Scientia Agricultura Sinica

Cloning and Analysis Expression Patterns of GhSPS1 Gene in Cotton

JIA Rong-Rong, LU Sha-Sha, JIANG Yuan, LIU Zeng-Ping, YU Jia-Ning

Scientia Agricultura Sinica. 2012, 45(6): 1031-1041. doi:10.3864/j.issn.0578-1752.2012.06.001

Abstract ( 636 )

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【Objective】 The GhSPS1 gene was cloned and analyzed the expression patterns under abiotic stresses treatments. In different tissues and at fiber developmental stages, transcriptional patterns of the gene were also detected. 【Method】 The DNA and cDNA sequence of GhSPS1 were cloned from cotton by genome-walking and over-lap PCR, respectively. The expression patterns of GhSPS1 were analyzed by using semi-quantitative PCR when the cotton seedlings were treated with abiotic stresses. The characteristics of four genes, GhSPS1, GhSUS3, GhINV and GhCESA4, were examined by real-time quantitative PCR at fiber developmental stages. 【Result】 Comparison GhSPS of DNA and cDNA sequence, the results showed that GhSPS1 is 4 545 bp, with 3 108 bp ORF encoding 1 035 amino acids, include 10 introns and 11 extrons. The results of semi-quantitative PCR exhibited that the mRNA levels of GhSPS1 increased when the cotton seedlings were treated with ABA and low temperature. In high temperature treatment, the expression of GhSPS1 decreased firstly, and then followed with a little increase. Analysis of the genes expression patterns by real-time quantitative PCR showed that GhSPS1 and GhSUS3 expressed in all tissues, but the highest expression was in 0 DAF ovules, 3 DAF and 18 DAF fibers. On the contrast, GhINV transcripts were expressed higher in 3-15 DAF fibers than in other tissues. And GhCESA4 transcripts had the highest expression level in 18 DAF fibers. 【Conclusion】 GhSPS1 belongs to SPSA family, which can be induced by using ABA, low and high temperature treatments. The expression of the gene might have close relationship with initial fiber development and secondary wall thickening.

Construction and Application of SSR Molecular Markers System for Genetic Diversity Analysis of Chinese Tartary Buckwheat Germplasm Resources

GAO Fan, ZHANG Zong-Wen, WU Bin

Scientia Agricultura Sinica. 2012, 45(6): 1042-1053. doi:10.3864/j.issn.0578-1752.2012.06.002

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【Objective】 The SSR molecular markers system was optimized and constructed for genetic diversity analyses of Chinese tartary buckwheat germplasm resources, which is helpful for evaluating Chinese tartary buckwheat collections. 【Method】 The SSR-PCR system was optimized by [L16(45)]orthogonal design, the optimized gel concentration of PAGE was confirmed, and the genetic diversity of 50 tartary buckwheat accessions was analyzed by 19 SSR primer pairs screened from 250 ones of different crops. 【Result】The optimized SSR-PCR system was as follows: 30 ng DNA template, 150 μmol•L-1 dNTP, 0.1 μmol•L-1 primer, 2.0 U•L-1 TaqDNA polymerase, 2.0 mmol•L-1 Mg2+, 1×Taq buffer and ddH2O then added up to terminal volume of 25 μL with 6% PAGE for testing. The primers screening efficiency was 7.6%, and the primers from common buckwheat were applicable. A total of 157 alleles were detected by 19 primers, with 2-11 alleles for each primer pair, and the average was 7.42. Moreover, the averaged PIC and DP values were 0.8881 and 5.684, respectively. Using Popgen Ver.1.31, 50 accessions were clustered into 5 groups at GS 0.578. The clustering results revealed that the genetic diversity of accessions of tartary buckwheat was not correlated to their geographic origins. The genetic diversity of tartary buckwheat from Sichuan was very rich as genetic parameters were the highest. The core primers could be used to identify the similar accessions.【Conclusion】 The SSR molecular markers system was effective for assessment of genetic diversity of Chinese tartary buckwheat germplasm resources. SSR primers of common buckwheat could be used in tartary buckwheat. TBP5 and Fes2695 were SSR core primers. It showed a high genetic diversity in 50 Chinese tartary buckwheat accessions which could be classified into 5 groups.

Effects of Increasing Aroma Cultivation on Aroma and Grain Yield of Aromatic Rice and Their Mechanism

HUANG Zhong-Lin, TANG Xiang-Ru, WANG Yu-Liang, CHEN Mu-Jiao, ZHAO Zheng-Kun, DUAN Mei-Yang, PAN Sheng-Gang

Scientia Agricultura Sinica. 2012, 45(6): 1054-1065. doi:10.3864/j.issn.0578-1752.2012.06.003

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【Objective】The purpose of this study was to investigate the effects of increasing aroma cultivation on aroma and grain yield of aromatic rice and their mechanism.【Method】A field contrast experiment was conducted with four conventional aromatic rice cultivars, Guixiangzhan, Meixiangzhan 2, Zhongxiang 1 and Raopingxiang, to evaluate the effects of increasing aroma cultivation(IAC) and conventional cultivation(CK) on aroma (2-acetyl-1-pyrroline) content in brown rice, biological characteristics and grain yield and its components of aromatic rice.【Result】The results showed that, compared with CK, increasing aroma cultivation could significantly increase 2-acetyl-1-pyrroline content in brown rice and milled rice, free proline content and proline oxidase activity in flag leaves and rice grains during post-heading and filling stages, and chlorophyll content(SPAD) in leaf (flag leaf) and leaf area index (LAI) at the main growth stages, dry matter translocation rate of stem-sheath, population photosynthesis potential during post-heading and filling stages, effective panicles, the total group of actual production, seed setting rate and yield, while it had no significant effect on 1 000-grain weight.【Conclusion】Increasing aroma cultivation could significantly increase free proline content and proline oxidase activity in flag leaves and grains of aromatic rice, then significantly increase the accumulation of 2-Acetyl-1-pyrroline(2-AP) in brown rice. Increasing aroma cultivation could also significantly enhance dry matter translocation rate of stem-sheath and population photosynthetic capacity during post-heading and filling stages, and then significantly increase the effective panicles the total group of actual production and seed setting rate, therefore, increasing aroma cultivation could increase not only aroma content but also the grain yield.

Isolation of Wheat Endophytic Diazotrophs and Determination of 1-Aminocyclopropane-1-Carboxylate Deaminase

QIN Bao-Jun, LUO Qiong, GAO Miao, HU Hai-Yan, XU Jing, ZHOU Yi-Qing, SUN Jian-Guang

Scientia Agricultura Sinica. 2012, 45(6): 1066-1073. doi:10.3864/j.issn.0578-1752.2012.06.004

Abstract ( 720 )

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【Objective】 The objective of this study is to determine the ammount of wheat endophytic diazotrophs and screen for ACC (1-aminocyclopropane-1-carboxylate) deaminase activity from the diazotrophs, determine the phylogenetic and classific position of selected strains and prepare strains for microbial fertilizer production. 【Method】 Surface sterilization and nitrogen-free medium were used to isolate diazotroph and ACC was used as sole nitrogen source to screen strains with ACC deaminase activity. Nitrogenase activity was determined with acetylene reduction assay. 16S rDNA was amplified with PCR and analysed with MEGA software. Strain identification was carried out based on the morphology, physiology, biochemical test results and 16S rDNA analysis. 【Result】 The ammount of endophytic diazotrophs at jointing stage of wheat was (0.2-17.8)×105 cfu•g-1 fresh weight. Sixty endophytic diazotrophs with nitrogenase activity ranging 1-36 nmol C2H4/h•mg protein were isolated from wheat. Nine of the 60 endophytic diazotrophs were ACC deaminase positive, the range of enzyme activity is 0.87-9.32 µmol α-ketobutryric acid/h•mg protein. New isolate 9136 with nitrogenase activity 1.82 nmol C2H4/h•mg protein and ACC deaminase activity 9.32 µmol α-ketobutryric acid/h•mg protein was identified as Pseudomonas sp.. 【Conclusion】 About 105 cfu•g-1 (fresh weight) endophytic diazotrophs naturally colonized field grown wheat, some of these endophytic diazotrophs could produce ACC deaminase. A few strains showed relatively high ACC deaminase activity, and they might play a role in crop resistance to enviromental stress.

Dynamic Characteristics of Summer Maize Yield Performance in Different Planting Dates and Its Effect of Ecological Factors

LI Xiang-Ling, LI Cong-Feng, HOU Yu-Hong, HOU Hai-Peng, GE Jun-Zhu, ZHAO Ming

Scientia Agricultura Sinica. 2012, 45(6): 1074-1083. doi:10.3864/j.issn.0578-1752.2012.06.005

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【Objective】Ecological factors play an important role in maize yield. In order to clarify the relationship between light, temperature, water in the growing period and yield performance of maize, provide a reference to the high yield of maize in the North China Plain. 【Method】 Three types of cultivars (YN 103, XY335, ZD 958 and DH 661) were used as materials, and three sowing dates (May 3rd, May 28th, and June 22nd) and four density treatments (45 000, 60 000, 75 000 and 90 000 plants/hm2) were designed. The dynamic of leaf area index, grain yield were measured and the growing period and ecological factors were recorded. 【Result】 Yield showed an order of XY335>ZD958> DH661>YN103, early sowing date>medium sowing date >late sowing date. Ecological factors had a regulatory effect on yield and yield performance indexes, effective temperature after silking affected the average leaf area index and mean net assimilation rate, daily average temperature affected the growth days and harvest index, total rainfall and sunshine hours affected grains number per spike and grain weight. Effective temperature of whole growth period, especially after silking was the most important ecological factors affecting grain yield. The order of correlation coefficient was that ，the effective temperature of whole growth period(0.64**)，the effective temperature after silking(0.55**), the daily average temperature after silking (0.51**), the daily average temperature of whole growth period(-0.49*), the rainfall after silking(-0.47*), the sunshine and silking(0.42*). The effective temperature was the main effective factor on yield. 【Conclusion】 Two-crop a year system is practiced in Huanghuaihai area, so medium maturing cultivars, such as ZD 958, should be selected, optimum early sowing should be practiced to ensure adequate and effective accumulated temperature of grain sufficient filling time for further enhancing corn yield in this area.

Soluble Expression and Purification of Fusarium graminearum β2-tubulin in Escherichia coli

XU Jian-Qiang, ZHOU Yu-Jun, ZHANG Cong, ZHOU Ming-Guo

Scientia Agricultura Sinica. 2012, 45(6): 1084-1092. doi:10.3864/j.issn.0578-1752.2012.06.006

Abstract ( 632 )

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【Objective】 The objective of this study is to express the β2-tubulin of Fusarium graminearum in E. coli in soluble form and purify it by HisTrap™ HP columns.【Method】 β2-tubulin gene contained in the plasmid (pET32a+-β2-tubulin) was amplified, cloned to the vector pET30a+ , and then transformed into the hosts Rossatta (DE3) pLysS and BL21 (DE3). After the positive clones were screened by the colony PCR and double enzymatic digestion, the induced fusion proteins were obtained and verified by SDS-PAGE and Western blot. In order to express the fusion protein in soluble form, the inducing factors, including temperature, induction time, IPTG (Isopropyl β-D-Thiogalactoside) concentration, cell density, medium composition and hosts were screened. 【Result】The positive clones which could express more fusion protein after induced were screened, however, the fusion proteins formed inclusion bodies. The molecular weight of fusion proteins were confirmed to be 51.6 kD by SDS-PAGE, which also showed specific activity to anti-6×His monoclonal antibody. After the optimization of imidazole concentration in binding and washing buffer, the soluble fusion protein was purified and its structural integrity was preserved through the purification process by the verification of Western blot. 【Conclusion】 The methods described here can be used to express and purify other recombinant proteins in soluble form in E. coli. The purified fusion tubulin can be used in the studies of tubulin target drug resistant mechanisms as well as high throughout screening of new fungicide.

Identification of QTLs Conferring Resistance to Late Blight in Solanum lycopersicoides LA2951 Introgression Line Population

ZHANG Chun-Zhi, LIU Lei, SUN Yu-Yan, ZHOU Long-Xi, YANG Yu-Hong, XIE Bing-Yan, LI Jun-Ming

Scientia Agricultura Sinica. 2012, 45(6): 1093-1105. doi:10.3864/j.issn.0578-1752.2012.06.007

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【Objective】The objective of this study is to explore the potential loci for resistance to late blight of introgression line (IL) population derived from a nightshade related species Solanum lycopersicoides LA2951. 【Method】 Detached-leaflet assay (DLA) was used for the artificial inoculation. The abaxial surface of each leaflet was inoculated with a drop of 10 μL sporangial suspension (2×104 sporangiums/mL), and the inoculated leaves were incubated at 19℃ with 70%-100% relative humidity. Lesion size (LS) of the leaf and disease incidence (DI) were measured 6 d after inoculation.【Result】The resistance of this wild species was controlled by quantitative trait loci (QTL). In total, 5 QTLs (Rpiq1b, Rpiq2b, Rpiq4b, Rpiq8a, Rpiq11) were identified after inoculation with race T1,2, which significantly reduced LS, and 3 QTLs (Rpiq1a, Rpiq2a, Rpiq8b) were indentified which clearly reduced DI. However, only 2 QTLs (Rpiq4a and Rpiq5) reducing DI were indentified after inoculation with more virulent race T1,2,4. It proved that QTLs derived from LA2951 showed a clear race-specific resistance. Based on tomato high density genetic linkage map, all QTLs detected in this paper were co-localized with those previously identified in other tomato wild species. In addition, 2 susceptibility QTLs (Spiq4 and Spiq10) increasing DI were identified after inoculation with both race T1,2 and race T1,2,4, which were located at the end of short arm of chromosome 4 and chromosome 10, respectively. 【Conclusion】Ten resistance QTLs and 2 susceptibility QTLs were identified, and they presented a race-specific behavior. The results obtained in this paper provided a theoretical basis for breeding of late blight resistance in tomato.

Construction of SSH-cDNA Libraries and EST Analysis in Roots of Xiaoli Black Bean in Response to Heterodera glycines Parasitization

WANG Fang, DUAN Yu-Xi, CHEN Li-Jie, WANG Yuan-Yuan, ZHU Xiao-Feng, WANG Dong

Scientia Agricultura Sinica. 2012, 45(6): 1106-1115. doi:10.3864/j.issn.0578-1752.2012.06.008

Abstract ( 635 )

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【Objective】The objective of this study is to research into the early stage root gene expression profile of G. max induced by H. glycines and to explore the resistant mechanism to the pathogen at molecular level. 【Method】SSH-cDNA libraries enriched with differentially expressed ESTs were constructed from SCN-challenged root tissues at pre-penetration and early infection stages from Xiaoli black bean (G. max) by suppression subtractive hybridization and reverse Northern blot. 【Result】 One hundred and sixty-six unique ESTs were identified and analyzed with Blastx and Blastn by comparing sequences in the GenBank. One hundred and ninteen unigenes accounted for 83% of all the unigenes showed high homology with the function known genes or proteins. The function-known ESTs were annotated into functional categories including signal recognition and transduction, energy and material metabolism, stress responses, transcriptional regulation, protein synthesis and/or modification, transport functions, cellular architecture.【Conclusion】Discreet gene tag clusters primarily including catalase, ubiquitin, chitinase, lipoxygenase, aquaporin, ripening related protein, metallothionein, plasma membrane intrinsic protein, cytochrome P450, glyceraldehyde-3-phosphate dehydrogenase were abundant in the SCN-infected roots. It is speculated that those genes played an important function in the incompatible interaction between Xiaoli black bean and H. glycines.

The Declining Process of Soil Phosphorus Availability and Comparison Between Agronomic and Environmental Indexes in Red Soil

WEI Hong-An, LI Yu-Yuan, YANG Rui, GAO Ru, SHI Hui, ZHANG Man-Yi, WU Jin-Shui

Scientia Agricultura Sinica. 2012, 45(6): 1116-1126. doi:10.3864/j.issn.0578-1752.2012.06.009

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【Objective】This study is to probe the key declining process of soil available phosphorus (SAP) and its relationship with P fertilizer application rate in red soil, and to verify the mathematical relationships among soil available P indexes in agronomy and environment. 【Method】 An laboratory incubation experiment was conducted with artificial fertilization and stable temperature of 25℃ using the material of red soil collected from subtropical area. The dynamic process of SAP was studied through testing SAP and phosphorus sorption index (PSI) in fresh soil samples in 1 h, 3 h, 12 h, 24 h, 3 d, 6 d, 9 d, 15 d, 30 d, and 60 d after fertilizer application into soil. Two types of test methods including Olsen- (OP), Bray 1-(BP), and Mehlich 3- (MP) extraction methods (agronomic type) and distilled water (HP) and 0.01M CaCl2 (CP) extraction methods (environmental type) were used. 【Result】 There was an obvious critical time (tc) during the declining process of SAP in red soil, ranging from 0.1 h to 18.7 h. The declining rate of SAP was rapid before tc but turned to be markedly slow since then. The impact of fertilizer application rate on tc was significant. The values of tc for OP, BP, and MP linealy delayed with the increment of fertilizer amount. However, the values of tc for HP and CP tended to delay in a power function pattern. Environmental index was nonlineally related to agronomic ones and there was a “change point” between them. Depending on this relationship, the threshold of soil P for environmental risk could be estimated. The thresholds of OP, BP, and MP based on CP were 49.97 mg•kg-1, 91.07 mg•kg-1, and 30.54 mg•kg-1, respectively, but these values based on HP were 60.78 mg•kg-1, 82.74 mg•kg-1, and 39.65 mg•kg-1, respectively. PSI ranged from 17.92 to 26.29 with the increasing fertilizer application rate and the value of PSI versus the threshold of OP for environmental risk was 23.46. 【Conclusion】There was an obvious critical time during the declining process of SAP in red soil. The value of critical time delays with increasing fertilizer application amount and subsequently the risk of soil P moving to the environment is more serious. There is a non-linear relationship between agronomic and environmental index. The critical value of SAP for environmental risk could be calculated according to this. The Olsen-P threshold of the tested red soil is 50 - 60 mg•kg-1and the relevant PSI is 23.46.

Influence of Calcium on Fruit Cracking of Jincheng Orange and Its Physiological Mechanism

WEN Ming-Xia, SHI Xiao-Jun

Scientia Agricultura Sinica. 2012, 45(6): 1127-1134. doi:10.3864/j.issn.0578-1752.2012.06.010

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【Objective】 The main purpose of this paper was to study the effect of mineral nutrition and enzyme activities on fruit cracking of Jincheng orange (Citrus sinensis Osbeck), and propose comprehensive prevention measures. 【Method】 Fruit cracking rate of Beibei 447 Jincheng orange was investigated to research the differences of N, P, K, Ca concentrations and enzyme activities in leaves, peel and pulp between cracked fruits and normal ones. The effects of calcium on pectin, malonaldehyde (MDA) and enzymes related to hydrolysis and reactive oxygen species clearing and polyphenol oxidase (PPO) of citrus peel were studied by spraying calcium nitrate before blossom and young fruit period of Beibei 447 Jincheng orange. 【Result】 The concentrations of Ca in leaves, peel and pulp of normal fruits were higher than those of cracked ones. Fruit cracking rate was negatively correlated to Ca concentration in citrus peel significantly. The activities of PPO, polygalacturonase ( PG ) and cellulase (CX) of cracked fruits were higher than those of normal ones, but those of catalase (CAT) were lower. Fruit cracking rate was positively correlated to PPO and negatively correlated to CAT and the concentration of protopectin (PP) significantly. The concentration of MDA, the activities of PPO, PG, CX of citrus peel at fruit rapid-expanding stages decreased by spraying calcium while the activities of superoxide dismutase (SOD), CAT and the concentration of protopectin (PP) increased significantly. 【Conclusion】 Low concentration of Ca in citrus peel resulted in increase of activities of enzymes (PPO, PG and CX) in cell-wall metabolism. The low protopectin concentration that might maintain intensity and extensibility of citrus peel was the main cause for fruit cracking. Spraying calcium on citrus trees decreased fruit cracking rate and could be regarded as an important measure to prevent fruit cracking of Jincheng orange.

Construction of Grape Core Collections

GUO Da-Long, LIU Chong-Huai, ZHANG Jun-Yu, ZHANG Guo-Hai

Scientia Agricultura Sinica. 2012, 45(6): 1135-1143. doi:10.3864/j.issn.0578-1752.2012.06.011

Abstract ( 1263 )

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【Objective】 Core collections of grape(Vitis vinifera) germplasm were constructed using SSR molecular markers based on the established grape primary core collections.【Method】The methods of M strategy (Core Finder and Power Core), genetic distance sampling (least distance stepwise sampling，LDSS and genetic distance optimization，GDOPT) and Core Hunter were used to construct core collections. The genetic diversity indexes of He, Ho and I and phenotypic indexes of mean difference percentage (MD%), variance difference percentage (VD%), coincidence rate of range (CR%) and variable rate of coefficient of variation (VR%) were used to evaluate the representativeness of core collections. The principal components analysis based on SSR and SRAP markers were also performed to validate the core collections. 【Result】 The core collections from M strategy have retained all the alleles of primary core collection, while those from genetic distance sampling have good representative. Core collections from M strategy, genetic distance sampling and Core Hunter were merged together in order to obtain maximum genetic distance and genetic diversity. At last, 48 materials of grape core collections retained 96.21% alleles of primary core collection and 92.90% genetic diversity of the original whole germplasm with 5.53% of the sampling ratio.【Conclusion】Core collections constructed in this study were proved to have good representative and enough genetic diversity according to the molecular and phenotypic statistic test. The method used in this study has important reference value for core collection construction of other plants.

Effects of Exogenous Divalent Cations Ca2+, Mg2+, and Cu2+ and Acriflavine on Ascorbate Biosynthesis in Rosa roxburghii Fruits

ZHANG Xue, YANG Man, AN Hua-Ming, HUANG Wei, LIU Wei

Scientia Agricultura Sinica. 2012, 45(6): 1144-1149. doi:10.3864/j.issn.0578-1752.2012.06.012

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【Objective】 The effects of divalent cations and organic inhibitors on GalLDH activity and AsA biosynthesis in Rosa roxburghii fruits were studied.【Method】Fruits of Rosa roxburghii Tratt cv. Guinong 5 was used to investigate the effects of divalent cations Ca2+, Mg2+, and Cu2+ and organic inhibitors acriflavine and lycorine on GalLDH activity and AsA biosynthesis, by using the methods of incubation in vitro and injection in vivo.【Result】The results showed that the activity of purified GalLDH was promoted by Ca2+ and Mg2+, but extremely inhibited by Cu2+. Even so, when flesh tissues of the fruit was incubated respectively in the mixed solution of the metal ions together with L-galactono-1,4-lactone (GalL), an immediate substrate of AsA biosynthesis, both Ca2+ and Mg2+ could not effectively improve the AsA biosynthesis, while the AsA content in the flesh tissues pre-incubated in presence of Cu2+ decreased markedly. AsA biosynthesis and accumulation in developing fruit was obviously promoted by injection of Ca2+ solution and inhibited by that of Cu2+, but not influenced by Mg2+. Acriflavine, suggested an organic inhibitor of AsA biosynthesis, was able to inhibit the activity of Rosa roxburghii GalLDH about 70%, but lycorine, another specific inhibitor of AsA biosynthesis, had a very slight influence on it. The similar inhibitory effect was detected in incubating fresh fruit tissues in the presence of the two inhibitors together with GalL respectively, although injection of acriflavine presented a visible inhibiting effect on AsA accumulation in developing fruit, and lycorine had no influence.【Conclusion】Exogenous Ca2+ could promote but Cu2+ or acriflavine inhibit AsA biosynthesis in Rosa roxburghii fruits.

Free and Bound Phenolic Contents and Antioxidant Activity of Five Varieties of Indica Rice Husk

LI Qing, ZHANG Ming-Wei, ZHANG Rui-Fen, WEI Zhen-Cheng, DENG Yuan-Yuan, TANG Xiao-Jun, ZHANG Ye-Hui, LI Wu, MA Yong-Xuan

Scientia Agricultura Sinica. 2012, 45(6): 1150-1158. doi:10.3864/j.issn.0578-1752.2012.06.013

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【Objective】 The contents and compositions of free and bound phenolics and antioxidant activity of husks of different rice varieties were compared. 【Method】 The contents of free and bound phenolics and flavonoids of rice husks of five different varieties were determined. High performance liquid chromatography (HPLC) method was used to measure the compositions and contents of phenolics in rice husks. The antioxidant activity was analyzed using the ferric reducing ability of plasma (FRAP) and ABTS radical cation scavenging activity assays. 【Result】 The free , bound and total phenolic contents of the husks of 5 tested indica rice varieties ranged from 42.8 to 123.0, 260.9 to 325.2 and 320.2 to 398.3 mg of gallic acid equivalents/100 g dry weight (DW), respectively. The average percentage contribution of bound phenolics to the total was 78.9%. The free, bound and total flavonoids contents of the husks ranged from 34.0 to 58.0, 47.9 to 64.4 and 82.0 to 115.7 mg of catechin equivalents/100 g DW, respectively. The average percentage contribution of bound flavonoids to the total was 56.4%. Gallic acid, protocatechuic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid and ferulic acid were detected in phenolic extracts of rice husks. Syringic acid and p-coumaric acid were the major free phenolics, with average of 25.09 μg•g-1 DW and 31.21 μg•g-1 DW, respectively. The most abundant bound phenolics was p-coumaric acid, and its average content was 2141.61 μg•g-1 DW. The free , bound and total FRAP antioxidant activity of the husks ranged from 37.7 to 106.0, 217.9 to 281.0, and 269.3 to 370.3 mg Trolox equivalents/100 g DW. The average percentage contribution of bound FRAP antioxidant activity to the total was 77.3%. The free , bound and total ABTS antioxidant activities of the husks ranged from 26.3 to 85.8, 67.2 to 111.9, and 93.4 to155.2 mg Trolox equivalents/100 g DW. The average percentage contribution of bound ABTS antioxidant activity to the total was 64.8%.【Conclusion】 Rice husk is rich in phenolics and possesses high antioxidant activity. Total phenolics and flavonoids, individual phenolics and antioxidant activity of 5 the tested rice husks were mainly contributed in bound form. Significant varietal differences were found. It is indicated that rice husk can be considered as a potential valuable source of the natural antioxidants.

Analysis of the Variety Composition and Quality Properties of Wheat in A Part of the Yellow-Huai River Zone

GUAN 二Qi, WEI Yi-Min, ZHANG Bo, GUO Jin-Kao, ZHANG Guo-Quan, LIU Yan-Jun, LUO Qin-Gui, BAN Jin-Fu

Scientia Agricultura Sinica. 2012, 45(6): 1159-1168. doi:10.3864/j.issn.0578-1752.2012.06.014

Abstract ( 670 )

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【Objective】This investigation was carried out to study the variety composition and quality traits of wheat from a part of Yellow-Huai River Zone by a follow-up survey and sampling analysis at the fixed locations, and to provide a theoretical basis for wheat breeding and novel product development of food industry. 【Method】 A total of 243 and 242 wheat samples were respectively collected in harvest periods in 2008 and 2009 from a part of the Yellow-Huai River Zone. The cultivars were collected from the farming field in each location. The variety composition was summed up, and wheat kernel quality and dough rheological properties were determined.【Result】The number of main varieties in production was too much (including 86 wheat varieties), and the proportion of wheat varieties with good quality was less (19.8%). The results indicated that the thousand kernel weight (39.5 g), bulk weight (787 g•L-1), kernel hardness (56%) and the protein content (14.0%) were higher, while the Zeleny sedimentation (26.7 mL) volume was lower. Except for Zeleny sedimentation volume and Farinograms, significant differences were observed in most quality traits of wheat in different years (P＜0.05).【Conclusion】Wheat from the studing areas of the Yellow-Huai River Zone was hard in kernel and high in protein content, and its gluten was medium-strong, and it is promising to grow strong-gluten wheat in the Yellow-Huai River Zone. More work should be done by governmental departments to strengthen the extension of wheat varieties with stable, high yield and good quality, which could improve the quality of commodity wheat in wheat production areas and promote the sustainable development of food industry in the traditional agricultural zones.

Evaluation of the Pelletizing Stability of Micro-encapsulated and Coated Enterococcus faecium

LIU Wen-Bin, MA Qiu-Gang, DENG Cheng-Jun, WEI Xiu-Lian, ZHANG Jian-Yun, JI Cheng

Scientia Agricultura Sinica. 2012, 45(6): 1169-1175. doi:10.3864/j.issn.0578-1752.2012.06.015

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【Objective】The viable count of the micro-encapsulated and coated Enterococcus faecium was compared with its normal freeze-drying powder under different heat treatment conditions to observe the protective effect of micro-encapsulation. And the pelletizing stability of the micro-encapsulated and coated E. faecium were evaluated through a laboratory heating trial. 【Method】Two probiotics products were treated at 65℃ or 85℃ heated air for 5, 10, 15, 30 and 60 min. The preparations of 0.01% micro-encapsulated and coated E. faecium were supplemented in piglet diets and being pelletized at 55℃and 65℃.【Result】The viable count of the micro-encapsulated and coated products, heated at 65℃ for 60 min, were higher (P=0.037) than the normal powder under the same condition. When the two products were heated at 85℃ for 30 or 60 min, the survival rates of the micro-encapsulated and coated ones were higher (P=0.002) than the normal powder. The Enterococcus faecium supplemented in piglet diet was more fragile than itself directly in heat air. The destruction of being pelletized in factory was much higher than being heated in the lab. The survival rate of E. faecium after being pelletized at 55℃ equivalent to that of being air-heated at 65℃ for 28 min or at 85℃ for 11 min in the same diet, while the survival rate after being pelletized at 65℃ equivalent to that of being air-heated at 65℃ for 48 min or at 85℃ for 23 min. 【Conclusion】The micro-encapsulation and coating process could improve the thermal stability of E. faecium in a certain extent. Directly air-heated treatment of E. faecium probiotics couldn’t represent their thermal stability during pelletizing, but it could be quickly evaluated through air-heated treatment to diets supplemented with them.

Studies on Expression Characteristics of EGF and Development Characteristics of Hair Follicle of Gray Goat

WANG Rui-Zhi, YIN Xun-He, WANG Hui, CHENG Zi-Qiang

Scientia Agricultura Sinica. 2012, 45(6): 1176-1182. doi:10.3864/j.issn.0578-1752.2012.06.016

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【Objective】 The relationship between the expression of epidermal growth factor and the development of hair follicle was studied.【Method】 The histology method, micro-observation approach and QRT-PCR were used to detect the relationship between the expression of EGF and the development of hair follicle of 1-day-old, 1-month-old, 2-month-old, 3-month-old, 4-month-old and 5-month-old Jining Gray goat. SAS9.0 was used to analyze the relationship between them.【Result】 The resuts indicated that the diameter of primary follicle increased, but not significantly (P＞0.05). The diameter of secondary follicle increased very significantly (P＜0.01). The diameter of secondary follicle reached peak of growth at 2-month-old. The expression of EGF increased after birth and achieved the highest at 2-month-old (P＜0.01). It decreased extremely significant after 2-month-old (P＜0.01).【Conclusion】 The primary follicle was completely developed before birth and the secondary follicle was developed after birth. The expression of EGF and the diameter of secondary follicle had a significant positive correlation before 2-month-old (P＜0.01). The results indicated that EGF is significantly positive to the development of secondary follicle.

Cloning of Dairy Goat Prm1 Gene CDS and Construction of the Eukaryotic Expression Vector

LI Ming-Zhao, LIU Chao, SUN Jun-Wei, ZHU Hai-Jing, CAO Hui, LU Xiao , CHOU Pu-Bin, HUA Jin-Lian

Scientia Agricultura Sinica. 2012, 45(6): 1183-1190. doi:10.3864/j.issn.0578-1752.2012.06.017

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【objective】The objective of the experiment is to study the Prm1 gene of dairy goat, and for further research on dairy goat spermatogenesis.【Method】The CDS of Prm1 gene was cloned by PCR from Guanzhong dairy goat testis tissue. The obtained sequence was constructed into the eukaryotic expression vector pIRES-GFP, and then transfected to GC1 cell line and human umbilical cord cells. PCR and immunohistochemistry were used to test the expression of Prm1 at different ages of dairy goat testis. 【Result】In dairy goats, the expression of Prm1 gene was high and specific in adult testis, located only in the head of sperm. The CDS of Prm1 gene of Guanzhong dairy goat was amplified and compared with the sequences of other species. The results showed that they shared a high homology, being 97.4% between dairy goats and bovine. The Prm1 gene was overexpressed in GC1 cells and human umbilical cord cells to confirm its function. 【Conclusion】 The complete CDS of dairy goat Prm1 gene and its expression patterns were obtained.

Influence of 5-HT Transporter Inhibitors on the 5-HT Content and the Proliferation of Smooth Muscle Cells in Pulmonary Arteriolar Media in Broilers with Pulmonary Hypertension

LI Ying, ZENG Jian-Ying, HAN Qian-Biao, TANG Zhao-Xin, GUO Jian-Ying, PAN Jia-Qiang

Scientia Agricultura Sinica. 2012, 45(6): 1191-1198. doi:10.3864/j.issn.0578-1752.2012.06.018

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【Objective】 An experiment was designed to detect the relationship between the content of 5-hydroxytryptamine (5-HT) in the media layer of pulmonary arterioles and the proliferation of pulmonary arterioles smooth muscle cells, with the purpose to clarify whether or not 5-HT transporter (5-HTT) was participated in the occurrence of pulmonary hypertension in broilers.【Method】Broilers at 20 day of age were randomly divided into non-disease inducing groups (intravenously injection of normal saline, including blank control group, Fluoxetine group, Citalopram group, 30 fowls in each group) and disease inducing groups (intravenously injection of cellulose particles, including medicine blank control group, Fluoxetine group, Citalopram group, 50 fowls in each group.). From day 21 on, the morbidity of pulmonary hypertension syndrome (PHS) of each group were recorded. Samples were randomly taken from each group on day 21, 28, 35 and 42, and the weight ratio between right ventricle and total ventricle (RV and TV) was determined. 5-HT content in pulmonary arteriolar media layer was detected by using the immunohistochemical technique, and the proliferation conditions of smooth muscle cells in pulmonary arterioles was detected by argyrophilic nucleolar organizer region proteins staining method (AgNOR) and proliferating cell nuclear antigen (PCNA) staining method.【Result】PHS morbidity and RV/TV value of disease-inducing groups were significantly higher than that of non-disease inducing groups (P＜0.05). The 5-HT content in pulmonary arteriolar media was increased in disease-inducing groups, as well as the argyrophilic particles on the smooth muscle cells and the PCNA positive cells. Fluoxetine and Citalopram (both are 5-HTT inhibitors) significantly reduced the morbidity of PHS and 5-HT contents in pulmonary arteriolar media in broiler, and inhibited the proliferation of smooth muscle cells. There were no significant difference between the two medicines.【Conclusion】During the occurrence of pulmonary hypertension in broilers, the increasing of 5-HT content in pulmonary arteriolar smooth muscle cells which was mediated by 5-HTT resulted in the excessive proliferation of smooth muscle cells, leading to the occurrence of PHS. 5-HTT is possibly a significant target molecular for the prevention and treatment of PHS in broilers.

Detection of High Pathogenicity Islands in Escherichia coli Isolated from Clinical and Subclinical Bovine Mastitis in Some Areas of China

XU Ji-Ying, YANG Zhi-Qiang, CHEN Hua-Qi, LIU Jun-Lin, XING Juan, LI Jian-Xi, LI Hong-Sheng

Scientia Agricultura Sinica. 2012, 45(6): 1199-1205. doi:10.3864/j.issn.0578-1752.2012.06.019

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【Objective】The objective of the experiment is to investigate the present distribution of high pathogenicity island (HPI) in E. coli isolated from cows with clinical and subclinical mastitis in China.【Method】Milk samples from 1 260 cows with clinical and subclinical mastitis in different farms and areas were used to screen Gram-negative bacteria by MacConkey agar. One hundred and ninety E. coli isolates was further identified from the Gram-negative bacteria by microbiological tests. Total DNA extraction from E. coli was used to detect HPI genes by PCR.【Result】The PCR results showed that 50 out of 190 isolates carried the irp2 gene, 36 isolates were positive for fyuA gene and 32 for intB genes. Sequence analysis of randomly selected PCR products showed that homology of genes irp2, fyuA, and asn_tRNA_intB were above 97.1%, 98.2%, and 97.2% identical to the published sequences. HPI+ isolates were associated with fyuA (72%) and asn_tRNA locus (64%).【Conclusion】 Yersinia HPI was widely distributed in the E. coli isolates from cows with clinical and subclinical mastitis in China. The prevalence of HPI is associated with special serotypes in E.coli from cows with clinical and subclinical mastitis.

Polymorphism of TLR4 and Function Analysis of C1027A in Suzhong Pig

LIU Xiao, FANG Xiao-Min, ZOU Xiao-Long, ZHAO Fang, REN Shou-Wen

Scientia Agricultura Sinica. 2012, 45(6): 1206-1214. doi:10.3864/j.issn.0578-1752.2012.06.020

Abstract ( 654 )

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As a significant pattern-recognition receptor, toll-like receptor (TLR) is essential for the natural immunity. Its polymorphism has a profound influence on responses to a wide range of pathogens and is associated with resistance and susceptibility to diseases.【Objective】This study speculated on the polymorphisms of TLR4 and function analysis of the mutation (C1027A) in its coding sequence in Suzhong pig. 【Method】 PCR-SSCP was used to search the mutations of TLR4 in 127 samples of Suzhong pigs. The expression levels of TLR4 and proinflammatory cytokines TNF-α and IL-1β were detected by real-time PCR in porcine alveolar macrophages (PAMs) that were induced by LPS. And the PAMs were grouped by their different genotypes (CC and AC) in 1027 bp of coding sequence (CDS) to discuss whether this mutation of TLR4 influenced its sensitivity to LPS. 【Result】 Three SNPs were found, including G962A, C1027A and G960A, and the first two of them were nonsynonymous SNPs and C1027A changed the character of amino acid. LPS stimulation markedly increased TLR4 and proinflammatory cytokines TNF-α and IL-1β mRNA expression. And PAMs had different sensitivities and response to LPS between genotypes CC and AC of C1027A. 【Conclusion】The coding sequence of TLR4 was relatively conservative in Suzhong pig and this population had low polymorphism and genetic variation. The mutation in C1027A can affect the binding ability to LPS. So, allele C in 1027 bp of TLR4 is the resistance allele to Gram-negative bacterial infections in Suzhong pig.

The Components and Changes of VOCs of Michelia champaca L. Flower at Different Developmental Stages

JIANG Dong-Yue, LI Yong-Hong, HE Fang, LIN Qi-Peng, PAN Hui-Tang

Scientia Agricultura Sinica. 2012, 45(6): 1215-1225. doi:10.3864/j.issn.0578-1752.2012.06.021

Abstract ( 759 )

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【Objective】The changes of the components and contents of volatile organic compounds (VOCs) of Michelia champaca flowers at different developmental stages were studied.【Method】The petals of M. champaca at different developmental stages were used as materials to investigate the components and their changes of volatile organic compounds (VOCs) by using the methods of headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS).【Result】Fifty-one volatile compounds were identified from petals during the whole flowering process of M. champaca, of which, 1,6-cyclodecadiene, 1-methyl-5-methylene-8-(1-methylethyl)-, [s-(E,E)]-, cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1- methylethenyl)-, [1S-(1.alpha., 2.beta.,4.beta.)]-，1,6-octadien-3-ol, 3,7-dimethyl-，benzoic acid, methyl ester , beta-pinene and Eucalyptol were the major volatile compounds emitted from M. champaca flower. The relative content of different volatile compounds emitted from M. champaca flower showed different trends during flowering. The relative content of cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1- methylethenyl)-, [1S-(1.alpha.,2.beta.,4.beta.)]-, benzoic acid, methyl ester and ylangene increased at first and then decreased, while the beta-pinene, 1,3,6-octatriene, 3,7-dimethyl- and eucalyptol showed crosscurrent, and the relative contents of caryophyllene, alpha -cubebene and 1,5-cyclodecadiene, 1,5-dimethyl-8-(1-methylethylidene)-, (E,E)- declined during the flowering process. 【Conclusion】The volatile compounds and their contents were distinctly different at different developmental stages of M. champaca flowers, the major components had officinal and health care effect. So, M. champaca is an excellent ornamental species for building plant landscape that is beneficial to people's health.

Cloning and Expression Analysis of A Stress-related Ta14S Gene from Wheat

REN Jiang-Ping, LIU Hai-Lun, WANG Xin-Guo, NIU Hong-Bin, LI Yong-Chun, WANG Xiang, CHEN Xin, YIN Jun

Scientia Agricultura Sinica. 2012, 45(6): 1226-1234. doi:10.3864/j.issn.0578-1752.2012.06.022

Abstract ( 607 )

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【Objective】In this study, the objective is to clone the stress resistance-related genes, and analyze the expression pattern of those genes which aim to investigate the underlying molecular mechanism of stress resistance and provide candidate genes for stress-resistance plant breeding.【Method】 Based on the analysis of up-regulated EST obtained by cDNA chip, a full-length cDNA sequence was cloned from wheat through rapid amplification of cDNA ends (RACE) method and proper bioinformatics softwares were applied for characterizing the cloned genes and the deduced proteins. The gene expression characters were analyzed by the real time PCR.【Result】The full-length cDNA sequence designated as Ta14S from wheat was 1 056 bp in length, contains a 792 bp open reading frame (ORF) encoding a 263 amino acid proteins, with 11 bp in the 5' UTR and 253 bp in the 3' UTR. Similarity alignment of plant 14-3-3 proteins by DNAMAN revealed four high conserved domains. These domains were a potential substrate for protein kinase C, annexin-like domain, domain to bind transcription factor and nuclear export signal peptide, respectively. This result suggests that the Ta14S belongs to the family of 14-3-3. Real time PCR analysis revealed that the expression level of Ta14S was the highest in root than in leave and stem. The expression of Ta14S was steadily up-regulated in any of the time points under PEG and low temperature stress and the relative expression level was significantly higher than those of control within 6 h of ABA and low temperature stress treatments. This result suggests that Ta14S may be involved in stress resistance-related response of ABA signaling pathways in wheat roots.【Conclusion】A full-length cDNA of Ta14S was cloned from wheat and the typical protein interact binding domains were found in the deduced proteins. The expression patterns of Ta14S gene under drought, high and low temperature and ABA treatments showed that Ta14S might play an important regulation role under stress in wheat. The results provided important information for further studies on molecular regulation mechanism underlying abiotic stress resistance in wheat.

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