Table of Content

01 April 2015, Volume 48 Issue 7

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Female Gametophyte Genotype Selection and Its Progeny Phenotypic Genetic Differentiation in Rice at Different Altitude Condition

LEI Wei, WEN Jian-cheng, PU Shi-huang, WANG Chang-jiang1, LIU Hua-ping, SUN Chao-hua, SU Jia-xiu, LI Zhen, XU Jin, TAN Ya-ling, JIN Shou-lin, TAN Xue-lin

Scientia Agricultura Sinica. 2015, 48(7):  1249-1261.  doi:10.3864/j.issn.0578-1752.2015.07.01

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【Objective】 Understanding of rice female gametophyte genotype selection effect and its progeny phenotypic genetic differentiation at different altitude condition could be given a reference for developing breeding method of female gametophyte genotype selection affected by altitude-based change. 【Method】 Four sites with obvious altitude difference were selected to produce and grow seven F₁ female gametophyte genotype selection (FGGS) populations and its 28 F₂ progeny segregation (PS) populations generated by crosses between two rice CMS-D1 lines and two cold tolerant japonica landrances, respectively, and then through analysis of the variation of morphological characters of these populations to learn whether the presentations of female gametophyte genotypic frequency variation and its progeny phenotypic genetic differentiation were associated with different altitude backgrounds. 【Result】 Eight morphological characters of the F₁ FGGS populations at the four altitude sites were detected that significant genetic segregation distortions caused by female gametophyte genotype selection which derived from the effect of different altitude conditions, and the influence of two altitudes at 2 200 m and 400 m was greater than that of 1 860 m and 1 250 m, respectively. However, the FGGS populations produced at the altitude of 1 860 m presented the greatest diversity index of morphological characters, and then the index decreased with increase or reduction of the altitude. Comparison of the character variations of these FGGS populations generated at the four altitudes showed a significant difference between at 2 200 m and the other three altitudes. In addition, significant phenotypic genetic differentiation, which resulted from selection of the F₁ female gametophyte genotypes affected by different altitude backgrounds, was screened in their all 28 F₂ PS populations grown at the four altitudes, and also found bigger segregation while the selection derived from the largely different altitude environments. There were 21 significant differences involved in seven characters of the PS populations derived from two extreme altitudes between the highest altitude of 2 200 m and the lowest altitude of 400 m, and were only six differences involved in three characters from the both middle altitudes 1 860 m and 1 250 m. An essential change was found that the populations selected by the highest altitude had the highest average seed setting rate and poor growth potential, and by the lowest altitude showed the opposite trend against that of the highest, and by the two middle altitudes appeared larger diversified index of morphological characters. 【Conclusion】 The effect of F₁ female gametophyte genotype selection caused by pressure of different growing altitude environments was detected, which is an important factor leading to show phenotypic genetic segregation of its progeny populations, and then to develop new features adapting to corresponding ecological environment. So it is feasible for developing breeding method of female gametophyte genotype selection caused by altitude condition difference.

Spatiotemporal Expression Pattern Analysis of NAM Transcription Factors Gpc-1 and Gpc-2 in Bread Wheat Cultivar Chinese Spring During Grain Filling

WU Dan, DONG Jian, YAO Yan-jie, ZHAO Wan-chun, GAO Xiang

Scientia Agricultura Sinica. 2015, 48(7):  1262-1276.  doi:10.3864/j.issn.0578-1752.2015.07.02

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【Objective】The objective of this experiment is to study the roles of no apical meristem (NAM) transcription factors Gpc-1 and Gpc-2 in early senescence and nutrient remobilization to the grain of bread wheat. 【Method】 Their spatiotemporal expression patterns were investigated during the grain-filling stage in wheat cultivar Chinese Spring. Their temporal expression dynamics were studied in penultimate leaf, flag leaf, peduncle, glume, rachis and the kernel using quantitative real time polymerase chain reaction (qRT-PCR). And the relative expression level was quantified using Pfaffl method with normalization against multiple verified reference genes. Applying mRNA in situ hybridization, the spatial expression pattern was explored in post-anthesis flag leaf, peduncle and the kernel only with digoxin-labeled oligonucleotide probes which were specifically targeting 5′ or 3′ untranslated regions (UTRs) of Gpc-1 and Gpc-2. 【Result】 Contrary to a previous report, the functional TaNAM-B1 rather than its dysfunctional paralog was found in Chinese Spring, and its nucleotide sequence was identical with the wild-type TtNAM-B1 in T. turgidum TaNAM-B2 and TaNAM-A1 were the highest in flag leaf among five NAM genes.TaNAM-A1, TaNAM-B1, TaNAM-B2 and TaNAM-D2 were expressed in all studied tissues before anthesis, whereas TaNAM-D1 was expressed only since the ?rst-?owering date. TaNAM-A1 exhibited the distinct expression patterns among the tissues. Its transcript abundance began to decline in penultimate leaf, rachis and the kernel at 15 DAA, which was preceded that in flag leaf (25 DAA) and other tissues. The transcription levels of TaNAM-B1 and TaNAM-D2 kept increasing till the late filling stage (25 DAA or 30 DAA) in most organisms except for advanced declines of TaNAM-B1 and TaNAM-D2 in the grain and the glume, respectively.(25 DAA) and the peduncle (20 DAA). Unlike other genes, a consistent growth until 25 DAA and 30DAA in transcript abundance of TaNAM-D1 was observed. 【Conclusion】 In conclusion, Gpc-1 and Gpc-2were closely associated with mineral translocation in the grain; however, no obvious relationship between five NAM genes and the PCD in wheat grain was observed. And the discrepant temporal expression dynamics suggested that their functions were not identical. In addition, the potential effect of these genes on senescence in vegetative tissues was still elusive. Combined with previous findings, it was proposed that Gpc-1 and Gpc-2 may directly regulate nutrient remobilization only or in parallel with the senescence during grain filling. The expression level of TaNAM-B2 decreased from 15DAA in most tested tissues other than the flag leaf var.dicoccoides. All the results showed that Gpc-1 and Gpc-2 were all widely expressed in studied tissues with the exception of the root in which only the transcript of Gpc-1was detected. The outcomes of mRNA in situ hybridization indicated that all five genes shared cell-type specificities. To be specific, no transcripts were distributed in leaf epidermal cells, pericarp and the seed coat; however, they mainly aggregated in leaf mesophyll cells, aleurone layer, embryo, and the tissues responsible for the mineral element transport (vascular bundle, pigment strand, nucellar projection and the transfer cell) in grain, in which the highest expression level was observed in embryo. In addition, lower expression level was detected in the peduncle and leaf vascular bundle as well. The results of qRT-PCR showed that the temporal expression dynamics of Gpc-1 and Gpc-2 differed upon the organs and the genes. According to the Ct value, TaNAM-D2 was expressed less than other genes in all tested tissues. And the most abundant gene in wheat kernel was TaNAM-D1,whereas that in peduncle, glume and the rachis was TaNAM-B2. Besides, the expression levels of

Advances in Research of the Regulation of Transcription Factors of Lignin Biosynthesis

GUO Guang-yan, BAI Feng, LIU Wei, BI Cai-li

Scientia Agricultura Sinica. 2015, 48(7):  1277-1287.  doi:10.3864/j.issn.0578-1752.2015.07.03

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Lignin is an important component of secondary cell wall in vascular plants and has important biological functions. Lignin, cellulose and hemicellulose are crosslinked in the cell wall and provide mechanical support for the plant cells and tissues. The hydrophobic property of lignin makes it impermeable to water, which facilitates the long-distance transport of water and nutrients in plant. Lignin and cellulose are natural physical barriers to various pathogens, which improve the defensive ability against biotic and abiotic stresses. While lignin also has some negative effects on the productive practice, e.g., in pulp and paper industry, many chemicals must be used to remove lignin, which increases the cost of pulping and pollution to the environment. High lignin content in the forage decreases the digestibility of livestocks and affect the nutritive value of forages. Higher lignin content also has a negative effect on the fermentation efficiency of biomass energy. Therefore, it is of great significance to improve the lignin degradability by genetic engineering. In higher plants, lignin can be synthesized by phenylpropanoid pathway and specific lignin biosynthesis pathway. Previous research has shown that NAC, MYB and WRKY transcription factors involved in the regulation of lignin biosynthesis pathway. In Arabidopsis, MYB26 can activate the transcription of NST1/NST2; WRKY12 can bind to the promoter region of NST2 and regulate its expression negatively. SND1 (NST3) and NST1 function redundantly in the regulation of secondary wall synthesis in fibers; NST1 and NST2 are redundant in regulating secondary wall thickening in anther walls; VND6 and VND7 mainly involved in xylem vessel differentiation. All these NAC transcription factors can bind to the downstream MYB transcription factors such as MYB83, MYB46 as well as (or) MYB58, MYB63, MYB85 and MYB103 to regulate lignin biosynthesis positively, whereas MYB75 regulates the lignin biosynthesis negatively. Most of the MYBs in this network can bind to the AC elements (I, II and III) in the promoters of the lingin biosynthesis pathway genes and therefore regulate their expression. Some studies also suggested the involvement of bHLH transcription factors in the regulation of lignin biosynthesis pathway. In this paper, advances in research of the regulation of transcription factors on lignin biosynthesis were reviewed, the major regulatory network of lignin biosynthesis in Arabidopsis was produced, some transcription factors related to lignin biosynthesis in other species (e.g. rice, wheat, maize, eucalyptus, pine and populus) were also summarized. With the development of high-throughput sequencing technology, key regulatory transcription factors will be discovered in more species, which will have an important reference to the lignin modification by genetic engineering.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY

Post-Anthesis Alternate Wetting and Moderate Soil Drying Irrigation Enhance Gene Expressions of Enzymes Involved in Starch Synthesis in Rice Grains

CHEN Ting-ting, XU Geng-wen, QIAN Xi-yang, WANG Zhi-qin, ZHANG Hao, YANG Jian-chang

Scientia Agricultura Sinica. 2015, 48(7):  1288-1299.  doi:10.3864/j.issn.0578-1752.2015.07.04

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【Objective】Grain filling in rice is a physiological process of transportation and conversion of photosynthetic assimilates into starch which determines the percentage of filled grains, grain weight, and rice quality. The process of grain filling could not only be affected by genetic factors, but also be regulated by environmental conditions such as temperature and moisture. Irrigation is an important technique in rice production and plays a vital role in regulating the formation of grain yield. However, little work has been done in the effect of postanthesis irrigation patterns on the expressions of genes encoding starch biosynthesis in rice grains. This study aims to investigate the effect of post-anthesis alternate wetting and drying irrigation on grain filling of rice and to understand its molecular mechanism.【Method】Two cultivars Liangyoupeijiu (two-line indica hybrids) and Yangjing 4038 (japonica hybrids) were used and grown in the cement tanks that were filled with soil. Three irrigation regimes were imposed from heading (50% of the panicles were protrudent the flag leaf sheath) to maturity: (1) conventional irrigation (CI), plots were kept a continuous shallow water layer until one week before harvest; (2) alternate wetting and moderate soil drying irrigation (WMD), the field was not irrigated until the soil water potential reached the threshold at -20 kPa, and then was flooded with 1-2 cm water depth, and was repeated this process again; (3) alternate wetting and severe soil drying irrigation (WSD), water in the plots was withheld until the soil water potential reached the threshold at -40 kPa, and then was flooded with 1-2 cm water depth, and this process was repeated again. The effect of irrigation regimes on grain filling rate, grain weight, activities of the enzymes involved in starch synthesis in superior and inferior spikelets, and expressions of the genes encoding these enzymes were determined using real-time quantitative PCR.【Result】The results showed that there were no significant differences in grain filling rate, grain weight, activities of enzymes involved in starch synthesis including sucrose synthase (SuS), adenosine diphosphate glucose pyrophosphorylase (AGP), starch synthase (StS), starch branching enzyme (SBE) and SuS2, SuS4, AGPL1, AGPL2, AGPL3, AGPS2, SSI, SSIIa, SSIIc, SSIIIa, SBEI and SBEIIb gene expressions in superior spikelets among the three irrigation treatments. Compared with CI, the WMD significantly increased the mean grain filling rate, grain weight, activities of the four enzymes involved in starch synthesis and their gene expressions in inferior spikelets except for AGPL1, whereas the WSD performed an opposite effect. The two trial cultivars behaved the same tendency. The correlation analysis showed that the mean grain filling rate and the grain weight of inferior spikelets were very significantly and positively correlated with activities of SuS, AGP, StS, SBE and gene expressions of SuS2, SuS4, AGPL2, AGPL3, AGPS2, SSI, SSIIa, SSIIc, SSIIIa, SBEI and SBEIIb. 【Conclusion】The results indicate that enhanced activities and gene expressions of the enzymes involved in starch synthesis in inferior spikelets under WMD contribute to their greater grain filling rate and grain weight through enhancing the starch synthesis and accumulation. The reduction in the grain filling rate and grain weight of inferior spikelets under WSD would be attributed to the decreased activities and gene expressions of the enzymes involved in starch synthesis.

Effects of Sowing Date on Formation of Branches and Spikelets in Indica-Japonica Hybrid Rice

ZENG Yan-hua, ZHANG Yu-ping, WANG Ya-liang, XIANG Jing, CHEN Hui-zhe, ZHU De-feng

Scientia Agricultura Sinica. 2015, 48(7):  1300-1310.  doi:10.3864/j.issn.0578-1752.2015.07.05

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【Objective】The objective of the study is to elucidate the effects of sowing date on characteristics of spikelets formation of hybrid rice with different compositions of indica and japanica.【Method】The inbred and hybrid varieties with different compositions of indica and japanica, Yongyou8 and Yongyou15, Qianyou2 and Zhejing88 were used in this study. The characteristics of differentiation and degradation of branches and spikelets were investigated, and the relationship between the composition of indica & japonica and the character of rice panicle was analyzed.【Result】The total number of differentiated spikelets per panicle of hybrid rice Yongyou 8 and Yongyou15 with indica-japonca blood were significantly higher than the indica rice Qianyou2 and japonica rice Zhejing88. The numbers of differentiated primary branches and spikelets on primary branches of Yongyou 8 and Yongyou15 were also significantly higher than the indica and japonica rice. The number of secondary branches, and spikelets on secondary branches of Yongyou15 and Qianyou2 was more than two others rice varieties. The number of differentiated spikelets decreased significantly with the sowing date delaying (ie. 28, June), especially the number of differentiated spikelets on secondary branches, but differentiated spikelets on primary branches on suitable sowing date (ie. 21, June) was more than other sowing date. More number of differentiated spikelets of Yongyou8 and Qianyou2 was found when the sowing date was ahead (ie.14, June). Degradation spikelets on primary branches of Yongyou varieties decreased significantly at suitable sowing date, and low spikelets degradation was observed for Yongyou8 in the middle-upper part of panicle and the base part or top part for Yongyou15, respectively, and that caused by higher primary branches differentiation. In all sowing date treatments, the number of differentiated secondary branches decreased from base panicle to top panicle in all varieties, and the secondary branches, spikelets on secondary branches showed an increased and then decreased tendency. The advantage was that low degradation of secondary branches, spikelets on secondary branches in the middle-top of panicle of Yongyou8 when the sowing date was ahead, and that was the base-middle of panicle for Qianyou2. The low degradation of spikelets was found in the top of panicle of Yongyou15 at suitable sowing date, and the advantage of sowing date delaying was that low degradation of spikelets happened in the base of panicle of Zhejing88. There was a significantly parabolic correlation between the japonica consanguinity component and the number of differentiated spikelets, primary, secondary branches and its spikelets, and the correlation coefficient was 0.8172, 0.9416, 0.9004, 0.8718 and 0.7988, respectively.【Conclusion】There were more branches and spikelets of indica-japonica hybrid rice than indica and japonica rice. Differentiated spikelets and secondary branches per panicle were reduced significantly with sowing date delaying, and it promoted the formation of spikelets and secondary branches when the sowing date was ahead for medium-maturing varieties, while there was a positive effect for late-maturing varieties by selecting suitable sowing date. There was a significantly correlation between the japonica consanguinity component and the branches, spikelets of panicle in all sowing date treatments.

Effects of Sub-Soiling Tillage on Wheat Field Water-Saving and Yield-Increasing in Canal Irrigation District of Haihe Lowland Plain

YIN Bao-zhong, ZHANG Yong-sheng, ZHEN Wen-chao

Scientia Agricultura Sinica. 2015, 48(7):  1311-1320.  doi:10.3864/j.issn.0578-1752.2015.07.06

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【Objective】The objective of this experiment is to explore the water-saving and yield-increasing effect of sub-soiling tillage in wheat field in canal irrigation regions, and aim to increase the yield potential and the water storage capacity.【Method】Using Liangxing 99, a winter wheat cultivar currently cultivated in Haihe low land plain as material, three tillage treatments including rotary tillage (RT), sub-soiling tillage (SRT), and moldboard plow tillage (MRT) were set up in a field experiment in canal irrigation region of Haihe lowland plain during 2011-2012 and 2012-2013. 【Result】 Sub-soiling tillage improved the soil water infiltration rate, in which the soil water infiltration rate of SRT treatment was 0.05 mm·s^-1 that was 2.50 and 1.67 folds of those in RT treatment and MRT treatment, respectively. Sub-soiling tillage was conducive to infiltration of the irrigated water in the soil, in favour of the water distribution in deep soil, and improvement of the storage capacity of surface water effectively. After 48 h of irrigation, the soil water storage content in SRT treatment was 158.5 mm in 0-180 cm soil layer, where it was 142.5 mm and 144.1 mm in the RT and MRT treatments, respectively. They were equivalent to that of 89.9% and 90.9% of the SRT treatment. Before wintering stage, the evaporation rate was the highest in MRT treatment, which was 1.15 and 1.35 times higher than the treatments of SRT and RT, respectively. After regreening stage, the evaporation rate in RT treatment was increased, and gradually increased to the date of spring irrigation with a value of 1.32 mm·d^-1. However, the evaporation rate in treatments of SRT and MRT were low, with values of 0.78 mm·d^-1and 0.85 mm·d^-1, respectively. The evaporation amount was the lowest in the SRT treatment (138.17 mm), which was equivalent to 86.9% and 89.7% of RT and NRT, respectively. The water storage content in 0-100 cm soil layer in RT treatment was more than those in SRT and MRT treatments from sowing date to jointing stage. Further analyses indicated that RT treatment had the highest water storage content in 0-20 cm soil layer, whereas SRT treatment had the highest water storage content in 20-80 cm soil layer. There were no significant differences in water storage content under the soil layer below the 80 cm layer among the three treatments. The total water consumption in SRT treatment was 419.1 mm across the whole growth stage, which saved water by about 6% in comparison with the treatments of RT and MRT. Of which, the consumption ratio of irrigation to precipitation in SRT treatment was 41.2% and 22.0%, respectively, which was significantly higher than those in MRT and RT treatments. The average yield in SRT treatment was 8 550 kg·hm^-2 across the two growing seasons. It had increases of 15.4% and 6.9% compared with those in treatments of RT and MRT, respectively. Moreover, the water use efficiency in SRT treatment increased by 22.9% and 14.0% compared with those in treatments of RT and MRT, respectively. 【Conclusion】 Sub-soiling tillage improved soil water infiltration rate, decreased the evaporation amount of irrigation and precipitation, and reduced the water consumption in winter wheat field. This tillage method could increase the water use efficiency and irrigation productivity efficiency and yield. Accordingly, sub-soiling tillage practice is recommended in winter wheat production in canal irrigation regions in Haihe lowland plain.

PLANT PROTECTION

Verification of Verticillium dahliae Pathogenicity of Glycometabolism Related Genes by Using Host-Induced Gene Silencing Method

ZHAO Yu-lan, SU Xiao-feng, CHENG Hong-mei

Scientia Agricultura Sinica. 2015, 48(7):  1321-1329.  doi:10.3864/j.issn.0578-1752.2015.07.07

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【Objective】 The objective of this study is to screen glycometabolism related genes from Verticillium dahliae, confirm the relationship between those genes and the growth and pathogenicity of V. dahliae, and to provide a theoretical basis for the control of plant Verticillium wilt. 【Method】 The glycometabolism related genes of V. dahliae were screened using host-induced gene silencing (HIGS) method. The Tobacco rattle virus vectors containing target genes were injected into tobacco for transient expression of those genes, then V.d991 was inoculatedto those transgenic tobacco using the root-dip approach, PDS would be whitened 7-10 days later, and their disease indexes were counted after 14 days. The fluorescence quantitative PCR was used to detect the amount of V.d991 biomass and the expression of VDPDF-1, VDEG-1, VDMAN-1and VDPD-1. 【Result】 Four glycometabolism related genes from V. dahliae (VDPDF-1, VDEG-1, VDMAN-1 and VDPD-1) were selected because of the great change of their disease indexes. Transgenic tobacco disease indexes of these four genes were reduced by 35%, 30%, 20%, and 10%, respectively, compared with the negative control. After injected with the mixture of VDEG-1/VDPDF-1, VDPD-1/VDPDF-1/ VDMAN-1, VDPD-1/VDMAN-1, VDPD-1/VDPDF-1,and VDEG-1/VDPD-1, transgenic tobacco disease indexes were reduced by 45%, 45%, 40%, 40%, and 35%. In addition to VDPD-1 transgenic tobacco, the transgenic tobacco disease indexes had significant differences compared with the control. After inoculation withV.d991, the V.d991 biomass and expression of target gene were decreased, mixed gene injection had better interference effect than the single gene. The most obvious biomass reduction wereEG/PDF (VDEG-1/VDPDF-1)and EG/PD (VDEG-1/VDPD-1), reached to 0.94, and the most significant interference effect was VDPDF-1, from PD/PDF (VDPD-1/VDPDF-1)，reduced by 0.91. 【Conclusion】 Four genes were selected from V. dahliae by HIGS method. When these genes were interfered, those plants had high resistance to disease, mixed injection of multiple gene vectors were more significant than the single gene. These genes were confirmed with the glycometabolism in V.d991, probably related with V. dahliae pathogenicity.

Isolation and Biological Properties of a Lytic Phage Infecting Ralstonia solanacearum

GAO Miao, YANG Jin-guang, LIU Xu, LIU Wei, SUN Hang-jun, SHEN Li-li, QIAN Yu-mei, YANG Qing-lin, YU Guang-hong, LI Xi-hong, WANG Feng-long

Scientia Agricultura Sinica. 2015, 48(7):  1330-1338.  doi:10.3864/j.issn.0578-1752.2015.07.08

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【Objective】In order to provide a new formulation of anti tobacco bacterial wilt, a lytic phageof Ralstonia solanacearum was isolated and purified from natural environments, and its biological characteristics were determined.【Method】Rhizosphere soil samples were taken from healthy tobacco plants growing in the disease field of tobacco bacterial wilt. Soil suspension after filtration was added into the R. solanacearum bacterial liquid to enrich the phage by co-cultivating. The existence of phage was detected using the way of two-layer plating method and a single clear of plague was obtained through purifying the single maximum phage repeatedly. The purified single phage was added into R. solanacearum bacterial liquid at logarithmic phase to propagate the phage culture. 20 μL concentrate of the phage particles by the conventional method was stained with phosphotungstic acid to observe their morphological characteristics by electron microscope. Meanwhile, the concentrate of phage’s fluid was used to detect the number and size of phage’s structure protein by SDS-PAGE electrophoresis, and confirm the size of genome fragment by agarose gel electrophoresis after extracting the total nucleic acid of the phage using the phage DNA extraction kit. The other biological properties include titer, MOI, one-step growth curve were measured by the conventional method, and the sensitivity to temperature, pH, ultraviolet ray and chloroform was tested by comparing the change in the OD₆₀₀ of the R. solanacearum bacterial liquid before and after adding the phage.【Result】A strain of lytic R. solanacearum phage named as ∈RS-1 was isolated and purified. Its plaques are circular, clear and transparent with smooth edge, showing 1-2 mm of diameter. The electron microscope result showed that the phage particles like tadpoles with an icosahedra head of 94 nm diameter, and a long flexible tail about 27 nm×100 nm. According to the classification standards of the international commission on virus classification, it is a lytic phage of Myoviridae in Caudovirales, and its nucleic acid is dsDNA. There were 25 protein bands showing relative molecular mass of 10-100 kD in the result of SDS-PAGE, meaning the phage had at least 25 structural proteins. The agarose gel electrophoresis showed that its genome size was greater than 48 kb, fitting to the characteristics of Myoviridae (31-317 kb). Measurement of biological properties showed that the multiplicity of infection (MOI) of the R. solanacearum phage was 0.01. Its incubation period was 30 min, burst phase time was 80 min, and the burst size was 156 when infecting and locating on the R. solanacearum. The activity of phage ∈RS-1 was strong from 28℃ to 50℃, reaching a supreme at 28℃, however showing inactivation at 60℃. And it had wide susceptibility to pH changes, but its activity was decreased when the pH was above 9. The phage ∈RS-1 was also insensitive to ultraviolet ray, its activity was stable when exposed to ultraviolet ray for 0-9 min and began to decline after 21 min, 24 min later it was inactivated. It was insensitive to chloroform, 5% concentration of chloroform had no effect on its activity. 【Conclusion】In this experiment, a lytic R. solanacearum’s phage was isolated belonging to Myoviridae in Caudovirales. With some good biological properties including short incubation period, strong sterilization ability and stable in wide temperatures and pH, it has a potential to be developed as a new preparation for control the R. solanacearum bacterial disease.

Comparative Analysis of the Competitiveness Between B and Q Biotypes of Bemisia tabaci Under Laboratory Conditions

LU Shao-hua, LI Jing-jing, LIU Ming-yang, BAI Run-e, TANG Qing-bo, YAN Feng-ming

Scientia Agricultura Sinica. 2015, 48(7):  1339-1347.  doi:10.3864/j.issn.0578-1752.2015.07.09

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【Objective】B and Q biotypes of Bemisia tabaci are two invasive cryptic species in China. The field investigation showed that Q biotype is becoming the dominant replacing B biotype in most areas of China in recent years. It is necessary to research the mechanisms and the influencing factor of the B. tabaci biotype replacement. Differences of feeding behaviors and competitiveness between B and Q biotypes of B. tabaci were compared under laboratory conditions, aiming to provide a basis for effective management of whiteflies. 【Method】 The stylet probing behaviors of B and Q biotypes of B. tabaci on Cucumis sativus were recorded and analyzed by using the electrical penetration graph (EPG) technique. The competitiveness of the two biotypes of B. tabaci was compared by investigating the population dynamics of the two biotypes in six successive generations on C. sativus. 【Result】 EPG results showed that B biotype B. tabaci had significantly more total number of probes (P=0.020) and longer total duration of probes (P=0.048) than Q biotype, and during the 2nd phase, the percentage of E was mainly lower (P=0.001) in B biotype than Q biotype, but there was no significant difference in percentage of probing duration between the two biotypes. Q biotype had significantly more total number of Pd (potential drop) (P=0.012) and longer total duration of Pd (P=0.016). For comparison of males and females in the same biotype, B biotype females were significantly lower than B biotype males in the percentage of Np (P=0.035) and the percentage of E (P＜0.001) during the 2nd phase, but females had significantly higher percentage of probe duration (P=0.011) than males. However, there was no significant difference in the duration of E(P＞0.05) in the 3rd phase between males and females of B biotype. For Q biotype, females not only had significantly higher percentage of probing duration (P=0.031) in the 2nd phase than Q biotype males, but also had significantly longer total duration of probes (P=0.039). Q biotype females had significantly shorter total duration of Np (P=0.038) than Q biotype males. There were no significant differences between males of B and Q biotypes for all EPG variables. For females whiteflies, B biotype had significantly more total number of individuals with E2＞10 min (P=0.036) than Q biotype, but Q biotype females had significantly higher percentage of E (P＜0.001) than B biotype in the 2nd phase. The results of the population dynamics of whiteflies on cucumber plants showed that the percentages of populations of the two biotypes became more and more divergent with the generations. In the 1st generation, the percentage of B biotype in the whole population almost reached 80%, and the percentage of the B biotype out of the whole population in the 5th generation and 6th already reached 98%. 【Conclusion】 Both the feeding ability and the competitiveness of B. tabaci B biotype were higher than those of Q biotype on cucumber without insecticide stresses in the laboratory, and those of females in either biotype of B and Q were higher than males.

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

Effects of Soil Profile Basic Properties on Water and Nitrogen Movement and Crop Yield

LIU Hai-tao, HU Ke-lin, LI Bao-guo, REN Tu-sheng

Scientia Agricultura Sinica. 2015, 48(7):  1348-1360.  doi:10.3864/j.issn.0578-1752.2015.07.10

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【Objective】The North China Plain (NCP) is one of the major winter wheat and maize production areas in China. Spatial variance of soil basic properties is one of the main reasons for the spatial variance in the grain yield. This study was carried out in order to quantify the effects of soil profile basic properties differences on water and nitrogen movement and crop yield in the farmland with the soil type of Alluvial Cambisols, and crop system of winter wheat and maize rotation in the NCP. Results of the study will give some suggestions on promoting grain yield, water and nitrogen use efficiency in NCP. 【Method】Three plots of field in Tai’an, Shandong Province with different soil profile basic properties and yield levels were selected as the three treatments and consistent field management strategies were applied in these fields. The soil profile properties such as soil particle size distribution, saturated hydraulic conductivity (K_s), field water capacity (FC), permanent wilting point (PWP), soil organic carbon (SOC) and soil total nitrogen (TN), soil moisture and nitrate content at the 0-160 cm soil profile, crop growth and grain yield were measured for three years. The water and N movement and balance were simulated using the Root Zone Water Quality Model (RZWQM).【Result】The dynamic of soil moisture, nitrate content, grain yield, aboveground biomass and LAI between Oct 2009 and Sep 2012 was precisely simulated by RZWQM, water and nitrogen balance was got from the model. The effects of basic soil properties on soil water and nitrogen movement, and yield was as follows: The maximum soil available moisture in 0-160 cm depth in the high yield field was 223 mm, which was 28 mm and 56 mm higher than the values in the intermediate yield field and low yield field, respectively. The saturated hydraulic conductivity at the ＞30 cm soil layers in the high yield field was lower than the values in the intermediate yield field and low yield field. These basic property differences resulted in the water loss (runoff + drainage) in the high yield field (150.3 mm) was 5.7 mm and 26.4 mm less than the intermediate yield field and low yield field. Thus less water stress occurred in most of time in the high yield field. The soil organic carbon and C﹕N ratio in the high yield field was higher than intermediate and low yield fields which resulted in higher organic matter mineralization rate (52.0 kg·hm^-2 and 82.6 kg·hm^-2 higher than the values in intermediate yield field and lower yield field). The nitrogen loss (ammonia volatilization + leaching + denitrification) in high yield field was 6.9 kg·hm^-2and 10.9 kg·hm^-2 less than the values in the intermediate yield field and low yield field. Thus less nitrogen stress occurred in most of time in high yield field. The water use efficiency (WUE) in the high yield field was 2.32 kg·m^-3, which was 12.1% and 6.87% higher than the values in the intermediate yield field and low yield field, respectively. That was caused by less nitrogen stress in the high yield field. There was no significant difference in nitrogen use efficiency (NUE) in the fields with different basic properties in this study.【Conclusion】The soil basic properties such as higher soil profile available water moisture, superior profile saturated hydraulic conductivity distribution, higher soil organic carbon and lower C﹕N ratio can promote water and nitrogen supply, decrease water and nitrogen loss. The farmland in the crop system of winter wheat and maize rotation in NCP with these soil basic properties suffers less water and nitrogen stress, and thus leading to high yield.

Effects of Different Straw Biochar on Nutrient and Microbial Community Structure of a Red Paddy Soil

LI Ming, LI Zhong-pei, LIU Ming, JIANG Chun-yu, WU Meng

Scientia Agricultura Sinica. 2015, 48(7):  1361-1369.  doi:10.3864/j.issn.0578-1752.2015.07.11

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【Objective】The various effects of different straw biochar on nutrient content and microbial community structure were studied in order to provide information for soil amelioration and proper management of straw residue.【Method】Through a 135-day laboratory incubation experiment and used a red paddy soil that originated from the Quaternary, the influences of rice and corn straw biochar that pyrolyzed at 300℃, 400℃ and 500℃ on soil pH, organic carbon, nutrient content, microbial biomass carbon and profile of microbial PLFAs community structure were investigated. The experiment consisted of seven treatments: control soil (CK), soil amended with 300℃ (RB300), 400℃ (RB400) and 500℃ (RB500) rice straw biochar, soil incorporated with 300℃ (CB300), 400℃ (CB400) and 500℃ (CB500) corn straw biochar. 【Result】 Feedstock type and pyrolysis temperature had a significant influence on the nutrient contents and chemical properties of biochar products. Compared with control, the two straw biochar amendments increased pH value by 0.16 unit and enhanced the contents of soil organic carbon, available P and available K by 26.1%, 20.6% and 281.8%, respectively. Under the same pyrolysis temperature, the application of rice straw biochar mainly promoted the level of available K while corn straw biochar improved the content of available P. Application of 300℃ straw biochar had no significant effect on soil available and mineral N contents. Compared with the control, soils amended with RB500 and CB500 were, respectively, 10.4% and 8.1% less in available N, while significantly increased by 63.6% and 100.7% in NO₃^--N concentration. Although the concentrations of microbial biomass carbon and total phospholipid fatty acids for soils amended with straw biochar were 63.4% and 47.5% higher than control soil, there was no significant difference between the control soil and soils with 300℃ straw biochar. Both the two types of biochar enhanced the contents of G-, G+, fungi and actinobacteria and shown as 300℃＜400℃＜500℃. Results of PCA indicated that rice straw biochar amendment had more effect on the structure of soil microbial community than corn straw biochar. The microbial community compositions of three rice straw biochar were separated from each other while no distinctive recognized among the three corn biochar. Results of CCA suggested that straw biochars can affect the composition of microbial community through altering soil chemical and nutrient properties, as soil available P, soil organic carbon and available had significant correlation with the distribution of soil microbial community. 【Conclusion】 Both the two straw biochars could ameliorate the acidity and nutrient content of red paddy soil, and enhance the level of soil microbial biomass. Soil microbial community structure had been affected in the presence of straw biochars and rice straw biochar had more effective influence than corn straw biochars.

HORTICULTURE

Analysis of Phenolic Compounds in Red Grape Varieties

ZHANG Juan, WANG Xiao-yu, TIAN Cheng-rui, ZHAO Qi-feng, MA Xiao-he, TANG Xiao-ping, MA Ting-ting, MA Jin-jin

Scientia Agricultura Sinica. 2015, 48(7):  1370-1382.  doi:10.3864/j.issn.0578-1752.2015.07.12

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【Objective】 Phenols are the most important substances of wine, which not only affect the sensory quality of wine, but also determine the function of the physiological activity of wine. The contents and types of phenolic compounds, as well as the differences of phenolic compounds in different parts of grape fruits in 20 red wine grape varieties, which were taken from areas of the Shanxi Loess Plateau, were studied in order to acquire more effective improvement in wine making process, and provide a theoretical basis for the breeding of wine grapes. 【Method】 Using 20 red grape varieties collected from Pomology Institute of Shanxi Academy of Agricultural Sciences, grape resources garden (Taigu) as materials, berry size and its shape index, mass and percentage of different parts, soluble solids content (TSS), titratable acid content, TPC, TFA, CGE and monomer phenol content were measured. 【Result】 The result indicated that the fruit shape index of 20 grape varieties were higher than 0.95, and the fruit shape was oval or conical. There was a significant difference between the proportion of grape peels, seeds and pulps. The ‘Cabernet Sauvignon’ with a higher quality was the first cultivar in China, its proportion of peels and seeds were the highest and reached 31.3%; whereas the proportion of peels and seeds of ‘Cabernet Gernischet’ was lower, only 10.62%, meanwhile the wine fermented by ‘Cabernet Gernischet’ had a lower content of polyphenols and a supple texture. Titratable acid content of 20 varieties of grapes was 3.97-9.00 g·L^-1 and the content of soluble solid was 14.5-25.4 g·L^-1. Different grape varieties and different parts of the grape fruit (skin, seed and pulp) were significantly different in the polyphenols content. TPC (total phenolic content) of grape peel was 12-125 mg·100g^-1FW (fruit fresh weight), TFA (total flavanol content) was 1-27 mg·100g^-1FW; TPC of grape seed was 17-456 mg·100g^-1 FW, TFA was 22-505 mg·100g^-1FW, TPC of grape pulps was 1.24-3.24 mg·100g^-1FW, that TPC in grape seed was 3-4 times of grape peel, TFA in the seed was 10 times more than the peel. Anthocyanin mainly existed in grape peels and the content of anthocyanin of grape peels was 1-34 mg·100g^-1FW. Among the 20 varieties of grape, ‘Cabernet sauvignon’ exhibited the highest anthocyanin content, which was followed by ‘Ruby cabernet’, ‘Marselan’ and ‘Cabernet gernischet’. The grape cultivars such as ‘Marselan’ and ‘Cabernet sauvignon’ had higher phenolic compounds, meanwhile the grape cultivars such as ‘Cabernet gernischet’, ‘Heihuxiang’ and ‘Cinsaut cehco’ had lower phenols content. The content of phenolic compounds in different parts of various grapes was significantly different. The order was that grape seeds was the highest, followed by the grape skins, and the pulp was the lowest. The phenolic compounds content in pulp was much lower than that in skins and seeds, and pulp mainly contained phenolic acids. A total of 12 kinds of monomeric phenols in grape pulps were identified by HPLC, including 4 kinds of flavonoids, 7 kinds of phenolic acids and resveratrol. 【Conclusion】 Characters of grape affected not only the quality of wine, but also the phenolic compounds in wine. The final content of phenolic compounds in wine was determined by the size of fruit, the proportion between peels and seeds, the phenolic content and variety of grape peels, seeds and pulps. By controlling the polyphenols content of the grape skin, seed and pulp through wine making technology, the quality of red wine can be improved. Based on the characteristics of grape polyphenols, a selective grape breeding work should be carried out.

The Influence of ‘Bingtang’ Sweet Orange or Citron C-05 on the Growth Characteristics of Xanthomonas axonopodis pv. citri

GE Hong-juan, LONG Gui-you, DAI Su-ming, LI Da-zhi, LI Na, DENG Zi-niu

Scientia Agricultura Sinica. 2015, 48(7):  1383-1391.  doi:10.3864/j.issn.0578-1752.2015.07.13

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【Objective】A new inoculation method was used to discuss the difference of the influence of ‘Bingtang’ sweet orange and C-05 on the growth characteristics of Xanthomonas axonopodis pv. citri (Xac)by comparing different influences of leaves on Xac.【Method】The fully expanded leaves with light green of ‘Bingtang’ sweet orange and citron C-05 were sterilized by 75% ethyl alcohol and 1% NaClO, cut and cultured with Xac in the same petri dish to determine the influence of ‘Bingtang’ sweet orange or citron C-05 on the growth of Xac in MT medium or the NYGA medium replaced the middle place of the MT medium (MA). And the leaves’ solution of ‘Bingtang’ sweet orange or citron C-05 were extracted and added with 25%, 50%, and 75% to MT medium to determine the influence on the growth of Xac; also these were added with different ratios to NYGB medium to determine the content of extracellular polysaccharide (EPS) of Xac,respectively. 【Result】The leaves of ‘Bingtang’ sweet orange promoted the growth of Xac on MT medium. The proliferation of the strain could be viewed after 1 week, the average diameter of Xac was 0.5 mm larger than CK. After 2 weeks, the average diameter of Xac was 5.27 mm, which was the 3 times of that of CK. From 2 to 3 weeks was the rapid growth stage of Xac, the average diameter of Xac was from 5.27 mm to 13.41 mm, which was more than 5 times of that of CK. After 3 weeks, the Xac cultured with the leaves of ‘Bingtang’ sweet orange in MT medium grew smoothly and steadily. Compared with that of CK, the diameter of Xac cultured with the ‘Bingtang’ sweet orange was very significantly different. The Xac cultured with the leaves of ‘Bingtang’ sweet orange in MA medium was 4.58 mm after 1 day, and that of CK was 6.19 mm. After 3 days of inoculation, the average diameter of Xac cultured with ‘Bingtang’ sweet orange was the same as CK, and there were no significant differences with each other. After 5 days, the Xac cultured with the leaves of ‘Bingtang’ sweet orange grew rapidly and the average diameter was 21.31 mm, which was very significantly different with CK. Also, the leaves’ extracted solution of ‘Bingtang’ sweet orange was able to increase the growth speed and the content of EPSof Xac. Compared with CK, they all reached very significant level. The leaves of citron C-05 could control the growth of Xac in MT medium. After 1 week, there were no significant differences with CK. After 2 weeks, the average diameter of Xac cultured with the leaves of citron C-05 was the half of that in MT medium. After 3 weeks, the average diameter of Xac in MT medium containing the leaves of citron C-05 was 2.06 mm, which was smaller than CK (2.62 mm), and the diameter of Xac cultured for 2 and 3 weeks all reached very significant differences when they compared with CK, respectively. The Xac cultured in MA medium containing the leaves of citron C-05 were growing slowly and the average diameter of Xac was all lower than CK in different point times and they all reached very significant level comparing with CK, respectively. There were no significant differences in the influence of the leaves’ extracted solution of citron C-05 on the growth and the content of EPS of Xac compared with CK, respectively. 【Conclusion】Some substance in the leaves of ‘Bingtang’ sweet orange could promote the growth and the content of EPS of Xac. The substance in the leaves of citron C-05, which was able to control the growth of Xac, might be induced by defensive reaction, when the leaves of citron C-05 and Xac were contracted with signals. ‘Bingtang’ sweet orange might be break this kind of reaction by some substance promoted the growth of Xac.

Cloning and Expression Analyses of R₂R₃-MYB Genes Related to Anthocyanin Biosynthesis in Rose

ZHAO Jia, LIU Rong, YANG Fan, LI Xin, LIU Hou-sheng, YAN Qian, XIAO Yue-hua

Scientia Agricultura Sinica. 2015, 48(7):  1392-1404.  doi:10.3864/j.issn.0578-1752.2015.07.14

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【Objective】Anthocyanin is a major factor conferring pink or red to rose flower. R₂R₃-MYB proteins are key transcription factors controlling anthocyanin biosynthesis. This work aimed to clone homologous genes of anthocyanin-related R₂R₃-MYB protein from rose flowers, and further to analyze their relatedness to anthocyanin biosynthesis and coloration in rose petals, which may lay a foundation for gene engineering improvement of flower.【Method】With a degenerate primer designed according to the conserved sequence of anthocyanin-related R₂R₃-MYB proteins from various plants, complete coding sequences of corresponding homologous genes were amplified and cloned by using 3′-RACE and Y-RACE methods. Multiple sequence alignment and phylogenetic analysis were performed using typical sequences from the 4th and 6th subfamily of plant R₂R₃-MYB proteins (Sg4 and Sg6) and Arabidopsis R₂R₃-MYB proteins related to secondary metabolism with deduced proteins of the cloned genes. To elucidate the relatedness of the cloned genes to anthocyanin biosynthesis and coloration in rose petals, anthocyanin contents and expression levels of the two cloned R₂R₃-MYB protein genes were determined in rose petals of different colors. 【Result】Two R₂R₃-MYB protein genes (RhMYBs4-1 and RhMYBs6-1, GenBank accession Nos, KJ664810 and KJ664811, respectively) were cloned from red petals of rose ‘Red Success’. Sequence analyses indicated that RhMYBs4-1 and RhMYBs6-1, both conserved in the R₂R₃-MYB domain, were homologous to Sg4 and Sg6 R₂R₃-MYB proteins, respectively. RhMYBs4-1 contained the C1, C2 repressors and zinc finger domains typical of Sg4 R₂R₃-MYB proteins, whereas RhMYBs6-1 had the signature motifs ((A/S/G)NDV and KPRPR(T/S)) of Sg6 R₂R₃-MYB subfamily. Expression analyses showed that both RhMYBs4-1 and RhMYBs6-1 were expressed at a high level in the red petals of rose ‘Red Success’, but at low levels in leaves and stamens. Among rose petals of different colors, red petals had the highest anthocyanin contents, and the highest expression levels of both RhMYBs4-1 and RhMYBs6-1 genes. In pink rose petals, the anthocyanin content was less than 10% of that in red petals and the RhMYBs4-1 expression level was very low, whereas the expression level of RhMYBs6-1 in pink petals was comparable to that in red petals. 【Conclusion】Results of this study indicated that the RhMYBs4-1 and RhMYBs6-1 genes encoded Sg4 and Sg6 R₂R₃-MYB proteins, respectively. Both RhMYBs4-1 and RhMYBs6-1 were highly expressed in red petals and might be important regulators of anthocyanin biosynthesis and coloration in rose petals.

STORAGE·FRESH-KEEPING·PROCESSING

Effects of High Hydrostatic Pressure on the Structural and Emulsifying Properties of Sugar Beet Pectin

PENG Xiao-yan, MU Tai-hua, SUN Hong-nan, ZHANG Miao, YU Ming, HE Wei-zhong

Scientia Agricultura Sinica. 2015, 48(7):  1405-1414.  doi:10.3864/j.issn.0578-1752.2015.07.15

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【Objective】 This study aimed to clear the effect of high hydrostatic pressure (HHP) on the structural and emulsifying properties of sugar beet pectin, and provide a theoretical basis for the applications of sugar beet pectin in food industry. 【Method】 1% (w/v) of sugar beet pectin solution was prepared, and treated under different pressures (0.1, 250, 350, 450 and 550 MPa) and pH 7 for 30 min; 450 MPa and pH 7 for different times (10, 20, 30 and 50 min); 450 MPa and different pH values (pH 3, pH 7 and pH 8) for 30 min. After that, the structural and emulsifying properties were compared with the untreated samples. 【Result】 When the sugar beet pectin solution was treated under different pressure conditions at pH 7, with the increase of pressure, the Mw of sugar beet pectin was reduced from 5.58×10⁵ Da (0.1 MPa) to 1.56×10⁵ Da (550 MPa), and the degree of esterification (DE) and acetylation (DA) were increased from 61.29% and 18.17% (0.1 MPa) to 68.24% and 21.72% (550 MPa), respectively. FT-IR spectra in 1 760-1 730 cm^-1 and 1 630-1 600 cm^-1 regions of SBP showed more clearly than untreated SBP, and FT-IR images spectra in 1 560-1 540 cm^-1 was appeared after the HHP treatment. After 250 MPa treated for 30 min, the emulsifying activity (EA) and the emulsifying stability (ES) of SBP were increased from 209 m²·g^-1 and 79 min to 230 m²·g^-1 and 97 min, the volume mean diameter (D_4,3) was decreased and the specific surface (Sv) was increased. However, the emulsifying properties of pectin were not significantly changed after continued to increase pressure. There were no significant differences in Mw, DA, DE and emulsifying properties between different processing times under 450 MPa. Mw of SBP under pH 3, pH 7 and pH 8 conditions were reduced from 5.88, 5.58 and 5.44×10⁵ Da to 2.38, 2.25 and 2.49×10⁵ Da after 450 MPa treated for 30 min; the DE of SBP did not change significantly under pH 3 and pH 7 conditions, the DA was increased from 19.35% and 18.17% to 21.70% and 24.84%, respectively; but the DA and DE were reduced from 70.13% and 19.53% down to 50.24% and 16.41% under pH 8 condition; the EA and ES of SBP were significantly improved after high pressure treatment at pH 3, pH 7 and pH 8, the EA was better at pH 3 and pH 7 conditions, and the ES was the best at pH 3. 【Conclusion】 These results suggest that HHP treatment reduced the molecular weight of sugar beet pectin, induced the protein exposure, and also improved the emulsifying properties of sugar beet pectin.

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

Food-Grade Induced Expression and Enzymatic Properties of Nitrite Reductase from Lactobacillus plantarum WU14 Under Nitrite Stress

YING Bi, CHANG Xiao-yu, LIU Zhi-wen, ZHOU Tong, CHEN Yao, ZHONG Ping-an, XU Bo

Scientia Agricultura Sinica. 2015, 48(7):  1415-1427.  doi:10.3864/j.issn.0578-1752.2015.07.16

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【Objective】The study aimed to explore the mechanism of nitrite reductase from Lactobacillus plantarum WU14 under nitrite stress so that lay a foundation for pure culture technology of lactic acid bacteria in fermented food. 【Method】 Growth density, pH and nitrite degradation quantity of L. plantarum WU14 were determined when the liquid medium contained sodium nitrite ranged from 0.02% to 0.16% under the condition of 37℃. The recombination strain Lactococcus lactic NZ9000/pRNA48- NirS was constructed followed the putative nitrite reductase gene from L. plantarum WU14 was amplified by PCR and then cloned into the food-grade cytoplasmic inducible expression vector pRNA48 of L. lactic NZ9000. After induced with 30 ng·mL^-1 nisin, the expressed target protein and the enzyme activity of nitrite reductase of the recombinant strains were analyzed by SDS-PAGE and Naphthyl ethylenediamine dihydrochloride spectrophotometric method. Using the bioinformatics software, the high level protein structure, membrane structure and hydrophobicity of nitrite reductase gene were predicted and analyzed. 【Result】L. plantarum WU14 could routinely grow in MRS medium containing less than 0.12% nitrite, along with degradation of nitrite. After the strain L. plantarum WU14 was cultured for 24 hours in the liquid medium containing 0.10% sodium nitrite, the nitrite reductase activity of L. plantarum WU14 was 2 347.5 U·mL^-1, and the degradation quantity was 56.34 μg·mL^-1 according to the analysis of its nitrite degradation ability. the NirS gene could express in the recombinant strain. Nitrite reductase gene encodes a kind of hydrophilic protein containing alpha helix and random coil, no signal peptide and transmembrane structure. The recombination strain could routinely grow in GM17 medium containing less than 0.10% nitrite, meanwhile,its enzyme activity reached 925.41 U·mL^-1 and the degradation quantity reached 22.21 mg·mL^-1 after 24 h fermentation in the 0.04% nitrite concentration medium. 【Conclusion】 Nitrite reductase from L. plantarum WU14 could degrade the high concentration nitrite, and NirS of food-grade induced expression possessed higher enzyme activity. The study laid a foundation for research of the mechanism of nitrite degradation and established a controllable system of nitrite degradation of fermented foods.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Study on Digital Network Platform of Large-Scale Dairy Farms

YANG Liang, Lü Jian-qiang, LUO Qing-yao, XIONG Ben-hai

Scientia Agricultura Sinica. 2015, 48(7):  1428-1436.  doi:10.3864/j.issn.0578-1752.2015.07.17

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【Objective】 The purpose of this study is to realize digital management of large scale dairy farms and to increase efficiency of breeding and genetic progress of dairy cows. 【Method】Based on the dairy cows’ production process from estrus, hybridization, pregnancy examination, gestation, calving, lactation, dry and to the next production cycle, also based on dairy cows’ growth stage from calves, heifers, fattening cattle to lactating cows, standards and specifications of essential information of dairy cows were set and dairy cows data management and analysis platform was constructed by using Microsoft. Net framework, SQL Server 2008 database technology and network mapping technology Fusion Charts.【Result】Data management and intelligent analysis platform for breeding, lactation and herd health were realized. The platform includes eight subsystems: system maintenance, cattle management, breeding management, milking management, feeding management, health management, statistical analysis and inner management, total 96 functions are achieved in these subsystems and they are 6, 10, 13, 14, 4, 10, 18 and 21, respectively. The platform realized the remote network database management of essential breeding and lactating process information or data, mainly including the record of whole breeding process of bull and cows, DHI data of different parities, online dynamic analysis of cattle card, directional sorting and output of data, also the average calving interval, parity structure, production performance, genealogy tracking or inbreeding coefficient could be calculated. The intelligent alert of various production events such as estrus, hybridization, pregnancy test, calving lactation, dry milk, lactagogue, elimination and calves weaning, etc. are included, as well as statistical and graphic analysis of various reproductive and lactating parameters like parities and yield distribution maps, comparison chart of annual milk yield, family tree diagram and lactation curves.【Conclusion】All these data mining and analysis expand the available value of production data and supply more convenience for the farm managers to make scientific decisions.

STORAGE·FRESH-KEEPING·PROCESSING

Selection of Reference Genes and Determination of Cytokines and Receptor mRNA Expression in Peripheral Blood of Piglets

WANG Ji-ying, WANG Yan-ping, GUO Jian-feng, WANG Huai-zhong, LIN Song, ZHANG Yin, WU Ying

Scientia Agricultura Sinica. 2015, 48(7):  1437-1444.  doi:10.3864/j.issn.0578-1752.2015.07.18

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【Objective】 Though Chinese indigenous breeds have stronger disease resistance and immunity than imported commercial breeds, little is known about the genetic basis and immune mechanism of their difference. It is supposed that the gene expression level of cytokines and receptors may be the reasons leading to the different disease resistances among breeds. In the present study, the mRNA expression levels of seven genes in piglets of Dapulian and Landrace were determined, including two pro-inflammatory cytokines (IL8 and TNFα), one anti-inflammatory cytokine (IL10), one interferon regulatory factor (IRF3), two interferon (IFNA and IFNG) and one pattern recognition receptor (TLR4), and their breed and sow effects in the two breeds were analyzed, which would benefit the understanding of the immunity levels of Chinese indigenous breeds and the commercial breeds. 【Method】 Blood samples of clinically healthy Dapulian (n=104) and Landrace (n=171) piglets of 35 days old were collected, and their total RNA was extracted and reverse-transcribed into cDNA. Based on the quantitative real-time PCR, firstly, the expression stabilities of the six candidate genes (ACTB, GAPDH, B2M, TBP, RPL4 and PPIA) were evaluated by the geNorm program to select the most stable reference gene. Then, the selected reference gene was used for normalization, and the gene expression levels of the above mentioned cytokine and receptor genes were quantified in piglets of Dapulain and Landrace. Finally, the breed and sow effects of their expression levels in the two breeds were analyzed by Least Square Means. 【Result】 The expression stability values (M) of the six candidate reference genes in blood were as follows: ACTB (0.595) ＞ GAPDH＞ PPIA＞ RPL4＞ TBP＞ B2MΔCt of the six cytokine genes were 9.23-14.25 and 5.83-8.73, respectively, indicating there were large difference among individuals of the two tested breeds. Compared with six cytokines, the D-values of TLR4 were smaller, 5.03 and 3.82 for Dapulian and Landrace respectively. Comparison between the two breeds, the expression levels of the six cytokines in Landrace were higher than that of Dapulian, while the expression levels of TLR4 in Dapulian was higher than that of Landrace. Additionally, the variation coefficients of all the cytokine and receptor genes in Dapulian (the average is 31.43%) were larger than those in Landrace (the average is 19.10%), which is consistent with the fact that Dapulian is one indigenous breed with less selecting strength compared with Landrace. The breed effect analyses indicated that, except IL10＜0.05) level or 0.01 level (P＜0.01). The sow effect analysis showed that it had significant influence on all the genes determined at 0.01 level (P＜0.01). 【Conclusion】 In the present study, the stably expressed reference genes were successfully selected which are used for the gene expression research of porcine peripheral blood, seven genes’ mRNA expression levels of peripheral blood in normally raised Dapulian and Landrace piglets were determined at population level and the breed and sow effects on their expression were analyzed. Results of the study would provide important information concerning understanding of the variation between the indigenous and commercial breeds and mining the characteristics of indigenous breed. , breed effect had significant influence on all the other genes determined at 0.05 (P (0.440). B2M, which had the most stable expression, was selected as the reference gene. In Dapulian and Landrace, the D-values between the maximum and minimum of (0.461) (0.466) (0.469) (0.571)

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Genomic Characterization of the Newly Emerged Canine Distemper Virus in Giant Panda

JIN Yi-peng, LIU Qiao-rong, SUN Ming, QIAO Yan-chao, QIAO Ming-ming, LIU Bo-hua, LIN De-gui, CHEN Xi-zhao

Scientia Agricultura Sinica. 2015, 48(7):  1445-1452.  doi:10.3864/j.issn.0578-1752.2015.07.19

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【Objective】 The objective of this study is to analyze the genetic variation of the newly emerged giant panda canine distemper virus by sequencing its full-length genome, thereby to track the source of infection and provide a theoretical basis for the prevention and control of giant panda CDV infection. 【Method】 According to the CDV genomic sequences published in GenBank, 17 pairs of oligonucleotide primers were designed, the overlapping fragments spanning whole genome of CDV were amplified by RT-PCR from lung exudate of the infected giant panda and then cloned into vector pMD19-T and sequenced, respectively, the determined sequences were spliced and assembled by using biological software DNAman. Based on above efforts, the first complete genome sequence of P-CDV isolated from giant panda was accomplished, and was further characterized by alignment with 20 published CDV whole-genome sequences from GenBank. Moreover, to track the source of infection, different phylogenetic trees were constructed according to complete genome as well as genes H, F, P and M, respectively. 【Result】 The genome of the giant panda CDV is 15 690 nucleotides in length (GenBank accession No. KP677502), and mainly encodes six genes in the order of 3′-N-P-M-F-H-L-5′. No nucleotides insert and deletion were observed in encoding and intergenic regions compared to other CDV complete genome sequences. Phylogenetic analysis of the giant panda CDV genome showed 91.5%-98.7% nucleotide identity with 20 representative CDV isolates. Along with virulent strains MKY-KM08 (accession no. HM852904), PS, HLJ1-06 (accession no. JX681125), Hebei (accession no. KC427278) and AC96I-H358 (accession no. AB753776), P-CDV belongs to one large homogeneous clade, and is phylogenetically most closely related to the isolate PS with 98.7% identity at nucleotide. It shares 95.7% nucleotide identity with wild strain A75-17, and exhibits the lowest nucleotide similarity with standard vaccine strain CDV3 (91.5%). Sequence analysis based on H gene indicates that the giant panda CDV is a virulent strain, it belongs to genotype Asia-I. There are a total of nine amino acid (aa) substitutions, among them three amino acid substitutions were firstly found and are unique to the giant panda CDV. Of these 3 aa substitutions, 2 substitutions (N/K208S, S215A) occur in F open reading frame (ORF), one substitution (Q58K) occurs in P ORF.【Conclusion】 The complete genomic sequence of this newly emerged giant panda CDV was successfully characterized, the key site mutations in F and P genes were recognized. The data will provide valuable theoretical reference for the prevention and control of giant panda CDV infection.

Appropriate Level of Tryptophan for Apis mellifera Worker Bee Larvae Feed

ZHAO Feng-kui, XU Bao-hua, WANG Hong-fang

Scientia Agricultura Sinica. 2015, 48(7):  1453-1462.  doi:10.3864/j.issn.0578-1752.2015.07.20

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【Objective】 The objective of this research is to study the appropriate levels of tryptophan in Apis mellifera worker bee larvae feed, and to provide a theoretical basis for the nutritional needs of tryptophan. 【Method】 A total of 1 008 one-day-old larvae were randomly divided into seven groups of three replicates of 48 larvae, and were fed different diets with tryptophan at the levels of 7.84, 8.84, 9.84, 10.84, 11.84, 12.84 and 13.84 mg?g^-1, respectively. The 5-day-old worker bee larvae were used for measuring TPH (tryptophan hydroxylase) gene and 5-HTR (5-hydroxytryptamine receptors) 1, 2α, 2β, 7 gene expression. The 6-day-old worker bee larvae as samples for determining total protein content, MDA (malondialdehyde), T-AOC (total antioxidant capacity), and the 6-day-old larval hemolymph was taken to be used for measuring the level of free tryptophan. The pupation rate was calculated at 7-day-old. Deposition amount of tryptophan was analyzed using 9-day-old pupa, and the worker bee eclosion rate was calculated at 21-day-old.【Result】Pupation rate and larvae total protein content at 6-day-old, tryptophan deposition at 9-day-old pupa and worker bee eclosion rate at 21-day-old were the highest when the tryptophan level was 10.84 mg?g^-1. The expressions of TPH, 5-HT receptors 1, 2α and 2β genes of larvae at 5-day-old and hemolymph free tryptophan content of larvae at 6-day-old were elevated significantly by 10.84-11.84 mg?g^-1 tryptophan (P＜0.05). However, 5-HT receptor 7 gene expression of 5-day-old worker bee larvae and MDA level of 6-day-old worker bee larvae fed 12.84-13.84 mg?g^-1 tryptophan were higher than worker bee larvae fed 7.84-11.84 mg?g^-1 tryptophan (P＜0.05). Dietary supplied with 10.84-13.84 mg?g^-1 tryptophan improved significantly 6-day-old worker bee larvae T-AOC compared with dietary with 7.84-9.84 mg?g^-1 tryptophan (P＜0.05). 【Conclusion】 The appropriate level of tryptophan for A. mellifera worker bee larvae is 10.84-11.84 mg?g^-1.