Table of Content

16 April 2015, Volume 48 Issue 8

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Cloning and Expression Analysis of A Stress-Related TaC2DP1 Gene from Wheat

XIAO Rui-xia, WANG Xin-guo, XIA Guo-jun, LI Yong-chun, NIU Hong-bin, WANG Xiang, YIN Jun, REN Jiang-ping

Scientia Agricultura Sinica. 2015, 48(8):  1463-1472.  doi:10.3864/j.issn.0578-1752.2015.08.01

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【Objective】The objective of this study is to clone the stress resistance-related gene, analyze its sequence features, evolutionary relationships and expression characteristics, investigate its biological function during the stress tolerance of wheat, and to provide candidate gene and a theoretical foundation for clarifying molecular mechanism of stress resistance.【Method】Using an up-regulated EST obtained by cDNA chip as a probe to search the wheat EST databases, filter out the ESTs sequences with the homology of 97% of the probe, a full-length cDNA sequence was cloned from wheat by in silico cloning and reverse transcription PCR (RT-PCR) method. The conserved domains and sequence features of the gene were analyzed by bioinformatics’ methods. A phylogenetic tree was constructed using the MEGA 6.0 software, and then the cloned geneORF was inserted into the expression vector pMAL-c2X by EcoR I and HindⅢ digestion. The recombinant plasmid was transformed into E. coli BL21 and expressed under the induction with 0.3 mmol·L^-1 IPTG for 1-5 h. The expression of the fusion protein was detected by SDS-PAGE. The expression profiles of the cloned genein various tissues and in response to cold, drought, heat and abscisic acid (ABA) treatment were investigated using quantitative real-time PCR (qRT-PCR).【Result】The full-length cDNA sequence designated as TaC2DP1 from wheat is 1 356 bp in length, contains a 1 209 bp open reading frame (ORF), with 50 bp in the 5' UTR and 97 bp in the 3' UTR. TaC2DP1was predicted to encode a 402 amino acid protein with a molecular mass of 43.41 kD and isoelectric point of 4.30, it belongs to the acidic protein. BLAST analysis revealed that the protein contains a C2-domain and was predicted to be a Ca²⁺ binding domain. Multiple sequence alignment and phylogenetic tree analysis showed that TaC2DP1 had the closest evolutionary relationship with a C2-domain protein in Triticum urartu with unknown function, and shares 91% identity in amino acids. Protein structure prediction showed that TaC2DP1 had not transmembrane helix and disulfide bondand might be localized in cytoplasm. The prokaryotic expression vector of TaC2DP1, pMAL-c2X-TaC2DP1,was successfully constructed. The expression of fusion protein was obtained by inducing with IPTG and its relative molecular weight was 90 kD, which was consistent with the theoretical value. Real time quantitative PCR (qRT-PCR) analysis revealed that TaC2DP1 constitutively expressed in all tested roots, stems, leaves, immature ears, immature seeds, embryo and endosperm, the expression level of TaC2DP1 in immature ears was the highest, while its expression level was very low in seeds at 5 days after anthesis (DAA). The expression profiling revealed that TaC2DP1 was induced by plant hormone ABA and the expression of TaC2DP1 was steadily up-regulated in any of the time point under drought stress. TaC2DP1 was rapidly up-regulated within 0.5 h of cold and heat stress treatments and the expression level was 21 and 17 times than those of control, respectively. These results revealed that TaC2DP1 might be involved in stress resistance-related response of ABA signaling pathways in wheat leaves.【Conclusion】A full-length cDNA of TaC2DP1 was isolated and the typical a Ca²⁺ binding domain was found in the deduced proteins. The expression of TaC2DP1 was all up-regulated under drought, high and low temperature and ABA treatments, showing that TaC2DP1 was involved in the ABA dependent stress-responding gene network.It was deduced that TaC2DP1 might play an important regulation role under stress in wheat.

Overexpression of Sucrose Transporter (TaSUT1A) Improves Drought Tolerance in Transgenic Wheat

HU Meng-yun, LI Hui, PANG Jian-zhou, LIU Qian, ZHANG Ying-jun, SUN Li-jing

Scientia Agricultura Sinica. 2015, 48(8):  1473-1483.  doi:10.3864/j.issn.0578-1752.2015.08.02

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【Objective】 Drought is one of the most important constraints resulting in large yield losses and limiting the average yield increase of wheat in China. The aim of this study is to develop and select stable TaSUT1A transgenic wheat lines with resistance to drought.【Method】The open-reading-frame sequence of TaSUT1A was synthesized and used to construct the gene transformation vector pUBI::cas-TaSUT1A, in which TaSUT1A gene was driven by maize ubiquitin promoter and should be highly expressed in monocot plants. Particle bombardment method was used to introduce TaSUT1A into wheat cultivar Kenong 199. After bialaphos screening, DNA PCR and RT-PCR methods were used to detect the presence and transcript levels of TaSUT1A in the transgenic wheat plants of T₀-T₄ generations. Subsequently, the resistance to water stress of transgenic wheat plants and non-transgenic wheat Kenong 199 at young seedling-stage was evaluated. Further, their determination of physiologic index related to abiotic stress was finished and the potential of improving abiotic stress tolerance in plant was elucidated. 【Result】The results indicated that the introduced TaSUT1A gene was stably inherited, and 220% PEG treatment could significantly induce the expression of TaSUT1A in root and leaf tissues of three transgenic wheat lines. The average germination rate of transgenic wheat lines was 85.53%, which was more higher than those of non-transgenic controls, and the average celoeptile and primary root length significantly higher than the non-transgenic plants. The results showed that the transgenic wheat significantly enhanced seed germination in 20% PEG treatment, as indicated by enhancement of the growth of celoeptile and primary root. Further, three pure lines with higher TaSUT1A expression were selected to elucidate physiologic index related to abiotic stress. In detail, in 20% PEG treatment, the transgenic lines overexpressing TaSUT1A increased sucrose and total soluble sugar content in leaf and root, with the average increase value of 42.95% and 36.56% in leaf, and 58.01% and 43.01% in root respectively, which was significantly higher than the non-transgenic plants. After treatment under water stress condition, the MDA content of transgenic plants only increased by 6.12 nmol·g^-1FW, however, the MAD content of the non-transgenic plants increased by 19.05 nmol·g^-1FW. Meanwhile, the transgenic plant showed better SOD activity, with the average increase value of 44.7 U·g^-1FW, which was significantly higher than the non-transgenic plants. 【Conclusion】It was concluded that TaSUT1A plays an important role in response to drought stress in plants, overexpression of TaSUT1A can improve significantly tolerance to drought stress in transgenic wheat plants.

Genetic Mapping of the Resistance Gene for the Race 4 of Sphacelotheca reiliana in Sorghum

YANG Hui-yong, ZHAO Wen-bo, WANG Hua-yun, ZHANG Fu-yao

Scientia Agricultura Sinica. 2015, 48(8):  1484-1491.  doi:10.3864/j.issn.0578-1752.2015.08.03

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【Objective】The objective of this study is to conduct mapping genes conferring resistance to physiological race 4 of head smut disease in sorghum and screen the molecular markers linked to the resistance genes, in order to lay a foundation for sorghum resistance breeding against the head smut in future.【Method】To build F2 populations, the material 961541 as a female, which is immune to physiological races 1, 2, 3, and 4, was crossed with V4B which is immune to physiological races 1, 2, and 3 and sensitive to race 4 and PI550607 which is sensitive to physiological races 1, 2, 3, and 4. The seeds were inoculated with soil containing suitable chlamydospores at sowing time, and the disease incidence of the parents, F₁ progenies and F₂ population was investigated at heading stage. The localization of resistance gene was studied by SSR technology and bulked segregation analysis (BSA) method using the 961541/V4B population.【Result】The results of field investigation showed that, for the cross combination 961541/V4B, the morbidities of 961541 and the F₁ progeny both were 0, and that of V4B and F₂ population were 21.5% and 7.25%, respectively. For another cross combination 961541/PI550607, the morbidities of PI550607 was 64.81%, and that of the F₁ progeny, F₂ population were 0 and 5%, respectively. The fitness test indicated that, for the two F₂ populations, the ratio of resistance quantities to sensitive quantities both were conformed to 15:1(χ²=0.201, 0.322, P>0.05), which implied that the resistance genes to head smut race 4 should be controlled by 2 non-allelic genes. The linkage analysis showed that, among the total 274 pairs of SSR primers, 53 pairs showed different amplification between the parents. Then screening of the specificity primers among the 53 pairs were conducted between the resistance pool and the sensitive pool, while only Xtxp325 on chromosome 1 was specific. The banding pattern of resistance pool was the same as 961541, and the sensitive pool was as the material V4B. Linkage mapping with the 5 pairs primers (Xtxp325, Xtxp302, Xtxp32, Xtxp340, Xtxp248) showed that the overall length of linkage map was 355.3 cM, the genetic distance between Xtxp325 and the resistance gene (Shs1) was 27.7 cM.【Conclusion】The resistances to physiological race 4 of sorghum head smut might be controlled by two mutual independent non-allelic genes. The linkage map was good correspondence with the public maps, and which the overall length was 355.3 cM. The resistance gene Shs1 of head smut race 4 is located on No. 1 chromosome of sorghum, and the genetic distance is 27.7 cM from the primer Xtxp325.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY

Effects of Deficit Irrigation on the Formation of the Yield in Rice (Oryza sativa) During Filling Period

CAI Yi-xia, LI Yang, ZHU Hai-tao, CAI Kun-zheng, HUANG Fei, WANG Wei

Scientia Agricultura Sinica. 2015, 48(8):  1492-1505.  doi:10.3864/j.issn.0578-1752.2015.08.04

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 【Objective】 This paper aimed to understand the characteristics of the translocation of dry matter stored in stems and grain filling in the single segment substitution line (SSSL) and its receptor parent with different drought resistances under water-deficit irrigation during filling period. 【Method】 The SSSL W27-3-5-2-4-3-3-5-7 that carries single segment genes encoding drought resistance of Brazilian upland rice IAPAR9 and its receptor parent Huajingxian74 were used as materials and grown in split-root system. Three soil water treatments, all roots well-watered (the two halves of the trunk were irrigated well water, W/W), half-root subjected to water deficit (one half of the trunk was irrigated with well water and the other was suffered from -30kPa—-40kPa of water deficit, W/D) and all roots subjected to water deficit (two halves of the trunk were suffered from -30kPa—-40kPa of water deficit, D/D), were imposed from 7th day after anthesis to mature period. The influences of different water deficits on the yield and its components of the SSSL and its receptor parent were studied, and the changes of ABA contents in stems and grains of rice were analyzed. So the effects of deficit irrigation on the catabolism and translocation of non-structural carbohydrates (NSC) in stem and sheath and the activities of key enzymes involved the grain filling were illuminated in this paper. 【Result】 Compared with W/W treatment, the increases of ABA contents in stems and grains of D/D treatment were found obviously. The relative water content, SPAD values and photosynthetic rate of flag leaves of D/D treatment were all decreased significantly during mid-late filling period. In D/D treatment, the activities of α, β starch enzymes in stem and sheath increased obviously, while the activity of sucrose synthase decreased. However, the activities of sucrose synthase (SS), acid invertase (AINV), soluble starch synthase (SSS) and Q enzymes in grains of D/D treatment decreased remarkably during early and mid filling period. Furthermore, the contents of ABA in stems and grain of W/D treatment increased, but no significant difference between W/W and W/D was found, and the similar results were gained for the relative water content, SPAD values and photosynthetic rate of flag leaves. But the activities of SS, AINV, SSS and Q enzymes in grains of W/D treatment increased remarkably during early and mid filling period. The differences between the SSSL and its receptor parent under the deficit irrigation condition during grain filling were found obviously. The remobilized rates of NSC in stem and sheath of the SSSL were higher than those of the Huajingxian74, and the activities of enzymes, dynamic changes of matters and endogenous ABA content of SSSL involved in the metabolisms of stems and grains were responded to water deficit more sensitively than those of Huajingxian74. And so there was no clearly decrease in the yield of the SSSL under two deficit irrigation treatments. The SSSL under deficit irrigation condition showed a strong drought resistance. 【Conclusion】 The moderate soil water-deficit could favor the increase of the ABA content in all organs of rice, avail the degradation and remobilization of NSC in stems and sheath, while the production and accumulation of photoassimilates of functional leaf during filling stage were not influenced significantly by water deficit, and the yield of rice would be decreased insignificantly under moderate water-deficit condition.

Response of Wheat Root Characteristics and Canopy Photosynthesis to Different Irrigation Methods in Lime Concretion Black Soil

ZHANG Xiang-qian, CAO Cheng-fu, QIAO Yu-qiang, LI Wei, CHEN Huan

Scientia Agricultura Sinica. 2015, 48(8):  1506-1517.  doi:10.3864/j.issn.0578-1752.2015.08.05

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【Objective】The aim of this experiment was to clarify the potential mechanism of the effects of different irrigation methods which was irrigated at early growth stage of wheat on root characteristics, canopy photosynthesis and yield at middle and late growth stages, and to reveal the relationship between root characteristics and canopy photosynthesis. 【Method】 The experiment was conducted in an artificial glass rainproof shed, and the designed ten irrigation treatments in this experiment were CK(adequate water was supplied at early growth stage), W1_20d (20 d after seedling emerged for 50 mm), W2_40d (40 d after seedling emerged for 50 mm), W3_60d (60 d after seedling emerged for 50 mm), W4_80d (80 d after seedling emerged for 50 mm), W5_100d (100 d after seedling emerged for 50 mm), W6_120d (120 d after seedling emerged for 50 mm), W7_20d+60d (20 d after seedling emerged for 25 mm＋60 d after seedling emergency 25 mm), W8_40d+80d (40 d after seedling emerged for 25 mm＋80 d after seedling emerged for 25 mm) and W9_60d+100d (60 d after seedling emerged for 25 mm＋100 d after seedling emerged for 25 mm). The effects of different irrigation methods which were irrigated at wheat early growth stage on root characteristics, canopy single leaf area, chlorophyll density, photosynthetic capacity, photosynthetically active radiation, chlorophyll fluorescence parameters and yield of wheat at middle and late growth stages were studied. 【Result】 To appropriate postpone the irrigation date at wheat early growth stage could help to increase total root length, total surface area, total volume, average diameter, total root tips and total root forks, and the difference between W5_100d (irrigation was carried out 100 d after seedling emergence) and CK (adequate water was supplied at early growth stage) was insignificant, but they were significantly higher than that of W1_20d (irrigation was carried out 20 d after seedling emergence) and W6_120d (irrigation was carried out 120 d after seedling emergence). The single leaf area of 1, 2 and 3 leaves counted from the top of CK was the highest at booting and flowering stages, but the difference between CK and W5_100d was not significant, and the canopy of single leaf area was not significantly affected by irrigation times under the same irrigation amount. When one time of irrigation was made at early growth stage of wheat, the canopy chlorophyll density had a change trend of initial increase and then decrease with the postponement of irrigation date, and the values of W5_100d were the highest, while of W1_20d were the lowest. The canopy photosynthetic rate of W5_100d at booting stage, flowering stage and middle of filling stage was significantly higher than that of CK, and was increased by 7.5%, 8.9% and 8.9%, respectively, but the canopy photosynthetic rate was not significantly affected by irrigation times. Under the condition of one time of irrigation, W5_100d had the highest values of canopy photosynthetically active radiation at booting stage, flowering stage and middle of filling stage, and when compared to W1_20d and W6_120d , the values were significantly increased by 18.7%, 9.7%, 11.0% and 5.7%, 4.9%, 4.3%, respectively. The chlorophyll fluorescence parameters of F_o, F_m, F_v/F_m, Φ_psII and ETR of W5_100d at booting and flowering stages were the highest, and the chlorophyll fluorescence parameters were not significantly affected by irrigation times. The differences in yield and harvest index between W5_100d and CK were not significant, and when compared to W1_20d and W6_120d they were increased by 15.4%, 22.1% and 3.2%, 9.2%, respectively.【Conclusion】Too early or too late irrigation at early growth stage had adverse impact on root growth, canopy light utilization and yield formation, while the irrigation date was appropriately postponed, the root characteristics, canopy photosynthesis and yield were better than or equivalent to the treatment of adequate water supply at wheat early growth stage, and the effects of irrigation times were not obvious under the same amount of irrigation water.

PLANT PROTECTION

Evaluation of Resistance to Stripe Rust in Eighty Abroad Spring Wheat Germplasms

ZHOU Xin-li, ZHAN Gang-ming, HUANG Li-li, HAN De-jun, KANG Zhen-sheng

Scientia Agricultura Sinica. 2015, 48(8):  1518-1526.  doi:10.3864/j.issn.0578-1752.2015.08.06

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【Objective】 Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide and resistance is the best control strategy. The objective of this study is to evaluate the resistance to current Chinese predominant stripe rust races in eighty abroad spring wheat germplasms, and these lines could be selected for their good resistance for genetic improvement of wheat stripe rust resistance breeding programs in the future in China. 【Method】 Eighty germplasms were tested with Chinese predominant stripe rust races CYR29, CYR31, CYR32, CYR33 and pathogenic types PST-HY8 and PST-V26 at seedling stage in greenhouse. Mingxian 169 and AvS as susceptible cultivars were also tested at same time. And field resistance evaluations were conducted at Yangling (Shaanxi province) and Tianshui (Gansu province) in 2013 and 2014, respectively. Based on the seedling and field reactions, the resistance of the germplasms was classified and assessed. 【Result】 The eighty germplasms could be classified into three groups. The first group consisted of eight lines that showed all-stage resistance. PI660067, PI660119 and PI660122 showed high level resistance at seedling stage and adult-plant stage in the field tests. The remaining five lines (PI660056, PI607839, PI591045, TA5602 and PI660064) showed susceptibility to individual races at seedling stage, and also showed susceptibility at adult-plant stage in different years and different test sites. The second group consisted of twenty-eight lines that showed adult-plant resistance. The lines in this group showed susceptibility to all tested races at seedling stage, and twenty-three lines showed resistance at adult-plant stage in the field tests. But PI660075, PI660083, PI660085, PI660097 and PI660107 showed susceptibility at adult-plant stage in different years and different test sites. The third group consisted of forty-four lines that showed adult-plant resistance and ineffective all-stage resistance to part of Chinese stripe rust races. The lines in this group show susceptible to at least one of the races in the seedling tests and thirty-seven lines showed resistance at adult-plant stage in the field. But PI660065, PI660076, PI660079, PI660080, PI660095, PI660096 and PI610750 showed susceptibility at adult-plant stage in different years and different test sites.【Conclusion】The majority of the eighty abroad wheat germplasms showed excellent resistance to most popular Chinese wheat stripe rust races. These germplasms should be useful in diversifying resistance genes that can be used in breeding programs for developing wheat cultivars with stripe rust resistance. Probably due to different new races popular in field in different years, causing some adult-plant resistance lines were susceptible in different years and different test sites, which indicated that adult-plant resistance might also have race-specific resistance.

A Universal RT-PCR Method for the Simultaneous Detection of the Viruses in Genera Comovirus and Fabavirus

YE Zhi-hong, LIAO Fu-rong, GUO Mu-jin, FANG Zhi-peng, CHEN Qing, CHEN Hong-yun, LIN Shi-ming, LIN Yi

Scientia Agricultura Sinica. 2015, 48(8):  1527-1537.  doi:10.3864/j.issn.0578-1752.2015.08.07

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【Objective】 The objective of the study is to develop a universal RT-PCR method for the simultaneous detection of the viruses from Comovirus and Fabavirus. 【Method】Analysis of the complete nucleotide sequence of comoviruses and fabaviruses was used to design a pair of degenerate primers for specific detection of members of the two genera. The sensitivity and specificity were evaluated, respectively. In order to direct the sequence, the non-complementary universal sequencing primer sequence RV-M and M13-47 were added, respectively, to the 5′ termini of the primers. Amplicons were directly sequenced to verify their identity. Finally, the method was used to detect viruses in Pseudostellaria heterophylla coming from Zherong, Fujian Province. 【Result】A generic PCR protocols was developed to detect the two virus genera in Comovirus and Fabavirus using degenerate primers designed to amplify part of the RNA-dependent RNA polymerase (RdRp) gene. An expected size product about 350 bp was amplified using the optimized PCR protocols from all 17 isolates of the 9 comovirus species and 2 fabavirus species tested including Andean potato mottle virus (APMoV), Broad bean stain virus (BBSV), Broad bean true mosaic virus (BBTMV), Bean pod mottle virus (BPMV), Cowpea mosaic virus (CPMV), Cowpea severe mosaic virus (CPSMV), Radish mosaic virus (RaMV), Red clover mottle virus (RCMV), Squash mosaic virus (SqMV), Broad bean wilt virus 1 (BBWV1) and Broad bean wilt virus 2 (BBWV2). When the RV-M and M13-47 primer sequences were added, respectively, to the 5′ termini of the primers, it was not only observed that the PCR’s amplicons could be directly sequenced by the universal sequencing primer, but also it could improve the detection sensitivity by 10-100 times. No cross-reaction was observed with either healthy plants or from isolates in the genus Nepovirus of the Comovirinae. Phylogenetic analysis using the generic PCR’s amplicons sequence showed that it could differentiate comoviruses and fabaviruses at the species level. The partial sequence of the RdRp gene of BBSV was firstly determined by this method, and was shown to have the closest relationship with RCMV. Using this method, BBWV2 was detected in P. heterophylla. 【Conclusion】 The described generic assay could be applied for the broad spectrum detection of members of the genus Comovirus and Fabavirus and, in combination with the PCR’s amplicons sequence, for the identification of species in the two genera. The assay may also be useful for the detection of new or uncharacterized species within the two genera.

Comparative Analysis of Rhopalosiphum maidis Feeding Behaviors on Eight Maize Hybrids (Inbreds)

ZHAO Man, TIAN Ti-wei, LI Wei-zheng, LUO Mei-hao, GUO Xian-ru, YAN Feng-ming

Scientia Agricultura Sinica. 2015, 48(8):  1538-1547.  doi:10.3864/j.issn.0578-1752.2015.08.08

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【Objective】 The objective of this study is to clarify the probing and feeding behaviors of maize aphid (Rhopalosiphum maidis) on eight maize hybrids (inbreds), and then select some proper electrical penetration graph (EPG) parameters as the indicators to classify the resistant levels of different maize hybrids (inbreds). Finally, the research will provide suggestion for the classification of maize’s resistant levels. 【Method】 The feeding behaviors of R. maidis on Xundan 20, Zhengdan 958, Liangyu 88, Xianyu 335, Han 21, 87-1, Pugai 340-1-1 and Qi 319 were observed, respectively, by using EPG technique and then compared and analyzed. The resistant levels of 8 maize hybrids (inbreds) were classified by using various EPG parameters, and the results were compared with the consequence which obtained in the maize field previously. Then the suitable EPG parameters were suggested to be used as parameters for screening the resistant maize hybrids (inbreds). 【Result】 The main EPG waveforms of R. maidis on maize hybrids (inbreds) were Np, C, Pd, E1, E2 and F, and the waveforms Np, E1, E2 and F were related with the maize’s resistant level. Before reaching the phloem, the total time and mean time of waveforms Np and F of R. maidis probing on Xundan 20, Zhengdan 958 and Liangyu 88 were significantly longer than those on other maize hybrids (inbreds). After reaching the phloem, the durable time of waveform E1 of R. maidis on Xundan 20 and Zhengdan 958 were longer, although the stylets of the aphids reached the phloem later. Meanwhile, the total time and durable time of E2 on Qi 319 and Han 21 were much longer when compared with other maize hybrids (inbreds), and they were significantly higher than Liangyu 88. The experimental 8 maize hybrids (inbreds) were categorized into three groups by cluster analysis according to the mean durable time of waveform Np, C, Pd, F and E, and the resistant levels were 1st classification (Xundan 20, Zhengdan 958 and Liangyu 88)>3rd classification (Pugai 340-1-1, Han 21 and 87-1)>2nd classification (Xianyu 335 and Qi 319). 【Conclusion】 The probing and feeding behaviors of R. maidis on 8 maize hybrids (inbreds) were different. There are some resistant factors in the leaf surface and phloem of these two high resistant maize hybrids Xundan 20 and Zhengdan 958. The resistant levels of all experimental maize hybrids (inbreds) obtained according to the mean durable time of waveform E and Np were approximately consistent with the resistant results of different maize hybrids (inbreds) to the maize leaf aphid obtained by the author’s laboratory in the maize field previously.

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

Effect of the Substitution of Straw Incorporation for K Fertilization in Different Rice Producing Regions of Hubei Province

LIU Qiu-xia, DAI Zhi-gang, LU Jian-wei, REN Tao, ZHOU Xian-zhu, WANG Zhong-liang, LI Xiao-kun, CONG Ri-huan

Scientia Agricultura Sinica. 2015, 48(8):  1548-1557.  doi:10.3864/j.issn.0578-1752.2015.08.09

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【Objective】The experiment was conducted to study the effects of potassium (K) fertilization and straw incorporation on rice yield, K uptake and K balance in different rice producing regions of Hubei province, in order to find how much chemical potassium can be replaced by straw potassium. The results would provide a scientific basis for K fertilization management under straw incorporation for different regions. 【Method】Field trials in 19 counties were carried out to study the effects of the substitution of straw incorporation for K fertilization in hilly regions of east (HEH) and center Hubei (HCH) and Jianghan Plain (JHP) in 2013. The designed six treatments were no K fertilization (NP), chemical K fertilization (NPK), straw incorporation (NP+S), straw incorporation with 50% of chemical K fertilizer (NP+1/2K+S), straw incorporation with 75% of chemical K fertilizer (NP+3/4K+S), and straw incorporation with 100% of chemical K fertilizer (NPK+S). 【Result】Both K fertilization and straw incorporation improved the rice yield and K uptake across the regions. Compared with NP treatment, the averaged rice yield increased by 0.36, 0.36 and 1.07 t·hm^-2 in NPK treatment in the regions of HEH, HCH, and JHP, respectively, with the increasing rate of 6.4%, 5.0% and 15.3%. For the NP+S treatment, rice yield increased by 0.57, 0.49 and 0.24 t·hm^-2 in HEH, HCH, and JHP regions, with the increasing rate of 7.6%, 6.9% and 4.0%, respectively. K fertilization (NPK) enhanced the K uptake by 20.1-61.9 kg K₂O·hm^-2, while the straw incorporation (NP+S) increased K uptake by 8.5-39.1 kg K₂O·hm^-2. Both yield and K uptake of NPK treatment were higher significantly than NP+S treatment in JHP area. However, no difference was observed between the NPK and NP+S treatments in the HEH and HCH areas. After calculating the apparent K balance, it was found that K lost (144.3-284.4 kg K₂O·hm^-2) occurred in all the treatments across the sites especially in the treatments without straw incorporation. Model was used to fit the relationship between K fertilization rate and rice yield, the optimum application rate of K fertilizer under straw incorporation for which could reach the yield under NPK treatment was obtained. The optimal K rate was 19.9, 14.9 and 54.2 kg K₂O·hm^-2 in the regions of HEH, HCH, and JHP, which saved chemical K fertilizer by 66.8%, 75.2% and 9.7% compared with the recommended amount (60 kg K₂O·hm^-2), respectively. Using the price of K fertilizer and rice in 2013, the economic optimum K rate (EOKR) could be obtained. The EOKR values were 27.4, 27.9 and 37.9 kg K₂O·hm^-2 in the HEH, HCH, and JHP, saved 54.3%, 53.5% and 36.9% chemical K fertilizer compared with the recommended amount, respectively. 【Conclusion】Chemical K fertilizer could be partly replaced by straw incorporation across different rice planting regions in Hubei province. In addition, straw incorporation can alleviate soil K loss, which plays an important role in sustainable potassium management on farmland.

The Time Loading Limitation of Continuous Cropping Maize Yield Under Different Plastic Film Mulching Modes in Semi-Arid Region of Loess Plateau of China

XIE Jun-hong, CHAI Qiang, LI Ling-ling, ZHANG Ren-zhi, NIU Yi-ning, LUO Zhu-zhu, CAI Li-qun

Scientia Agricultura Sinica. 2015, 48(8):  1558-1568.  doi:10.3864/j.issn.0578-1752.2015.08.10

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【Objective】 The objective of this study is to evaluate the impact of continuous maize cropping under different plastic film mulching modes on the yield, water consumption, and water use efficiency (WUE) of maize, uncover the mechanism of increase production and water use efficiency under different plastic film mulching patterns. It is also to ascertain time limitation for continuous maize cropping for sustainable and efficient water use. 【Method】 Based on the field experiment, quantization of soil water balance between years and maize yield stability was determined. Maize continuous cropping time limitation was determined by continuous high yield and no appearance of soil desiccation in harvesting period.【Result】Under 3-year continuous cropping conditions, the results showed that completely film mulched alternating narrow and wide ridges with furrow planting had significant effects on increasing maize yield and WUE, and was of benefit to the water demand of maize crop at the key growth and development stage. Compared with half-mulching and flat-planting treatment, maize yield and WUE was increased by 41.8% and 33.4%, respectively. Maize biomass, WUE_b, WUE_y, total output value, net output value, output per rainfall value and input-output ratio increased by 21.8%, 12.3%, 31.2%, 27.8%, 21.1% and -3.2%, respectively; they increased by 24.9%, 39.1%, 225.5%, 1423.9%, 212.4% and 93.5% compared to flat-planting without mulching treatment, respectively. Film mulching increased the amount of water consumption in the maize whole growth period by 15.5%-29.2%, 10.0%-20.8% and 4.2%-12.6% for completely film mulched alternating narrow and wide ridges with furrow planting, complete mulching and flat-planting, half-mulching and flat-planting treatment compared to flat-planting without mulching treatment. High water consumption in the crop growing period made the soil water storage under three film mulching treatments decreased by 37.3%, 33.5% and 30.9% lower than the beginning of the experiment in the second year, and it was 29.6%, 27.5% and 23.9% lower in the third year, this continuous soil water decrease practice resulted in soil water deficiency. With the prolongation of the time of continuous cultivation, soil desiccation appeared and has a negative effect on soil water sustainable use.【Conclusion】Under the same rainfall conditions, the completely mulched alternating narrow and wide ridges with furrow planting had a significant effect on increasing the yield, benefit and the WUE. Taking the sustainable use of water into account, 2 years is a suitable period of continuous cropping for the completely mulched alternating narrow and wide ridges with furrow planting, and 3 years is a suitable period of continuous cropping for complete mulching and flat-planting, half-mulching and flat-planting, flat-planting without mulching, respectively, in semi-arid region of Loess Plateau.

HORTICULTURE

Variation in Nitrogen Uptake and Utilization Efficiency of Different Cucumber Varieties in Northern China

ZHAO Chun-bo, SONG Shu-yao, ZHAO Jing, ZHANG Xue-mei, ZHANG Yue, ZHANG Song-ting

Scientia Agricultura Sinica. 2015, 48(8):  1569-1578.  doi:10.3864/j.issn.0578-1752.2015.08.11

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【Objective】The objective of the experiment was to probe the variety variation of nitrogen uptake and utilization efficiency among cucumber genotypes in northern China for providing reference for nitrogen efficient varieties screening.【Method】 Cucumber varieties of North China ecotype was used as the test materials in a hydroponic experiment, the split plot design was employed with main plot of nitrogen nutrition levels (3.5 mmol·L^-1 and 11.0 mmol·L^-1), sub-plot of cucumber varieties. Content, assimilate and utilization efficiency of nitrogen was measured, nutrition efficiency type was divided. 【Result】 The indicators related to nitrogen uptake and utilization efficiency at two nitrogen levels were significantly different among cucumber varieties. Plant dry weight, nitrogen uptake efficiency and nitrogen utilization index showed a significant varietal variation under two nitrogen levels, the coefficient of variation were CV _N3.5 21.49 % and CV _N11 18.51 %, CV _N3.5 19.90 % and CV _N11 19.94%, CV _N3.5 25.49 % and CV _N11 19.25 %, respectively. Plant dry weight was very significantly correlated to stem-leaf N accumulation, plant N utilization indexes (P< 0.01), and correlation coefficient were r_N3.5 0.933^** and r_N11 0.925^**, _N3.5 0.964^** and r_N11 0.941^**, respectively. Nitrogen uptake was significantly positively correlated with stem-leaf N accumulation, plant N utilization index, and the correlation coefficients were r_N3.5 0.986^** and r_N11 0.963^** , r_N3.5 0.809^** and r_N11 0.768^**, respectively; Nitrogen utilization efficiency was significantly negatively correlated with stem-leaf nitrogen content, and the correlation coefficients were r_N3.5 -0.909^** and r_N11 -0.886^**. Pant dry weight average as a standard under two nitrogen levels to classify nitrogen nutrition efficiency of cucumber varieties, the plant dry weight of efficient type was more than the average, and that of the inefficient type was less than the average. Varieties were classified into four nitrogen types, such as efficient-efficient, efficient-inefficient, inefficient-efficient, and inefficient-inefficient types. The least was inefficient-efficient type, which was 15.6% of these varieties. The path coefficient analysis revealed that nitrogen use efficiency was dominated by nitrogen uptake efficiency at two nitrogen levels, the path coefficient with nitrogen use efficient was q_N3.5 1.069 and q_N11 0.931, q_N3.5 0.347 and q_N11 0.361, respectively. 【Conclusion】 Nitrogen use efficiency of seedlings of different cucumber varieties showed a significant varietal variation. Plant dry weight can be used as the first index for evaluation difference in nitrogen use efficiency of seedlings at the same N level, stem-leaf nitrogen accumulation, stem-leaf N content and nitrogen utilization index can be used as the secondary indexes. Nitrogen uptake efficiency is a major factor for high N use efficiency of seedling. A hydroponic experiment can effectively reflect nitrogen nutrition efficiency variation of cucumber seedlings of different varieties, and it provides a possibility for massive screening of nitrogen use efficiency of cucumber seedlings and rapid identification.

Effect of Root Border Cells on Reactive Oxygen Metabolism and Root Activity of Cucumber and Figleaf Gourd Seedlings Under Cinnamic Acid Stress

QIAO Yong-xu, ZHANG Yong-ping, GAO Li-hong

Scientia Agricultura Sinica. 2015, 48(8):  1579-1587.  doi:10.3864/j.issn.0578-1752.2015.08.12

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【Objective】The aim of this paper was to clarify the defensive mechanism of root border cells (RBC) against cinnamic acid (CA) stress by analyzing the reactive oxygen metabolism and root activity of seedling RBC in cucumber (Cucumis sativus L.) and figleaf gourd(Cururbita ficifolia) under CA stress. 【Method】 The 5 mm length of aeroponicly cultured roots of cucumber cultivar Zhongnong No. 16 andfigleaf gourd cultivar bouche were divided into two groups: one group was used to investigate reactive oxygen metabolism by spraying with 0 and 0.25 mmol·L^-1 CA at 1 h interval, another group rinsing root tips of distilled water once every 4 h firstly and RBC were removed, then spraying 0 and 0.25 mmol·L^-1 CA every 1 h. The metabolic index of active oxygen was measured after treatment for 0, 12, 24 and 36 hours of CA stress, root fresh weight, root respiration rate and root activity of the seedlings were measured after 24 hours. 【Result】The root fresh weight and physiological metabolism were not affected significantly by RBC without CA stress. If the RBC were not removed, the root fresh weight and root activity decreased and the level of reactive oxygen species (ROS) and malonaldehyde (MDA), total respiration rate, CN-resistant respiration rate, the activities of superoxide dismutase (SOD), catalase (CAT) and guaiacol-dependent peroxidase (POD) increased significantly in cucumber under CA stress. However, the root fresh weight, root activity, the level of ROS and MDA, total respiration rate, CN-resistant respiration rate, the activities of SOD, CAT and POD were not affected significantly in figleaf gourd seedlings. If RBC were removed, the effect of CA stress on figleaf gourd was similar with cucumber, but significantly than RBC not removing. 【Conclusion】 RBC possibly attenuated the CA toxicity to the roots of cucumber and figleaf gourd through decreasing ROS and MDA content. The defensive ability of RBCs against CA on figleaf gourd was stronger than that of cucumber. Removal of RBCs could lead to great damage to the roots of cucumber and figleaf gourd significantly under CA stress.

STORAGE·FRESH-KEEPING·PROCESSING

Effect of Exogenous Spermine on Chilling Injury and Sucrose Metabolism of Post-Harvest Vegetable Soybean

LIU Chun-quan, SONG Jiang-feng, WANG Yuan, LI Da-jing

Scientia Agricultura Sinica. 2015, 48(8):  1588-1596.  doi:10.3864/j.issn.0578-1752.2015.08.13

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【Objective】 Mechanism of exogenous spermine in delaying chilling injury and regulating sucrose metabolism of vegetable soybean during cold storage was investigated to provide a theoretical basis for the preservation of post-harvest vegetable soybean. 【Method】 ‘Xindali No. 1’ vegetable soybeans used as the experimental materials were immersed in 0.5, 1 and 2 mmol·L^-1 spermine for 20 min, respectively, distilled water as a control, then stored at 1℃ for 10 weeks under the environmental condition of relative humidity of 85%-90%. The chilling resistance related physiological indexes, soluble sugars (sucrose, fructose and glucose) contents and the activities of sucrose metabolism related enzymes were all determined every week.【Result】 The chilling injury (CI) symptoms of vegetable soybean aggravated with the extension of storage period, and it appeared during the first two weeks of storage. Spermine treatment not only delayed the increase of CI index and postponed the symptoms until the 3rd week, but also inhibited cell membrane permeability, especially after 5 weeks of storage, both 0.5 and 1 mmol·L^-1 spermine treatment groups reached significant levels (P<0.05) compared with the control group. In addition, during cold storage, the membrane lipid peroxidation product, including malondialdehyde (MDA) gradually accumulated in vegetable soybean, low concentration of spermine could reduce the lipid peroxidation and decrease MDA accumulation, while high concentration of spermine had a toxic effect. Compared with the control group, 1 mmol·L^-1 spermine significantly improved the activities of antioxidant enzymes (CAT, POD and SOD) of vegetable soybeans during storage. And meanwhile, spermine treatment also significantly (P<0.05) reduced the sucrose loss of vegetable soybean, fructose and glucose contents in spermine-treated vegetable soybean grains were lower than the control group, but the concentration differences among all treatment groups were not significant (P>0.05) during the whole storage period. Through the correlation analysis between soluble sugars contents and sucrose metabolism related enzyme activities in vegetable soybean grains during cold storage, it indicated that sucrose content had a significant negative correlation (P<0.01) with AI activity, but a significant positive correlation (P<0.01) with SPS activity, and no significant correlation with NI and SS activities, which meant AI and SPS played a key role in sucrose degradation of vegetable soybean during cold storage, exogenous spermine effectively inhibited the sucrose loss by regulating the activities of sucrose-metabolizing enzymes in vegetable soybean.【Conclusion】 Spermine effectively delayed the chilling injury of vegetable soybean during storage at 1℃, and a dose of 1 mmol·L^-1 could be an effective treatment. Sucrose loss might cause the aggravated chilling injury, while spermine maintained higher sucrose content by simultaneously inhibiting the increase of AI activity and the decrease of SPS activity in vegetable soybean during cold storage.

The Optimal Composite Enzymatic Hydrolysis Process on Defatted Rice Bran and Its Nutritional Evaluation

WEN Wei, LIU Lei, ZHANG Ming-wei, ZHANG Rui-fen, WEI Zhen-cheng, TANG Xiao-jun, MA Yong-xuan

Scientia Agricultura Sinica. 2015, 48(8):  1597-1608.  doi:10.3864/j.issn.0578-1752.2015.08.14

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【Objective】The conditions of composite enzymatic hydrolysis of defatted rice bran were established in order to give some technical guidance for the high value utilization of defatted rice bran , and the nutritive characteristic change of its enzymatic extract was evaluated.【Method】 In order to prepare a high nutritional value enzymatic extract, defatted rice bran was used as the raw materials, they were firstly treated by gelatinization and liquidation, and then was simultaneously hydrolyzed by the composite enzyme (glucoamylase, acid cellulose, and acid protease). The reduced sugar yield and protein extraction yield were chosen as the evaluation criteria, as there are differences among parameters for reduced sugar yield and protein extraction yield, a fuzzy comprehensive evaluation model was established to optimize the parameters for the composite enzymatic hydrolysis of defatted rice bran. Freeze drying method was used to prepare the hot water extracts and the enzymatic hydrolysate extracts. The contents of solid, carbohydrate, soluble fiber and protein in defatted rice bran were also determined by using the government standard methods. The efficiency of composite enzymatic hydrolysis was evaluated by the nutriment differences compared by the hot water extraction and composite enzymatic hydrolysis. Amino acids contents of the three preparations were measured by high-speed amino acid analyzer. Nutritive evaluation for the three preparations’ proteins was assessed by using the WHO/FAO method.【Result】The optimum conditions for composite enzymatic hydrolysis established by fuzzy comprehensive evaluation model were that the composite enzyme concentration was 3.5%, glucoamylase was 0.5%, acid cellulose was 1.5% and acid protease was 1.5%, the composite enzymatic hydrolysis was carried out at pH 4.1, temperature 57.4℃, material to water ratio 1:5, and 190 minutes. The raw material utilization ratio, carbohydrate conversion ratio, soluble dietary fiber extraction yield and protein extraction yield by the composite enzymatic hydrolysis were 48.43%, 65.33%, 6.68% and 58.43%, the percentage of essential amino-acid in the composite enzymatic extract protein was 36.93%. Compared with the hot water extracting, the raw material utilization ratio, carbohydrate conversion ratio, soluble dietary fiber extraction yield and protein extraction yield was increased by 118.73%, 90.74%, 284.22% and 257.14% as compared with those of hot water extracting, the percentage of essential amino-acid increased by 276.33% (P＜0.05). Compared with the raw material, the soluble dietary fiber increased by 13.62%, the content of essential amid acid in per solid increased by 14.78%, which of the lysine, methionine, threonine and valine increased by 35.38%, 37.75%, 40.06% and 7.70%, respectively (P＜0.05), the ratio of essential amino acid to nonessential amino acid was 0.59, which was closely to the FAO/WHO recommended value 0.6. 【Conclusion】The raw material utilization ratio increased significantly through the composite enzymatic hydrolysis process, compared to the hot water extracting, the soluble solid content, soluble carbohydrate, soluble dietary fiber and protein content raised significantly, thus it has provided a dependable approach for preparation of a defatted rice bran enzymatic extract with potential use as a functional food.

ANIMAL SCIENCE·VETERINARY SCIENCERE

Impact of Putrescine and Proline on Suckling Piglet Jejunum Villus - crypt Axis Epithelial Polyamine Metabolism and Wnt Signal Pathway

WANG Xiao-cheng, XIONG Xia, YANG Huan-sheng, GAO Wei, GONG Min, YIN Yu-long

Scientia Agricultura Sinica. 2015, 48(8):  1609-1615.  doi:10.3864/j.issn.0578-1752.2015.08.15

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【Objective】The aim of this paper is to study the effects of putrescine and proline on polyamines (putrescine, spermidine and spermine) metabolites, relative mRNA expression levels of Wnt signaling molecules along the villus-crypt axis in the jejunum of sucking piglet. 【Method】 Eighteen 0-day old newborn crossbred (Landrace × Yorkshire × Duroc) piglets were randomly divided into three groups: control group, putrescine group and proline group. The piglets in control group were administered an equal volume of saline, and that in other two groups received putrescine (5 mg·kg^-1 body weight ) and proline (25 mg·kg^-1 body weight ), respectively. Piglets were weaned at 14-day old and slaughtered on 3 days postweaning. The jejuna epithelial cells along the crypt-villus axis were isolated by Krebs Henseleit (KH) solution and yield 3 “cell fractions” (Fraction 1, 2 and 3). The concentration of polyamine in F1, F2 and F3 was determined by high performance liquid chromatography, and relative mRNA expression of related genes of polyamine metabolic and wnt signaling pathways were measured by real time PCR. 【Result】 The concentration of putrescine, spermine and spermidine in F1 were significantly increased (P＜0.05) in proline group compared to control group, while there was no significant difference in putrescine group (P＞0.05). The concentrations of spermine and spermidine in F2 of proline or putrescine groups were significantly higher than that of control group (P＜0.05). the concentrations of putrescine, spermine and spermidine in F3 of three group were not changed significantly (P＞0.05). The expression of ornithine decarboxylase (ODC) gene in F1 of putrescine group was significantly higher than that of proline group and control group (P＜0.05). The expression of arginase in F2 of proline group was significantly higher than that of putrescine group and control group (P＜0.05). The expression of SFRP3 in F2 of proline group was significantly higher than that of putrescine group (P＜0.05); the expression of sFRP4 in F3 of putrescine group is significantly higher than that of control group and proline group (P＜0.05). 【Conclusion】 Exogenous putrescine and proline increased the concentration of polyamine in differentiated epithelial cells of jejunum in sucking piglet and promoted the polyamine metabolism, however, no statistically significant effect was observed on crypt cell. In addition, exogenous putrescine and proline promote differentiation in the intestinal epithelial cells by Wnt signaling pathways.

Study on the Association Between the Expression of Candidate Genes in Different Waves and Hair Follicle Characteristics

NI Rong, SUN Wei, YIN Jing-feng, Lü Xiao-yang, WANG Qing-zeng, SU Rui, CHEN Ling, WU Wen-zhong, XU Hou-sheng, LI Yong, CHEN Jia-zhen, LIU Wei-zhong

Scientia Agricultura Sinica. 2015, 48(8):  1616-1623.  doi:10.3864/j.issn.0578-1752.2015.08.16

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 【Objective】The article aims to study the relevance between the expression of 5 candidate genes in different patterns and hair follicle characteristics, in order to learn more about the correlation between candidate genes and hair follicle characteristics and screen the genes for later functional verification. 【Method】The article detected the expression of 5 genes between different groups by RT-PCR, combined histology and microscopic observation techniques, analyzed the correlation between 5 genes and hair follicles. 【Result】 The results showed that: Hu-sheep hair follicle was distributed in groups, and it was mainly three follicles in a group, the larger diameter was the primary hair follicles, and the relatively small diameter was primary follicles.The diameter of primary follicles in large waves was significant differences with medium and small waves (P＜0.01) , and there was no significant differences between small and medium waves(P＞0.05). The diameter of secondary follicles in medium waves was significantly differences with large and small waves(P＜0.01), but there were no significant differences between small and large waves(P＞0.05).The number of primary follicles was no significant differences among large, medium and small waves(P＞0.05), but in the same vision,the number of primary follicles in large waves was more than that of medium and small waves.The number of hair follicles in medium waves was significant differences with large and small waves(P＜0.01), but there were no significant differences between large and small waves(P＞0.05);The expression of MMP2 among medium waves was highly significant differences with large and small waves(P＜0.01) and showed no significant differences between large and small waves(P＞0.05). The expression of BMP7 and SFXN1 among small waves was significant differences with large and medium waves(P＜0.05) and showed no significant differences between large and medium waves; The expression of remaining genes was not significant differences among large, medium and small waves(P＞0.05). The relative expression of MMP2 had a significant positive correlation with secondary follicle in large waves(P＜0.05). The relative expression of BMP7 was significantly correlated with primary follicle diameter in small waves, with the number of small waves’ secondary follicles showed a highly significant positive correlation(P＜0.01), and negatively correlated with medium waves’ primary follicle diameter, the number of secondary follicle(P; The relative expression of SFXN1 was negatively correlated with large waves’ primary follicles(P＜0.05), showed significantly positively related and highly significant positive correlation with small waves’ primary and secondary follicle diameter, and in medium waves’ primary follicles showed significant negative correlation. Under the condition of the same expression of BMP7 gene, the primary follicles’diameter was more than medium waves, the number of secondary follicles was more than medium waves, it was consistent with biopsy results. With the same expression of MMP2 gene , the number of large waves was more than small waves and medium was the least. It was consistent with biopsy. In addition, with the same expression of SFXN1 gene, the diameter of small waves was the largest and medium was the least. It was not match with the biopsy results, but the number of secondary follicles was slightly difference, the trend was the same, the results were basically consistent. Although other genes associated with hair follicles, their expression in large, medium, small waves were not significant.【Conclusion】 BMP7、MMP2、SFXN1 can be used as an important candidate gene for early breeding.＜0.05)

Effect of HOPX Gene Overexpression on Chicken Preadipocyte Proliferation

SHI Hong-yan, HE Qi, CHENG Min, SUN Ying-ning, LI Hui, WANG Ning

Scientia Agricultura Sinica. 2015, 48(8):  1624-1631.  doi:10.3864/j.issn.0578-1752.2015.08.17

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【Objective】The objective of this study was to construct the eukaryotic expression vector of chicken full-length HOPX gene and investigate the effect of HOPX gene overexpression on chicken preadipocytes proliferation.【Method】Using Primer Premier 5.0 software, a pair of primers was designed to amplify the full-length coding sequence (CDS) of chicken HOPX. The full-length coding sequence (CDS) of chicken HOPX gene was PCR amplified from the cDNA from the abdominal fat tissues of AA broiler chickens and cloned into pCMV-HA vector. Chicken preadipocytes were isolated from the abdominal fat tissues of 12-day-old AA broiler chicken by collagenase digestion, cultured and transfected with pCMV-HA-HOPX and pCMV-HA empty vector, respectively. Cell proliferation was assayed by microscopic examination and Cell Counting Kit-8 (CCK-8). Gene expression was measured by Western blotting and quantitative Real-time RT-PCR. 【Result】 The sequencing results showed that the full-length coding sequence of chicken HOPX gene is222bp and identical with NCBI reference sequence (NM_204556). Western blotting analysis showed that pCMV-HA-HOPX could correctly express the HA-tagged HOPX. The microscopic examination showed that the numbers of the preadipocytes transfected with pCMV-HA-HOPX were less than those of the preadipocytes transfected with empty pCMV-HA vector at 24 h and 48 h after of cell adhesion. CCK-8 analysis showed that the OD values of the preadipocytes transfected with pCMV-HA-HOPX were significantly lower than those of the preadipocytes transfected with empty pCMV-HA vector at 24 h, 48 h and 72 h after of cell adhesion (P＜0.01). Consistently, at 24 h after of cell adhesion, the mRNA expression of CyclinD1 was significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P＜0.05); at 48 h after of cell adhesion, the mRNA expression of PCNA was significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P＜0.05); at 72 h after of cell adhension, the mRNA expression of CyclinD1 and PCNA were extremely significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P＜0.01) 【Conclusion】 HOPX gene overexpression in vitro inhibits chicken preadipocyte proliferation.

AGRICULTURAL ECONOMY

Driving Mechanism and Regional Differentiation of Cultivated Land Non-Agricultural-Transformation

ZHANG Guang-hong, CUI Xu-feng

Scientia Agricultura Sinica. 2015, 48(8):  1632-1640.  doi:10.3864/j.issn.0578-1752.2015.08.18

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【Objective】 The purpose of this study is to reveal the cultivated land non-agricultural-transformation and its regional differentiation. 【Method】 Firstly, the cultivated land non-agricultural-transformation in China was analyzed based on the method of fixed-effect panel data model and by using Chinese cultivated land non-agricultural-transformation, growth of town populations, investment in fixed assets, level of economic development, cultivated land resource endowment panel data in 2001-2008. In order to compare the difference of different areas, test the models using eastern region data, midland data, and western region data, respectively, the differences among the three regions in cultivated land non-agricultural-transformation were comparative analyzed. 【Result】 Model test results show that the economic development level and its quadratic term, cultivated land resource endowment show a significant state based on the control of urban population growth and fixed asset investment. There are differences among eastern, central and western regions. Investment in fixed assets and cultivated land resource endowment show a extreme significance in eastern, central, and western regions respectively. Elastic coefficient of investment in fixed assets in central regions is bigger than western regions.【Conclusion】 The cultivated land non-agricultural-transformation and economic development show a significant reverse diagram "U" rule of Environmental Kuznets Curve. The cultivated land non-agricultural-transformation in China is significantly influenced by the urbanization growth and investment in fixed assets. Urban population increase (or decrease) of each 1%, the quantity of cultivated land conversion will increase (or decrease) of 0.1 percentage, investment in fixed assets increase (or decrease) of each 1%, the quantity of cultivated land conversion will increase (or decrease) of 0.7 percentage. Cultivated land non-agricultural-transformation driving factors have significant difference among eastern, central and western regions. Cultivated land resource endowment is a significant affecting factor of non-agricultural-transformation in eastern China, cultivated land resource rich regions tend to higher speed of cultivated land non-agricultural-transformation. Investment in fixed assets is a significant affecting factor of non-agricultural-transformation in central and western China, driving effect of investment in fixed assets in central regions is greater than western regions.

Spatial Evolution, Mechanism, Effect and Policies of Vegetable Production Agglomeration in China

WU Jian-zhai, SHEN Chen, WANG Sheng-wei, ZHANG Jian-hua, KONG Fan-tao

Scientia Agricultura Sinica. 2015, 48(8):  1641-1649.  doi:10.3864/j.issn.0578-1752.2015.08.19

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【Objective】 In recent years, vegetable supply and demand pattern characterized by “large market, great circulation” has been basically formed in China. And the change of the spatial agglomeration in vegetable industry is remarkable. In this paper, the characteristics, effects, and mechanism of the spatial agglomeration evolution in China’s vegetable production from 1995 to 2012 were systematically discussed by taking 31 provinces (cities) as research units, and the policy suggestions were also proposed. This research will provide a support for optimizing production arrangement and ensuring effective supply. 【Method】 The share of n largest provinces (cities) in total production was calculated based on the index of concentration ratio. In order to reveal the characteristics of spatial agglomeration in vegetable production industry from 1995 to 2012, the spatial concentration on provincial level was measured by using Gini coefficient model. To show the effect of production agglomeration on vegetable industry development, the model based on modified Cobb-Douglas production function was built to analyze the significance of the inputs such as vegetable production agglomeration level, vegetable price, labor per unit, and investment per unit. 【Result】 The characteristic of China’s vegetable production spatial distribution clustered in Southwest, mainly in eastern China, and less in western China at the beginning of the study period was significantly changed in the research period. During 1995 to 2012, the ratio of output in western China increased 6%. The gap gradient in output between eastern and western China diminished obviously. In the vertical direction, the ratio of output in northern China increased 5% which indicated that the gravity of output had moved towards the north. During 1995 to 2012, CR₄ and CR₈, the two indexes of concentration ratio, were increased slightly with constant fluctuation at first, and were decreased a little to steady state at the later stage, lingering between 0.4 and 0.6. The peaks of the two indexes were hit in 2001 and 2004, and the bottoms were reached in 1995 and 1997, respectively. The Gini coefficient increased at first and decreased later then. It reached its highest level of 0.5045 in 2004 and gradually decreased after that. The spatial agglomeration of vegetable production was affected by a variety of factors such as nature, society, and economy, which could lead a space reconstruction and was also a process of industrial optimization. It was more dependent on light, temperature, water, soil, and other natural factors at the beginning, and then the economic and policy factors played more important roles in the evolution process. The modified effect evaluation equation passed F-test which meant that it had a relatively high capacity in interpretation. Gini coefficient at 10% level of significance passed t-test. The result of model showed that per unit vegetable production would increase by 2.48% when Gini coefficient increased by 1%. 【Conclusion】 From 1995 to 2012, the gap gradient of China’s vegetable production between eastern and western area diminished notably. The gravity of production moved towards the north and the characteristics of spatial distribution changed obviously, especially the volatility showed at the beginning. The impact to vegetable production due to the spatial agglomeration is significant, and strengthening the agglomeration could effectively promote the production of vegetable. In view of optimizing the spatial pattern of vegetable production, strengthening the construction of the main production areas, promoting large-scale and specialized production, and focusing on the commencement of industrial nodes collaboration should be carried out in the future to promote healthy development of vegetable industry.

RESEARCH NOTES

Cloning and Activity Analysis of the Promoter of Sulfate Transporter Gene GmSULTR1;2b

ZHOU Xiao-qiong, DING Yi-qiong, ZUO Li, YU De-yue

Scientia Agricultura Sinica. 2015, 48(8):  1650-1659.  doi:10.3864/j.issn.0578-1752.2015.08.20

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【Objective】 Following the nitrogen, phosphorus and potassium, sulfur is the fourth nutrient necessary for the plants. Sulfur-containing organic compounds involved in many important physiological and biochemical reactions in plants, which play an important role in withstanding environmental stress and growth and development of plants. Sulfate transporters (sulfate transporter, SULTR) participate in absorption and transportation of the exogenous sulfate(SO₄^2-). The sulfate transporter gene GmSULTR1;2b of soybean is specifically expressed in the root, which plays a role in transporting sulfate from the environment. Cloning the promoter of GmSULTR1;2b, and studying on its activity and tissue expression will contribute to understanding the regulatory mechanism of GmSULTR1;2b. It can also provide a molecular foundation for improving the content of sulfur amino acid in soybean.【Method】According to the sequence of GmSULTR1;2b in the NCBI, the predicted 2 259 bp upstream was analyzed and predicted as the promoter. The online debases PLACE and Plant-CARE were used to prognose the regulatory elements of the sequence. The sequence was obtained by PCR through taking the soybean cultivars Nannong N2899 DNA as template. The sequence was fused with GUS to construct the plant expression vector pSULTR1;2b::GUS. The binary vector constructs were transformed into Agrobacterium tumefaciens EHA105 by the freeze-thaw method. The transient expression assays were carried out in soybean by Agrobacterium tumefaciens-mediated method, and the activity of the promoter was analyzed by using GUS as the reporter gene. In addition, hairy root transformation experiment was carried out by transforming the binary vector constructs into Agrobacterium rhizogenes K599. By analyzing the transverse section of the hairy roots under the stereoscope, its expression was observed. Finally, the GUS activity was implemented to test the activity of the promoter, which was based on the positive transformed hairy roots.【Result】The cloned promoter sequence of GmSULTR1;2b from Nannong N2899 was basically in line with the sequence in NCBI. Through online prediction analysis of regulatory elements, it was found that the promoter contained not only TATA-box, which was the necessary component of eukaryote, but also contained hormone response element ERE (ethylene response element), ABRE (abscisic acid response element), stress response element TC - rich repeats (diseases and insect pests stress and drought stress), the AT - rich element (the DNA of AT - rich and protein binding sites) and MYB, etc. The successful construction of the recombination vector pSULTR1;2b::GUS was confirmed via PCR and sequencing appraisal. The X-gluc dyeing conducted on the transient expression of soybean showed blue where the soybean was infected by the recombinant vector pSULTR1;2b::GUS. It indicated that the promoter could drive GUS expression downstream. After staining the transformed hairy roots, the transverse section of the positive hairy roots was analyzed under the stereoscope. The GUS was mainly found in the root hair, root epidermis and the stele, which manifested the promoter mainly expressed in the root hair, root epidermis and the stele. The GUS activity test of the transformed hairy roots attested weaker activity than the promoter of CaMV35S.【Conclusion】GmSULTR1;2b promoter was cloned. It could drive GUS in the downstream, and express in the root hairs, root epidermis and the column.

Responses of Antioxidant System to Cold Water Stress in Weedy and Cultivated Rice with Different Chilling Sensitivity

WANG Guo-jiao, WANG Jia-yu, MA Dian-rong, MIAO Wei, ZHAO Ming-hui, CHEN Wen-fu

Scientia Agricultura Sinica. 2015, 48(8):  1660-1668.  doi:10.3864/j.issn.0578-1752.2015.08.21

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【Objective】 Rice is most sensitive to temperature from booting stage to heading and flowering stages, so cold damage during this stages severely limited rice growth and the further expansion of acreage in Northeast China. Study on the physiological mechanism of rice cold tolerance will provide an important safeguard for the rice production in Northeast China. The aim of this study was to understand the changes of reactive oxygen species metabolism, antioxidant enzyme activities and antioxidants contents in flag leaves of weedy and cultivated rice with different chilling sensitivity at flowing stage under cold water stress, and provide a theoretical basis for the breeding and cultivation of rice tolerance to cold. 【Method】 Two chilling-tolerant genotypes, weedy rice WR03-45 and cultivated rice Lijiangxintuanheigu, and two chilling-sensitive genotypes, weedy rice WR03-26 and cultivated rice Xiuzinuo, were used and grown in plastic pots. The time of booting stage of main spike was calculated according to length of the flag leaf collar. The tested materials were subjected to low temperature in cold water pool at booting stage, and the control was the tested materials without cold water treatment. In order to study  the effects of cold water stress on reactive oxygen species (ROS) metabolism, antioxidant enzyme activities and antioxidants contents of flag leaves at flowing stage, the flag leaves of tested materials were clipping and saved in ultra-low temperature freezer at -85 ℃, which were used to measure superoxide radical () generating rate, hydrogen peroxide (H₂O₂) content, malondialdehyde (MDA) content, activities of antioxidant enzymes of superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and non-enzymatic antioxidants contents of ascorbate acid (AsA) and reduced glutathione (GSH). 【Result】 Under cold water stress, only CAT and GR activities in flag leaves of chilling-sensitive weedy rice WR03-26 increased significantly at flowering stage, and SOD, CAT and GR activities in flag leaves of chilling-sensitive cultivated rice Xiuzinuo increased significantly at flowering stage. There were no significant difference in contents of AsA and GSH and ratio of AsA/DHA and GSH/GSSG in flag leaves of WR03-26 and Xiuzinuo compared with their controls.  generating rate and MDA content increased significantly, and H₂O₂ content increased very significantly in flag leaves of WR03-26 and Xiuzinuo. SOD activity increased significantly, and POD, APX, CAT and GR activities increased very significantly in flag leaves of chilling-tolerant weedy rice WR03-45 at flowering stage. SOD, POD and APX activities increased significantly, and CAT and GR activities increased very significantly in flag leaves of chilling-tolerant cultivated rice Lijiangxintuanheigu at flowering stage. Antioxidants (AsA and GSH) contents and AsA/DHA ratio and GSH/GSSG ratio also increased significantly in flag leaves of WR03-45 and Lijiangxintuanheigu under cold water stress.  generating rate, H₂O₂ content and MDA content in flag leaves of WR03-45 and Lijiangxintuanheigu had no significant difference compared with their controls. 【Conclusion】 Under cold water stress, the chilling-tolerant rice could raise the antioxidant enzyme activities and antioxidants contents, decrease the levels of ROS and the membrane lipid peroxidation and improve the tolerance to cold water stress. Chilling tolerance in rice is associated with the capacity of antioxidative system removing ROS under chilling stress. Weedy rice WR03-45 is useful as a germplasm resource of strong chilling tolerance for improving chilling tolerance in cultivated rice.