concentrations ranged from 85.8% to 109.4% (RSDs, 0.86%-6.06%) and 88.4% to 112.7% (RSDs, 1.05%-5.23%), respectively. The limits of detection (LODs, S/N=3) ranged from 0.001 to 0.09 mg·kg^-1. The proposed method was applied to the determination of phenolic compounds in citrus samples including five categories, which were mandarin orange (sugar orange and ponkan), sweet orange (Newhallnavel orange), pomelo grapefruit (cocktail), citron lemon (lemon) and kumquat. There were different flavonoids and phenolic acids and their different contents in various types of citrus. The total contents of the phenolic compounds in ponkan and cocktail grapefruit peels were the highest, ponkan was about 1.5 times of other species, 5 times of kumquat and the peel was about 3-5 times of the pulp. The content of the flavonoids in cocktail peel with 1 813.22 mg·kg^-1 was the highest, followed by ponkan, Newhallnavel orange, sugar orange, lemon and kumquat. The variation trend of the flavonoid content in the pulp was similar to the peel with the peel significantly higher than the pulp. Flavanones including eriocitrin, naringin, hesperidin, neohesperidin, didymin, narirutin, naringenin and hesperetin are the main flavonoids in citrus. The content of the flavanones in cocktail peel with 1 491.8 mg·kg^-1 was the highest, followed by lemon and Newhallnavel orange and kumquat was the least with the pulp significantly lower than the peel. The total content of the phenolic acids in sugar orange peel with 515.21 mg·kg^-1 was the highest, followed by ponkan, lemon, cocktail and Newhall navel orange and kumquat was the least. The phenolic acid contents of the pulp and peel had a consistent trend and the content of peel was about 3-5 times of pulp. Chlorogenic and ferulic acids were the main acid in citrus.【Conclusion】 A rapid method for the simultaneous detection of the major phenolic and flavonoid substances by UPLC was established. This method was efficient, accurate, low-loss, environment friendly and authenticated by the actual citrus samples including five categories. The flavonoids and phenolic acids were detected in different varieties of citrus and their contents and types. This method can be used for the conventional analysis of the fast and simultaneous detection of flavonoids and phenolic acids in citrus.

ANIMAL SCIENCE·VETERINARY SCIENCERE

Effects of CZKJKL on Stress-Related Factors of Nerve - Endocrine - Immune System of Rats After Immunological Induction

SUN Yao-gui, CHENG Jia, LI Hong-quan, WANG Jun-dong

Scientia Agricultura Sinica. 2014, 47(23):  4718-4725.  doi:10.3864/j.issn.0578-1752.2014.23.016

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【Objective】“Piglet early weaning stress syndrome” brought huge economic losses to the pig industry, immunological stress is one of the important factors. Chaizhukangji particles (CZKJKL) were prepared through modern pharmaceutical technology, total saikosaponins (TSS, the composition of the main therapeutic effects of Bupleuri Radix), polysaccharide of Atractyles macroceohala koidz (PAM, the composition of the main therapeutic effects of Atractylodis Macrocephalae Rhizoma), total glycosides of paeony (TGP, the composition of the main therapeutic effects of Paeoniae Radix Alba)were also selected, and proved that the preparation has a significant mitigation effect on stress of early weaned piglets and stress of early weaning piglets after immunological stress by lipopolysaccharide. The experiment was conducted to investigate the effects of CZKJKL on stress-related neurotransmitters, cytokines and hormones in HPA axis of nerve-endocrine-immune system of early weanling rats after LPS immunological stress, to further explore the anti-stress effects of CZKJKL.【Method】Total 72 early weanling wistar rats at the age of 18 days were randomly divided into 3 treatments of 24 rats each, with 6 replicates in each group and 4 rats in each replicate. The 3 treatments were group Ⅰ(control group), group Ⅱ (LPS group) and group Ⅲ (LPS+CZKJKL group). In the period of 18-24 days of age, the rats in group Ⅲ were fed with 5 000 mg·kg^-1BW CZKJKL daily by gavage, the rats in group Ⅰ and group Ⅱ were fed with equivalent amount of sterile saline. At the age of 21 days, the rats in group Ⅱ and group Ⅲ were injected intraperitoneally with 4 mg·kg^-1BW of LPS, the rats in group Ⅰ were injected with equivalent amount of sterile saline. Blood and tissue samples were obtained at the time of 6 h after injecting LPS. The following indicators were determinated: The contents of NE,5-HT of Hippocampus; the contents of β-EP, CRF of Hypothalamus; the contents or activities of β-EP, ACTH, COR, IL-1β, IL-6, TNF-α, NO, and iNOS of serum.【Result】Compared with groupⅠ, the contents of NE and 5-HT in hippocampi of groupⅡ were significantly increased (P＜0.01), the levels of β-EP in hypothalamus were significantly increased (P＜0.01), the contents of β-EP, IL-1β, IL-6, TNF-α, NO, ACTH, and COR in serum were significantly increased (P＜0.01), and the activity of iNOS in serum was significantly enhanced (P＜0.01). Compared with groupⅡ, the contents of NE and 5-HT in hippocampi of group Ⅲ were significantly decreased (P＜0.01), the levels of β-EP in hypothalamus were significantly decreased (P＜0.01), the contents of β-EP, IL-1β, IL-6, TNF-α, NO, ACTH, and COR in serum were significantly decreased (P＜0.01), and the activity of iNOS in serum was significantly reduced (P＜0.01).【Conclusion】The results indicate that CZKJKL can play a role of anti-immunological stress by efficiently alleviating the disorder of stress-related neurotransmitters, cytokines and hormones in HPA axis of nerve-endocrine-immune system of early weanling rats after LPS immunological stress.

Development of an ELISA Method for Multi-Residue Detecting of Fluoroquinolones

LI Xin-peng, JIANG Jin-qing, QIAN Ai-dong, WANG Zi-liang, FAN Guo-ying, SHAN Xiao-feng, KANG Yuan-huan, LI Yi

Scientia Agricultura Sinica. 2014, 47(23):  4726-2735.  doi:10.3864/j.issn.0578-1752.2014.23.017

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【Objective】Fluoroquinolones(FQs) are widely used in veterinary medicine for the treatment and prevention of bacterial infection. With the increasing use, FQs residues in animal edible tissues have caused serious public health problems and attracted serious attention by research scholars all over the world. The objective of this study wasto produce class-specific monoclonal antibodies (mAbs) against fluoroquinolones (FQs), establish competitive indirect enzyme linked immunesorbent assay (icELISA), and in order to lay a foundation for detection of multi-residue FQs in animal foods.【Method】 The aminobutyric acid was introduced to carboxyl of ciprofloxacin as hapten (CPFX-A) and was proved by (+) ESI-MS spectrum, which was conjugated to bovine serum albumin (BSA) as immunogen (CPFX-A-BSA) by the N,N'-Dicyclohexylcarbodiimide (DDC) method, and to ovalbumin (OVA) as coating antigen(CPFX-A-OVA) by mixed anhydride method, respectively, which were then identified by infrared ray (IR) and ultraviolet (UV). Balb/c mice immunized by CPFX-A-BSA were selected for cell fusion, which was identified by ELISA and icELISA. Under the effect of PEG-1500, NS0 cells and spleen cells were fused at the ratio of 1﹕5. Hybridoma lines that secrete mAb against FQs were selected and their immunological traits were characterized by titer, subtype, sensitivity and cross reaction rate, which ascites were carried out by injecting 10⁸ hybridoma cells in vivo, and icELISA standard curve was established and optimized. High titer, class-specific monoclonal antibody was used to detect 10 FQs in chicken for calculating recovery rate and variation coefficient. The data were also compared with that of HPLC, and SPSS 17.0 software was used to conduct the significant difference analysis.【Result】 The hapten and artificial antigen were synthesized successfully and antiserum titers of three mice were higher than 1﹕1.28×10⁴, in which the titer and IC₅₀ of No.2 mouse were1﹕2.56×10⁴ and 12.92 ng·mL^-1. Three hybridoma cell lines named 2H5, 3D11, and 4F4 were screened after 4 times subclone, which titers were 1﹕1 600, 1﹕1 600, and 1﹕800 in supernatants and 1﹕1.6×10⁶, 1﹕8.2×10⁵, and 1﹕8.2×10⁵ in ascites, respectively. The icELISA procedure was optimized at a concentration of CPFX- A-OVA for 1 μg·mL^-1 at 4℃ package overnight by 5% negative serum of pig, monoclonal antibody and GaMIgG-HRP were diluted 1﹕40 000 and 1﹕8 000, respectively. Under the reaction temperature of 37℃, reaction time of standard substance and monoclonal antibody was 15 min, and 25 min after adding GaMIgG-HRP, also 10 min for termination reaction. Cell line named 2H5 showed a good sensitivity and class-specific toward 10 FQs, the linear regression equation was y= -28.022x+56.219，R²=0.9 782，with an IC₅₀ value of 1.67 ng·mL^-1 for ciprofloxacin, 1.82 ng·mL^-1 for norfloxacin, 1.97 ng·mL^-1 for pefloxacin, 1.54 ng·mL^-1 for enrofloxacin, 2.79 ng·mL^-1 for danofloxacin, 3.38 ng·mL^-1 for lomefloxacin, 5.50 ng·mL^-1 for ofloxacin, 4.40 ng·mL^-1 for marbofloxacin, 11.76 ng·mL^-1 for sarafloxacin, 13.60 ng·mL^-1 for difloxacin, and the lowest detectable limits (LODs) of 0.09 ng·mL^-1-0.64 ng·mL^-1 and cross- reactivity (CR) of 12.3%-108.4%, no cross-reactivity to other compounds was found. The recovery ranges of 10 FQs spiked in chicken using icELISA were 80.5%-91.8 %, 85.1%-95.7% for HPLC, both of the coefficient variations (CVs) were below 10.0%, and no significant difference (P＞0.05) was observed.【Conclusion】The high-sensitivity and class-specific mAb against FQs was prepared, which laid a solid foundation for FQs multi-residue detection.

ANIMAL SCIENCE·RESOURCE INSECT

Sequencing and Phylogenetic Analysis of the RPB1 Gene of Nosema sp. PA

YAN Wei1, SHEN Zhong-yuan, TANG Xu-dong, XU Li, LI Qian-long, XIAO Sheng-yan, YUE Ya-jie, FU Xu-liang

Scientia Agricultura Sinica. 2014, 47(23):  4736-4744.  doi:10.3864/j.issn.0578-1752.2014.23.018

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【Objective】 The objective of this study is to clarify the taxonomic status of Nosema sp. PA and provide a new foundation for the further study of its biological function by cloning the RPB1 (largest subunit of RNA polymeraseII) gene of Nosema sp. PA and analyzing the gene sequence by bioinformatics methods. 【Method】 Six pairs of homologous primers were designed by Primer Premier 5.0 software for the RPB1 gene of Nosema sp. PA based on the RPB1 gene of Nosema bombycis. Partial sequence of the RPB1 gene of Nosema sp. PA was cloned by PCR amplification. Then, bioinformatics analysis on the RPB1 gene of Nosema sp. PA and its encoding protein were conducted by bio-softs as GSDS, SMART, DNAstar and MEGA4.1. 【Result】 Partial sequence of the RPB1 gene of Nosema sp. PA was cloned by PCR amplification (GenBank accession number KJ728831). The partial sequence of the RPB1 gene of Nosema sp. PA had 2 933 nucleotides which contained an ORF with 2 922 bp encoding a polypeptide of 974 amino acids with a molecular weight of 109.38 kD and an isoelectric point of 7.087. The structure of the partial sequence of the RPB1 gene was a single exon. The encoded protein contained four domains: RPOLA_N, RNA_pol_Rpb1_4, RNA_pol_Rpb1_5 and RNA_pol_Rpb1_6. RPOLA_N domain is a very important domain both in prokaryotes and eukaryotes among the four domains. The encoded protein contained four main secondary structures: α-helix, random coil, β-turn and extended strand. The proportion of α-helix and random coil was quite high. Extended strand was mainly located between the α-helix and random coil. Sequence comparison and phylogenetic analysis showed that the encoded protein of Nosema sp. PA was 81.9%-99.6% identity and 0.004-0.049 divergence with those of N. bombycis, N. trichoplusiae, Nosema sp. CPP, N. fumiferanae, N. disstriae, N. tyriae and N. granulosis. Nosema sp. PA and other seven kinds of “true’’ Nosema species above-mentioned clustered in the same clade. The encoded protein was 99.6% identical to N. bombycis and had a close relationship with N. bombycis. 【Conclusion】The partial sequence of the RPB1 gene of Nosema sp. PA is successfully cloned. The results confirmed that Nosema sp. PA is a member of Nosema species in the aspect of molecular biology.

AGRICULTURAL ECONOMY & MANAGMENT

Regional Differences and Influential Factors of the Development of China’s Green Food Industry

HUANG Man-yu, PENG Hu-feng

Scientia Agricultura Sinica. 2014, 47(23):  4745-4753.  doi:10.3864/j.issn.0578-1752.2014.23.019

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【Objective】 This paper is to evaluate the development level of green food industry of the 30 provinces, autonomous regions, municipalities directly under the central government of China, and to study the regional differences. It also aims to analyze the different influential effects of the factors on the level of the development of green food industry in different regions. The conclusions of the paper help to comprehensively understand the regional development of green food industry and give references to the formulation of regional policies that aim to promote the development of green food industry. 【Method】 The principal component analysis was used to evaluate the regional development level of green food industry. Clustering analysis was adopted to categorize the regions on the basis of the development level of green food industry. Grey correlation analysis was implemented to calculate the influential degree of the influential factors on the development of green food industry in different regions. 【Result】 The 30 provinces, autonomous regions, municipalities directly under the central government were categorized into three groups. The developed group includes Shandong and other 10 regions, the developing group includes Shaanxi and other 7 regions, and the under-developed group includes Chongqing and other 10 regions. The development level of green food industry relates closely to the development degree of regional economy, the correlation index amounts to 70%. The influential effect of market demand and industry structure decreases with the decreasing of the development level of green food industry, the grey correlation grade decreases from 0.298 to 0.189 and 0.233 to 0.125, respectively. While the influential effect of resource conditions and policy support increases with the decreasing of the development level of green food industry, the grey correlation grade increases from 0.125 to 0.218 and 0.183 to 0.220, respectively.【Conclusion】The development of green food industry relates closely with the development level of regional economy. The influential effect of the five factors (market demand, resource condition, industry structure, related industry and policy support) on the development of green food industry is various in different regions, and it proves that the focus of policy should be different in different regions according to the influential differences.

RESEARCH NOTES

Regional Analysis of Transcription Activity and Screening of Interaction Proteins of AtMYB73 Transcription Factor in Arabidopsis

FAN Jin-tao, JIA Jiao, JIANG Chen-xi, WANG Guan-yu, ZHANG Jing, XING Ji-hong, DONG Jin-gao

Scientia Agricultura Sinica. 2014, 47(23):  4754-4762.  doi:10.3864/j.issn.0578-1752.2014.23.020

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【Objective】 The objective of this study is to analyze the transcription activity region and screen interaction proteins of Arabidopsis resistance related transcription factor AtMYB73 and the study will lay a foundation for clarifying the regulation mechanism of the AtMYB73 gene in Arabidopsis resistance in the future. 【Method】 The bait vector pAS1-AtMYB73 was constructed and introduced into yeast Y190 by PEG/LiAC mediated transformation method. The self-activation and cytotoxicity of pAS1-AtMYB73 were detected in this paper. The transcription activity domain of the AtMYB73 was analyzed through detecting the transcription activity of the N-terminus and C-terminus of the AtMYB73. The AtMYB73 was used as bait to screen Arabidopsis cDNA library by the yeast two-hybrid system. The positive clones were screened on SD/-Ade/-His/-Leu/-Trp plates, identified by PCR and sequenced, and function of the interaction proteins were analyzed using TAIR database. The pGBDT7-AtMYB73 and pGADT7-F12F1.4 vectors were constructed and co-transformed into yeast AH109. The interaction relationship between AtMYB73 and F12F1.4 was analyzed by yeast two-hybrid system.【Result】The bait vector of the AtMYB73, pAS1-AtMYB73, was successfully constructed and transformed into yeast Y190. The pAS1-AtMYB73 yeast could grow on SD/-His/-Trp/ and SD/-Ade/-Trp plates with different concentrations of 3-AT, suggesting that AtMYB73 has higher self-activation activity. The OD600 of the pAS1-AtMYB73 yeast cultured for 24 h in SD/-Trp/Amp liquid media was greater than 0.8, showing that the bait vector has no cytotoxicity. Vectors of pAS1-AtMYB73-N and pAS1-AtMYB73-C were successfully constructed and transformed into yeast Y190, respectively. The pAS1-AtMYB73-N yeast was colorless, but the pAS1-AtMYB73-C yeast was blue. These results indicated that the C-terminus of AtMYB73 has obvious self-activation activity and the N-terminus of AtMYB73 has no self-activation activity. Eight candidate interacting proteins of AtMYB73 were obtained by screening Arabidopsis cDNA library using yeast two-hybrid system. Function annotation showed that these candidate interacting proteins were related to photosynthesis, defense reaction and resistance. The interaction relationship between AtMYB73 and F12F1.4 was determined by yeast two hybrid system. 【Conclusion】 Transcription factor AtMYB73 has higher self-activation activity and the transcription activity region of AtMYB73 was localized in its C-terminus. Eight interaction proteins related to photosynthesis, defense reaction and resistance were obtained by screening Arabidopsis cDNA library. Transcription factor AtMYB73 interacting with F12F1.4 was determined by yeast two hybrid system.

Study on Viremia of the Ducks Infected with DHOV-HB Strain

XIE Jia, HAN Chun-hua, LIN Jian, DUAN Hui-juan, YANG Zhi-yuan, ZHAO Ji-cheng, PAN Jie, LIU Yue-huan

Scientia Agricultura Sinica. 2014, 47(23):  4763-4768.  doi:10.3864/j.issn.0578-1752.2014.23.021

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【Objective】 The objective of the study is to illustrate the viremia of ducks challenged with duck hemorrhagic ovaritis virus (DHOV-HB Strain) , to gain insight into the pathogenesis of DHOV and provide data for diagnosis and vaccine development.【Method】A group of twenty-four 250-day-old Peking ducks with implanted chip were infected with 100-fold diluted DHOV-HB (3×10⁴ELD₅₀) orally, 15 ducks as negative control were kept in isolation unit under the same condition, clinical signs were observed daily. On 1-10 day post inoculation (DPI), 10 serum samples were collected from each group via wing vein daily for virus-isolation and antibody determination (select the same duck as possible). The serum samples of 1-10 DPI were inoculated into 6-day-old SPF chicken embryos via yolk-sac route. Five SPF chicken embryos were inoculated for each sample at the inoculum of 0.1 mL per embryo. Then they were hatched at 37℃ further. The chicken embryos were observed regularly twice a day. Reap and count the death chicken embryos during 24-168 h in time. Then the death chicken embryos were cut into small pieces, and then centrifuged the grinding homogenate. The viral nucleic acid was detected by RT-PCR in the supernatant. If there are more than one (including one) death chicken embryos, and the nucleic acid testing was positive, then it was concluded that virus isolation was positive. Using the classical neutralization test method to detect the serum antibody of 4-10 DPI, 5 SPF chicken embryos were inoculated for each serum dilution. The inoculation method and criteria of embryos’ specific death were performed as previously described. The determination standards are as follows: results with serum protect more than 80% (4/5) chicken embryos, the serum antibody was recognized as positive, 20%-60%(1/5-3/5) were as doubtful, and 0%(0/5) as negative.【Result】The viremia and antibody responses were closely related after infection with DHOV. The viremia could be detected as early as 1 DPI, peaked at 1-3 DPI. By this time, the positive isolation rate of serum were all 100% (10/10). On 4-6 DPI, the positive isolation rate of serum began dropping which were 90% (9/10), 70% (7/10), and 30% (3/10), respectively, and no virus was recovered anymore on 7-10 DPI. The results of the neutralization test indicated that there were low titer antibodies in the serum while the virus isolation positive rate showed a decreasing trend on 4 DPI, showed up as the death time of the embryos postponed while the antibodies of the inoculated ducks were all negative temporarily. It was 80%(8/10) negative, 20%（2/10） doubtful on 6 DPI , 70% (7/10) positive, 10% (1/10) negative, 20% (2/10) doubtful on 7 DPI, 90% (9/10) positive 10% (1/10) doubtful on 9 DPI, and the antibodies of the inoculated ducks were all positive on 10 DPI. Virus isolation (1-10d) and antibody test (4-10d) results were all negative in the negative control ducks. 【Conclusion】The duration of viremia stage of ducks infected with DHOV-HB was short, the virus was recovered in 100% (10/10) on 1-3 DPI, decreased on 4 DPI and disappeared on 7 DPI. The antibody-positive rate was 70% (7/10) on 7 DPI and reached 100% (10/10) on 10 DPI. Further analysis showed that the quickly absence of viremia was closely related to specific antibody response. The antibody-positive rate was 70% (7/10) on 7 DPI and reached 100% (10/10) on 10 DPI.