Scientia Agricultura Sinica

Polymorphism of the Amy32b Gene and Its Effect on the α-Amylase Activity in Barley

JIANG Xiao-Dong, ZHANG Jing, GUO Gang-Gang

Scientia Agricultura Sinica. 2012, 45(5): 823-831. doi:10.3864/j.issn.0578-1752.2012.05.001

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【Objective】 The objective of the experiment is to study the polymorphism of the Amy32b gene in Chinese barley and dissect its relevance to the α-amylase activity 【Method】 Three pairs of specific primers covering the whole sequence of the gene were designed to locate single nucleotide polymorphism (cSNP) and other In/Del mutations. The complete cDNA of Amy32b was cloned from barley via RT-PCR. Full-length cDNA carrying alleles（G/A）and truncated cDNA of Amy32b were cloned into expression vector pET-28a(+). Their expression products were purified, characterized, and compared. 【Result】 According to the result of amplified DNA sequences of Amy32b (x05166), there was a single nucleotide polymorphism cSNP(G/A) and an insertion/deletion of A were found in the coding region of the gene, located in 2 269 bp and 2 403 bp, respectively. The SNP(G/A) caused an E355K amino acid substitution in the enzyme. And the In/Del variation of A insertion formed an in-frame stop codon, resulting in the deletion of a 48 bp fragment in cDNA as well as a β5 sheet in carboxyl terminal of the α-amylase. The full-length of Amy32b cDNA is 1 314 bp, while the truncated cDNA is 1 266 bp in length. The enzymatic activity assay showed that rAmy32b_A and rAmy32b_G exhibited normal and similar amylase activities. But for the truncated Amy32b gene, the mutant enzyme (rΔAmy32b) did not produce detectable actvity. 【Conclusion】 The In/Del mutation formed an early translation stop codon, resulting in the losses of 48 bp sequence fragment and a β-strand in C-terminal end of α-amylase, and seriously affected enzymatic activity, while the G/A transition only resulted in amino acid substitution and did not alter enzymatic activity.

Cloning and Expression Analysis of a Chalcone Synthase (CHS) Gene from Purple Potato (Solanum tuberosum)

HU Chao-Yang, ZHOU You-Feng, GONG Yi-Fu, JIN Si, WANG He-Yu, ZHAO Qun-Fen

Scientia Agricultura Sinica. 2012, 45(5): 832-839. doi:10.3864/j.issn.0578-1752.2012.05.002

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【Objective】 The aim of this study was to clone the full length cDNA of chalcone synthase from purple potato, to analyze its expression at different organs and the relationship between CHS expression and anthocyanin accumulation after treated with different concentrations of GA3 and sucrose. 【Method】 The full-length cDNA of CHS was isolated from Solanum tuberosum by RT-PCR and RACE. Semi-quantitative RT-PCR was used to analyze the expression levels of StCHS in different organs, and the expression levels induced by GA3 and sucrose were adopted by semi-quantitative RT-PCR. The anthocyanin content of purple potato was measured by spectrophotometer method. 【Result】 The cloned full-length CHS was 1 490 bp in length, containing a 1 170 bp open reading frame (ORF), which encodes 389 amino acids. The deduced amino acids contained Cys164, Phe215, His303, and Asn336 active sites, which constitute the catalytic center of CHS. CHS is expressed at different levels in different organs, which was found to be expressed in stems, leaf stalks and leaves but not in roots, tubers or rachises. The anthocyanin accumulation was slightly induced by GA3 as well as the expression of CHS. The anthocyanin accumulation was strongly induced by sucrose, but the CHS expression was not notably induced. 【Conclusion】CHS gene was cloned from purple potato and its expression is tissue-specific. StCHS is a rate-limited gene in the flavonoid synthesis pathway of purple potato.

Effects of Endogenous Hormones on Tiller Development Process of Different Maize Varieties

WANG Ru-Fang, ZHANG Ji-Wang, LU Peng , DONG Shu-Ting, LIU Peng, ZHAO Bin

Scientia Agricultura Sinica. 2012, 45(5): 840-847. doi:10.3864/j.issn.0578-1752.2012.05.003

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【Objective】 This experiment was done to study the changes of endogenous hormones during tillers growth and death of different maize varieties, and to investigate the regulation of endogenous hormones in maize tillering.【Method】Maize varieties with different tillering capacities were used as experimental materials. On the basis of tillers development regularity investigated, the endogenous hormones in basal stem and functional leaves were measured during the tillers’ emergency and death. 【Result】The tillering capacity of different maize varieties was controlled by both the content and the ratio of endogenous hormones in basal stem and functional leaves. During the tillering stage, the ZR (zeatin riboside) content was higher and appeared an increasing trend, however, the IAA (indole -3-acetic acid), ABA (abscisic acid) and GA (gibberellin acid) contents were lower and appeared a decreasing trend in basal stem and functional leaves. The ratios of IAA/ZR and ABA/ZR were lower while tillering and higher at the end of tillering stage. The ZR content of MXG with the strongest tillering ability was high and IAA, ABA, GA, IAA/ZR, ABA/ZR were low respectively.【Conclusion】Higher ZR content was benefit to the tillering of maize. Higher content of IAA , ABA and GA suppressed tillering and accelerated the death of tillers. Tillers development was also determined by the balance among endogenous hormones which took effects in an interaction way. The lower ratios of IAA/ZR and ABA/ZR improved tillering, and the higher ones suppressed tillering and promoted tillers’death.

Role of Heterotrimeric G Protein in UV-B-Induced Arabidopsis Stomatal Closure

MA Xian-Ge, HE Jun-Min

Scientia Agricultura Sinica. 2012, 45(5): 848-853. doi:10.3864/j.issn.0578-1752.2012.05.004

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【Objective】 The role of heterotrimeric G protein in UV-B-induced stomatal closure was analyzed to provide valuable evidence for further elucidating the mechanism of UV-B signaling transduction in plants.【Method】By using Arabidopsis (Arabidopsis thaliana) wild type, Gα-subunit-knockout mutants gpa1-1 and gpa1-2, wGα (a gpa1 overexpression line) and cGα (overexpression constitutively active form mutant of GPA1) combined with pharmacological analysis, the role of heterotrimeric G protein in 0.5 W•m-2 UV-B-induced stomatal closure was explored via stomatal bioassay.【Result】Pharmacological results showed that pertussis toxin (PTX), an inhibitor of G protein α subunit, inhibited UV-B-induced stomatal closure significantly. Meanwhile, cholera toxin (CTX), an activator of G protein α subunit, induced stomatal closure of leaves under light as well as UV-B radiation. Genetic data showed that UV-B-induced stomatal closure was completely impaired in the mutants gpa1-1 and gpa1-2, but not in the overexpression lines of wGα and cGα. In contrast, cGα showed not only smaller stomatal aperture than wild type under light, but also faster stomatal closure induced by UV-B than they did in the wild type.【Conclusion】 Results of this study strongly suggest the involvement of heterotrimeric G protein α subunit in the signaling transduction pathway of UV-B-induced stomatal closure in Arabidopsis.

Molecular Cloning and Analysis of a Novel Pectate Lyase Gene Hg-pel-5 from Soybean Cyst Nematode

PENG Huan, PENG De-Liang, HUANG Wen-Kun, HE Wen-Ting, HU Xian-Qi

Scientia Agricultura Sinica. 2012, 45(5): 854-866. doi:10.3864/j.issn.0578-1752.2012.05.005

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Abstract:【Objective】The objective of this study is to clone and analyze the pectate lyase genes from soybean cyst nematode (Heterodera glycines), understand the parasitism and pathogenicity of plant parasitic nematodes, and to develop new control strategies for soybean cyst nematode. 【Method】A noval pectate lyase gene Hg-pel-5 was cloned from H. glycines by EST analysis and RACE. The tissue localization and developmental expression of pectate lyase were analysed at different life stages of H. glycines by in situ mRNA hybridization and RT-PCR amplification, and the copy number of the gene was identified by Southern blot. 【Result】 A novel parasitism gene encoding pectate lyase, named Hg-pel-5, was cloned from H. glycines. The full length cDNA of Hg-pel-5 consists of 957 bp, with a 681 bp ORF encoding a 227 amino acid residues. The structure of Hg-pel-5 genomic DNA includes 2 extons and 1 intron. The predicted protein includes a signal peptide of 20 amino acids on the N-terminal and 4 conserved regions characteristic of class Ⅲ pectate lyases of microbes. In situ mRNA hybridization analyses showed that the transcripts of Hg-pel-5 was expressed in subventral oesophageal gland cells of H. glycines. RT-PCR analysis confirmed that Hg-pel-5 transcriptions were mainly expressed at the pre-parasitic and early parasitic second-stage juveniles. Southern blotting confirmed that Hg-pel-5 was of nematode origin and a member of a small multigene family. 【Conclusion】 A novel pectate lyase gene was cloned and analyzed from H. glycines, and these results imply that the Hg-pel-5 might play an important role during the early stages of plant parasitism.

The Host Range of Phytophthora parasitica var. nicotianae

MA Guo-Sheng, GAO Zhi-Mou, CAO Shun

Scientia Agricultura Sinica. 2012, 45(5): 867-876. doi:10.3864/j.issn.0578-1752.2012.05.006

Abstract ( 976 )

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【Objective】The objective of this study is to explore the host range of Phytophthora parasitica var. nicotianae, aiming to provide a theoretical basis for related researches in P. parasitica var. nicotianae, and to provide a decision-making basis for integrated control of tobacco blank shank and other diseases caused by P. parasitica var. nicotianae.【Method】The host range of P. parasitica var. nicotianae was systematically studied with big samples through inoculating 61 kinds of crops and weeds with the methods of mycelium dish wounded and unwounded inoculation under the condition of manual simulation of the infection approaches in tobacco field, and then the lesions were identified through Koch’s postulates.【Result】The pathogenic microorganism could infect 51 kinds of plants from 41 genera in 21 families under the wounded condition, and could infect 16 kinds of plants from 15 genera in 10 families in unwounded inoculating condition. It was discovered for the first time that P. parasitica var. nicotianae could infect 47 kinds of plants from 40 genera in 21 families by the artificial wounded inoculation, and could infect 12 kinds of plants from 12 genera in 10 families under the condition of mycelium block unwounded inoculation. 【Conclusion】 The host range of P. parasitica var. nicotianae is wide, some of the hosts are common crops and familiar weeds in tobacco field. These plants might be infected under the conditions that are wounded during farming or by insects, or even unwounded in high temperature and high humidity in the field. Tobacco could not be rotated or intercropped with these crops. Moreover, it might be positive for the integrated management of tobacco blank shank and other diseases caused by P. parasitica var. nicotianae to control these weeds in field in time.

mRNA Expression and Physiological Function of Chitin Synthase1 from Oxya chinensis (Thunberg)

YU Zhi-Tao, LIU Xiao-Jian, MA 恩Bo, GUO Ya-Ping, ZHANG Jian-Zhen

Scientia Agricultura Sinica. 2012, 45(5): 877-884. doi:10.3864/j.issn.0578-1752.2012.05.007

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【Objective】The objective of this study is to analyze the mRNA expression characteristics and physiological function of chitin synthase1 gene (OcCHS1) from the Chinese rice grasshopper, Oxya chinensis (Thunberg), and to provide a theoretical basis for effective pests control based on RNAi.【Method】Based on the known partial conservative cDNA fragment of OcCHS1 (GenBank accession number: HM214491), primers were designed for mRNA expression analysis. The developmental and tissue-specific expression patterns of OcCHS1 were determined by RT-PCR and qPCR. RNAi experiment was performed to explore the physiological function. 【Result】 OcCHS1 was expressed at all the developmental stages. Tissue-specific analysis showed that OcCHS1 was highly expressed in the integument, followed in the trachea, and lowest in other tissues. RNAi-based gene silencing experiment showed that the fourth day of the 4th instar nymphs injected by OcCHS1-speci?c dsRNA could repress the transcript levels of OcCHS1 by 70.8% compared to those in the control nymphs. Moreover, dsRNA injections signi?cantly increased abnormalities, such as stunted development, uncompleted molting, crimpy abdomen and eventually died. Consequentially, 85.2% of mortalities were observed, while the mortality in the control nymphs was only 7.4%. 【Conclusion】OcCHS1 is essential for development and mainly responsible for chitin synthesis in cuticle and trachea of O. chinensis. Moreover, silencing of chitin synthase 1 gene by RNAi is lethal to O. chinensis.

Effects of Long-Term Different Fertilization Managements on Changes of N in Soil and Its Uptake by Wheat on Dryland

LIANG Bin, ZHAO Wei, YANG Xue-Yun, ZHOU Jian-Bin

Scientia Agricultura Sinica. 2012, 45(5): 885-892. doi:10.3864/j.issn.0578-1752.2012.05.008

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【Objective】Organic fertilizer application can improve the biological function and affect the fates of inorganic N in soil. 【Method】 The microplot method was used to compare the effects of 19-year different fertilization managements (NF, no application of fertilizer; NPK, inorganic NPK fertilizer; MNPK, manure plus inorganic NPK fertilizer) on changes of mineral N and soil microbial biomass N (SMBN) and its uptake by wheat on dryland. 【Result】 Addition of urea-N significantly (P＜0.05) increased mineral N content in the NF soil by 239%, 70%, and 62% at elongating, flowering, and harvesting stage, respectively; and about half of added N was leached below 30 cm soil layers after harvesting wheat. Addition of urea-N also enhanced the mineral N in the NPK soil at elongating and flowering stage, but had no effect on mineral N in the MNPK soil. Addition N had no effect on SMBN in the NF soil, but significantly (P＜0.05) increased SMBN by 1.8 times in the NPK soil and 3.4 times in the MNPK soil at elongating stage. Soil microbial biomass N in the NPK and MNPK soils at vigorous stages of wheat (from elongating stage to flowering stage) decreased by 49% and 63%, respectively. The use efficiency rate of added N in the MNPK soil was 69%, which was significantly (P＜0.05) higher than in the NPK (40%) and NF (5%) soils. 【Conclusion】 It was concluded that long-term application of manure plus chemical fertilizers increased the buffer capacity of soil to N fertilizer, and synchronized N retention in soil and take up by crops.

Effects of Water and Fertilizers Applied on the Soluble Solid Content in Tomato Under Alternate Partial Root-Zone Irrigation

NIU Xiao-Li, ZHOU Zhen-Jiang, LI Rui, HU Tian-Tian

Scientia Agricultura Sinica. 2012, 45(5): 893-901. doi:10.3864/j.issn.0578-1752.2012.05.009

Abstract ( 1136 )

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【Objective】 The effects of irrigation amount and fertilizer rates of nitrogen, phosphorus, potassium and manure on the soluble solid content of tomato were investigated in a pot experiment under alternate partial root-zone irrigation. 【Method】Quadratic orthogonal regressive rotation design with five factors was used. A regression model on water and fertilizer and soluble solid content in tomato was established. Single and coupling effects were analyzed. 【Result】 The results showed that if other factors were zero codes, the soluble solid content in tomato decreased linearly with irrigation amount, while increased linearly with the fertilizer rates of N, K or manure, but it was not affected by P fertilizer rate. The interaction effects between N fertilizer and manure rate, P fertilizer rate and irrigation amount, K fertilizer or manure rates were presented positive correlation, while they were negatively correlated with N and phosphorus fertilizer rates, in which the interaction effect between P and K fertilizer rate was the largest. 【Conclusion】 It was concluded that heavy irrigation was not of benefit to the soluble solid content in tomato, and proper application of fertilizers of N, P, K, or manure could raise the soluble solid content in tomato effectively.

Effects of Ca2+ Signal Inhibitors on Oxalate Effluxes by Ectomycorrhizal Fungi Under Aluminum Stress

WANG Ming-Xia, ZHOU Zhi-Feng, YUAN Ling, HUANG Jian-Guo

Scientia Agricultura Sinica. 2012, 45(5): 902-908. doi:10.3864/j.issn.0578-1752.2012.05.010

Abstract ( 1018 )

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【Objective】 An experiment was carried out to study the mechanism of oxalate effluxes by ectomycorrhizal fungi (ECMF) under Al stress and to establish a theoretical foundation for alleviating the Al toxicity of trees, guide the selection of Al-resisted ECMF and preserve forest health. 【Method】 The growth and the oxalate effluxes by ectomycorrhizal species, Suillus luteus (Sl 08 and Sl 14 ) and Boletus sp. (Bo 15), which were isolated from acid forest soil, were studied in pure culture in liquid media with variable concentrations of Al3+ and Ca2+ signal inhibitors. 【Result】 Compared to the control without Al3+, when Al3+ concentration was 0.4 mmol•L-1, the growth of Sl 14 was inhibited significantly. But the growth of Sl 08 and Bo 15 was inhibited significantly in 1.2 mmol•L-1 Al3+ culture. It was showed that Sl 14 was sensitive to Al; Sl 08 and Bo 15 were resistant to Al. Meanwhile, the oxalate efflux by Sl 14 was few and invariable with the increase of Al3+ concentration in culture. But the oxalate effluxes by Sl 08 and Bo 15 increased with the increase of Al3+ concentration in the range of 0.2-0.8 mmol•L-1 Al3+ culture. It showed that the oxalate effluxes by ECMF were closely related with the tolerance of ECMF to Al. In the culture without Al3+, compared to without Ca2+ signal inhibitors, there was no significant change in the oxalate efflux by Bo 15 in the respective presence of verapamil (VP), ruthenium red (RR), trifluoperazine (TFP) and ethylene glycol tetraacetic acid (EGTA), while the oxalate efflux by Sl 08 was decreased significantly in the respective presence of VP, TFP, and EGTA. So Ca2+ signals could regulate oxalate efflux by Sl 08 but not by Bo 15 in culture without Al3+. Under Al stress, however, the oxalate effluxes by Sl 08 and Bo 15 were reduced significantly by four Ca2+ signal inhibitors. It was showed that Ca2+ signals could regulate oxalate efflux by Sl 08 and Bo 15 under Al stress. Oxalate efflux by Bo 15 was most sensitive to TFP, which led oxalate effluxes to a most greatly reduction, and Sl 08 was most sensitive to RR and TFP, compared to any other Ca2+ signal inhibitors.【Conclusion】 These results showed that Ca2+ signal participated in oxalate effluxes to accommodate Al stress in the fungi.

Evaluation of Productivity and Light Quality in Two High Density Dwarf Rootstock Apple Orchards in Central China

GAO Deng-Tao, GUO Jing-Nan, WEI Zhi-Feng, FAN Qing-Jin, YANG Chao-Xuan

Scientia Agricultura Sinica. 2012, 45(5): 909-916. doi:10.3864/j.issn.0578-1752.2012.05.011

Abstract ( 769 )

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【Objective】The objective of the experiment is to investigate the productivity and light quality in two high density M26 dwarf rootstock apple orchards in central China and give some management guidance for the high density dwarf rootstock apple planting. 【Method】 The technical parameters of individual trees, group parameters as shoots number and composition, the percentage of the land covered by canopy were determined, and the light quality in the canopy, crop production and fruit quality were investigated.【Result】 Slender spindle (SS) orchard has 54 thousands shoots per 667m2. Coverage rate is 76%. The ratio of long，medium and spur shoot is 1﹕1﹕8. The fruit yield is 3 263 kg/667m2. The light interception is 58%. The ratio of canopy which has good illumination is 65%. The first grade fruit accounts for 85%. Modified slender spindle (MSS) orchard has 93 thousands shoots per 667m2 and the coverage rate is 77%. The ratio of long, medium and spur shoot is 1﹕2﹕7. The fruit yield is 3 931 kg/667m2. The light interception is 73%. The ratio of canopy which has good illumination is 35%. The first grade fruit accounts for 85%.【Conclusion】Apple orchard with M26 dwarf rootstock trained as SS and MSS tree form may be useful to the management of the similar orchards in central China.

Molecular Cloning of Waterless-Related Genes in Ponkan Mandarin Using Suppression Subtractive Hybridization

YANG Ming, WANG Ri-Kui, ZHOU Lian, GE Wen-Dong, JIAO Yan-Xiang

Scientia Agricultura Sinica. 2012, 45(5): 917-925. doi:10.3864/j.issn.0578-1752.2012.05.012

Abstract ( 1049 )

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【Objective】The aim of this experiment is to reveal the molecular mechanism of the waterless citrus, to explore related genes, and to lay a foundation for the citrus waterless prevention.【Method】A suppression subtractive hybridization library was constructed using cDNA synthesized from RNA extracted from normal pulp as driver and waterless pulp as tester.【Result】 A total of 300 positive clones were selected for sequencing, and a total of 260 EST sequences were obtained. After comparison with GenBank using the online software of the BLAST, 197 ESTs, involving in 52 genes, were found to share considerable homology with known genes while the rest 63 ESTs had low or even no homology with known genes. The expressions of the β-tubulin, senescence-associated protein, cytochrome c, cysteine protease, phosphoenolpyruvate carboxykinase, trafficking protein, aspartic protease precursor, WRKY transcription factor 21 genes were studied by real-time quantitative PCR. The eight genes were significantly up-regulated in waterless pulp. These differentially expressed genes were related to numerical biological processes such as aging, stress-tolerance, chitin and cell wall macromolecule catabolic and proteolysis. 【Conclusion】 Some genes related to waterless were obtained, while the suppression subtractive hybridization library was constructed. These genes reflected the regulation of pulp to waterlessness, and can be used to prevent the disorder of waterlessness as candidate genes.

cDNA-AFLP Analysis of White Rust Response Genes in Chrysanthemum morifolium ‘Zihe’

HUANG He, WANG Shun-Li, DAI Si-Lan

Scientia Agricultura Sinica. 2012, 45(5): 926-935. doi:10.3864/j.issn.0578-1752.2012.05.013

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【Objective】An experiment was conducted to study the molecular mechanism of Chrysanthemum morifolium ‘Zihe’ response to the white rust, and to screen the white rust resistance genes. 【Method】 Infections were conducted by spraying the C. morifolium ‘Zihe’ leaves with suspension of 106 white rust sporangia per mL pure water for 3, 6, 12, 24, 48, 72, 96 h. cDNA-AFLP was used to moniter the gene expression under the white rust infections, and RT-PCR was used to validate the gene expression patterns. RACE method was used to get two fully cDNA sequences encoding 14-3-3 proteins, and phylogenetic analysis was also performed. 【Result】 Seventy-six primer combinations were used to investigate 4 950 cDNA fragments. After sequencing of 80 ESTs, the nucleic acid sequences of 51 ESTs were obtained, and among them, 18 ESTs were obtained, and showed homologous to the resistance-related genes of other species. These 18 ESTs were sorted into six functional categories: disease resistance protein, signal transduction, photosynthesis and photorespiration, retroelement polyprotein-like, senescence and metabolism pathways. RT-PCR showed that the cDNA-AFLP results was accurate and believable. Furthermore, two fully cDNA sequences of genes encoding 14-3-3 proteins were obtained: CmGFR01, CmGFR02. These two genes were 1 062 and 1 098 bp, encoded ORFs of 260 and 271 amino-acid residues. Phylogenetic analysis showed that the CmGFR01 and CmGFR02 might belong to a new subfamily of the higher plants 14-3-3 genes family. 【Conclusion】Gene expression profiling in response to white rust and differentially expressed genes of C. morifolium ‘Zihe’ were identified via cDNA-AFLP analysis. These genes could help us to understand the mechanism of response of plants to white rust. Furthermore, the isolated two genes, CmGFR01 and CmGFR02, could be used for the gene enigeering of the cultivated chrysanthemum resistance to the biotic stresses.

The Role of Reactive Oxygen Species in Heat Treatment-Induced Chilling Tolerance in Banana Fruit

WANG Hai-Bo, PANG Xue-Qun, HUANG Xue-Mei, ZHANG Zhao-Qi

Scientia Agricultura Sinica. 2012, 45(5): 936-942. doi:10.3864/j.issn.0578-1752.2012.05.014

Abstract ( 935 )

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【Objective】The objective of this study is to investigate the role of reactive oxygen species (ROS) in heat -induced chilling tolerance in banana fruit.【Method】 In this study, 10 μmol•L-1 diphenylene iodonium (DPI), a NADPH oxidase specific inhibitor, was used to inhibit ROS generation in hot water treated fruits. After heat treatment (52℃ for 3 min), banana fruits were stored at 7℃. The chilling injury index, Fv/Fm, contents of H2O2 and , activities of catalase (CAT) and ascorbate peroxidase (APX), gene expressions of MaCAT and MaAPX genes were determined.【Result】Compared with heat-treated fruits, fruits treated with DPI prior to heat treatment exhibited higher chilling injury index and lower values of Fv/Fm, Moreover, DPI treatment prevented heat-triggered H2O2 and accumulation, and inhibited the increase of the activities of CAT and APX, and the expressions of MaCAT and MaAPX genes. 【Conclusion】 It is suggested that pre-treatment with DPI prevented the rapid accumulation of ROS induced by heat treatment, which counteracted heat treatment-induced chilling tolerance accordingly had a negative effect on the chilling tolerance induced by heat treatment and showed serious chilling injury symptoms. The reactive oxygen species induced by heat treatment might serve as signal molecules involving in chilling resistance of banana fruits.

Effect of Lactobacillus casei Zhang Administration on Liver Lipids Metabolism of High-Fat Diet Induced Hypercholesterolemia Rats

ZHANG Yong, DU Rui-Ting, HE Qiu-Wen, LI He, ZHANG He-Ping

Scientia Agricultura Sinica. 2012, 45(5): 943-950. doi:10.3864/j.issn.0578-1752.2012.05.015

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【Objective】 The therapeutic effect of different doses of L. casei Zhang (2.0×1010 CFU/d, 2.0×109 CFU/d and 2.0×108 CFU/d) and its fermented milk beverage (2.0×108 CFU/mL/d) on liver lipids of hypercholesterolemia rats was investigated. 【Method】Hypercholesterolemia rats were induced by high fat feeding. Liver TC and TG levels, fecal total bile acid and cholesterol levels as well as serum ApoAⅠand ApoB levels were dertermined by commercial kits. 【Result】 The results showed that liver TC and TG levels were significantly decreased in all probiotic treatment groups compared to hypercholesterolemia model group (P＜0.05 or P＜0.01). In addition, fecal total bile acid levels and serum ApoAⅠlevels were significantly promoted and serum ApoB levels significantly reduced in all probiotic treatment groups compared to model group, while fecal cholesterol levels were changed with no significance (P＞0.05). 【Conclusion】 It is suggested that enhancing elimination of bile acids and regulating apolipoprotein levels could be mainly contributed to liver lipids ameliorated by probiotic L. casei Zhang in hypercholesterolemia rats.

Effect of Intermittently Monochromatic Light Stimuli During the Embryogenesis on Breast Muscular Growth and Meat Quality in Male Broiler Chicks

ZHANG Lin, ZHANG Hai-Jun, WU Shu-Geng, YUE Hong-Yuan, YAO Jun-Hu, QI Guang-Hai

Scientia Agricultura Sinica. 2012, 45(5): 951-957. doi:10.3864/j.issn.0578-1752.2012.05.016

Abstract ( 919 )

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【Objective】 LED (light emitting diodes) as light sources was used in this study to investigate the effect of intermittently monochromatic light stimuli during the embryogenesis on breast muscle growth, chemical composition, and meat quality of male broiler chicks.【Method】 Fertile broiler eggs (Arbor Acres, n=1 480) were preweighed and randomly assigned into one of the three treatment groups in 3 modified incubators: 1) monochromatic green light group (560 nm, n=490), 2) monochromatic blue light group (480 nm, n=490) and 3) control group (in dark condition, n =500). Light intensity was 15 lx at the eggshell level, and was intermittent at 15 min on and 15 min off. After hatching, 120 male chicks from each group were placed in 6 replicates with 20 birds each. All the birds were housed under normal white light (30 lx at bird-head level) with a light schedule of 23L﹕1D.【Result】 No significant differences in the hatchability, birth weight and feed intake among all groups were observed (P＞0.05). Compared to birds in blue or dark group, BW in green light group was significantly increased at 21 d and 42 d of age (P＜0.05). The breast muscle weight and breast muscle percentages in birds incubated under green light were significantly elevated by 38.3 g, 0.67%, and 44.6 g, 0.78% than those in the dark condition or blue group at 42 d of market age (P＜0.05), respectively. In green light group, feed conversion ratio during 0-42 d was lower than that in the dark condition or blue group (P＜0.05). No significant differences in the contents of breast moisture, CP, crude fat or crude ash among all groups were observed (P＞0.05). There was a trend toward green light stimuli increasing drip loss (P=0.10) and cooking loss (P=0.07). In addition, blue light induced a higher b* value of 24-h meat color than that in the dark condition or green light group (P=0.05).【Conclusion】Intermittently green light (15 lx) stimuli during the embryogenesis enhanced the posthatch BW of male broilers, increased breast muscle growth, and improved feed conversion ratio, but did not cause any significant changes in breast chemical composition, or any noticeable changes in overall meat quality characteristics except for a decreasing trend in water-holding capacity.

Integration Sites and Their Characteristic Analysis of piggyBac Transposon in Cashmere Goat Genome

BAI Ding-Ping, FANG Kun, YANG Ming-Ming, QU Lei, CHEN Yu-Lin

Scientia Agricultura Sinica. 2012, 45(5): 958-965. doi:10.3864/j.issn.0578-1752.2012.05.017

Abstract ( 1149 )

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【Objective】 The objective of the experiment was to construct piggyBac (PB) transposons vectors and integration site in Cashmere goat genome of PB was also studied. 【Method】 Donor plasmid of PB-CMV-EGFP-Neo and helper-dependent plasmid of pcDNA-Trans were constructed and transferred into Cashmere goat fetal fibroblasts by lipofectamine 2000. Cell clones stably transfected were obtained after screening by G418. Transgenic cell genome was obtained and the integration site of PB transposons were detected by r-PCR.【Result】Transposition system was constructed and the integration of foreign gene in Cashmere goat fibroblasts was successfully mediated. Transgenic cell line was also obtained. There were 21 integration sites of PB transposon in Cashmere goat genome after r-PCR detection. Analysis of the composition of the five bases, which was close to the side of the PB integration sites TTAA, showed that PB 3′ tended to insert into region rich in AT (58.35%) and the PB 5′ tended to insert into region rich in GC (57.8%). 【Conclusion】 The transposition of foreign PB transposons in Cashmere goats was highly efficient. The integration site information which is acquired from this research will provide theoretical references for Cashmere goats study by PB transposons.

Cloning and Analysis of MC1R Gene in Silky Fowl and Its Prokaryotic Expression

CHI Liang, LI Lan, PAN Qing-Jie, LIU Huan-Qi

Scientia Agricultura Sinica. 2012, 45(5): 966-972. doi:10.3864/j.issn.0578-1752.2012.05.018

Abstract ( 841 )

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【Objective】 MC1R (melanocortin 1-receptor) gene was analyzed and expressed in prokaryotic expression system.【Method】The CDs of silky fowl melanocortin 1-receptor gene was amplified and cloned from total RNA of silky fowl by RT-PCR according to the corresponding sequence in Gallus gallus. The biological information analysis was performed with it. The prokaryotic expression vector pET32a-MC1R was constructed and expressed in E. coli BL21 (DE3).【Result】The ORF of silky fowl MC1R gene consisted of 945 nucleotides and encoding 314 amino acids. The prokaryotic expression system of recombined vector pET32a-MC1R was constructed successfully and the protein MC1R was expressed in vitro by inducing with IPTG. 【Conclusion】 The MC1R gene of silky fowl was closed successfully and its relationship with melanin traits and its genetic relationship with other kinds of chicken were analyzed by constructing phylogenetic tree. At the same time, the MC1R protein of silky fowl was optimizedcy expressed in E. coli.

Isolation and Identification of an Antagonistic Bacterial Strain Against Ascosphaera apis from Honeybee Larvae Infected with Chalkbrood Disease

LI Jiang-Hong, ZHENG Zhi-Yang, HONG Shuang-Yan, QI Xiang-Feng, LIANG Qin

Scientia Agricultura Sinica. 2012, 45(5): 973-980. doi:10.3864/j.issn.0578-1752.2012.05.019

Abstract ( 959 )

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【Objective】 The objective of this study is to explore Ascosphaera apis antagonistic bacteria for biological control of honeybee chalkbrood disease.【Method】An antagonistic bacterial strain was isolated from honeybee larvae suffered from chalkbrood disease on PDA medium plate. It was identified by combining of morphology, gram stain and 16S rDNA sequence analysis. Its inhibiting activity to Ascosphaera apis was determined in vitro and in vivo. 【Result】 The bacterium was identified as Bacillus cereus. It could inhibit the growth of Ascosphaera apis in PDA medium, but its fermentation solution could not. The death rate and development of the honeybee larvae was unchanged when feeding diet containing the bacteria suggesting that the strain was not pathogenic to honeybee. By inoculating together with the spore of Ascosphaera apis to honeybee larvae, the bacterium could not inhibit the growth of Ascosphaera apis at early infecting stage, but could effectively degrade the hyphae soon after. Ascosphaera apis could not form the offspring spore, thus lost the capability of spreading. 【Conclusion】This study provides a theoretical basis for biological control of honeybee chalkbrood disease.

The Association Between the SLA Microsatellites Typing and the Immune Parameters of Introduced Pig Breeds

YU Hui, LIU Rong-Hui, ZUO Qi-Zhen, LI Yan, LI Hua

Scientia Agricultura Sinica. 2012, 45(5): 981-989. doi:10.3864/j.issn.0578-1752.2012.05.020

Abstract ( 1054 )

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【Objective】 The polymorphisms and the haplotypes of the swine leukocyte antigen complex (SLA) were investigated in Landrace and Yorkshire pigs using microsatellites (MS). The association between haplotypes and immune parameters was analyzed in order to provide references for disease resistance breeding in pigs.【Method】The genetic diversity parameters were calculated by thirteen microsatellites, which were in SLA region, the haplotypes in class Ⅰ and class Ⅱ were typed by some selected microsatellites typing, and the association between haplotypes and immune parameters were studied by microsatellites in class Ⅰand class Ⅱ regions.【Result】The expected heterozygosity ( =0.70) and polymorphism information content ( =0.65) of Yorkshire pigs were higher than those（ =0.67， =0.62）of Landrace pigs, respectively. The multiple comparison results of six haplotypes and immune parameters in SLA Ⅰ showed that the mean corpuscular hemoglobin concentration (MCHC) of H2 was higher than that of H3 (P＜0.05) and H5 (P＜0.01), respectively. The platelet distribution width (PDW) of H2 was lower than that of H3 (P＜0.05). The results of multiple comparisons between twelve haplotypes and immune parameters in SLAⅡ showed that the mean of red blood cell count (RBC) of H1′ was lower than that of H6′ and H9′ (P＜0.05), respectively. The value of lymphocytes ratio (LYM) of H1′ was lower than that of H7′ (P＜0.05), and extremely lower than that of H6′, H9′ and H11′ (P＜0.01), respectively. The mean of MCHC of H6′was lower than that of H7′ (P＜0.05), H1′ and H11′ (P＜0.01), while the value of MCHC of H9′ was lower than that of H1′, H7′ and H11′ (P＜0.05), respectively. The mean corpuscular volume (MCV) of H9′ was lower than that of H1′, H6′ and H11′ (P＜0.05) and extremely lower than that of H7′ (P＜0.01), respectively. The value of monocytes ratio (MONO) of H11′ was significantly lower than that of H6′, H7′ (P＜0.05) and H1′ (P＜0.01), while the value of H6′, H7′and H9′ was lower than that of H1′ (P＜0.05, P＜0.01), respectively.【Conclusion】The polymorphisms of Yorkshire pigs were higher than those of Landrace pigs, and the associations were detected between some haplotypes of SLA-MS and immune parameters, which were PDW, MCHC, RBC, MCV, LYM and MONO. These results could be used in evaluation of health index in future.

Comparison of the In vitro Antiviral Activity Between the Chicken Mutant-Type and Wild-Type Mx Proteins

FU De-Zhi, SUN Min, ZHENG Meng-Meng, GAO Bo, ZHANG Ya-Ni, DOU Tao-Cun, WANG Ke-Hua, LI Bi-Chun, SUN Huai-Chang

Scientia Agricultura Sinica. 2012, 45(5): 990-998. doi:10.3864/j.issn.0578-1752.2012.05.021

Abstract ( 995 )

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【Objective】This experiment was conducted to assess the relationship between the 631 amino acid variations of chicken Mx protein and the disease resistance. 【Method】 Mutant pcDNA3.0-MMx and wild-type pcDNA3.0-Mx plasmids of Mx protein gene of Chinese Langshan chicken were constructed successfully, then the CEF and NIH-3T3 cells were transfected by mutant pcDNA3.0-MMx and wild-type pcDNA3.0-Mx plasmids, respectively. RT-PCR was used to identify the expression of mutant MMx and wild-type Mx genes and the mini-cytopathic effect inhibition assay was used to evaluate the antiviral activity for Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV). 【Result】 The recombinant Mx protein protected the CEF cells from NDV infection up to 48-h incubation while the NIH-3T3 cells transfected by pcDNA3.0-MMx were not infected by 60-h incubation with VSV, however, the CEF and NIH-3T3 cells transfected by pcDNA3.0-Mx developed pathological changes during 24-h incubation with the viruses. 【Conclusion】 Recombinant Mx protein could delay the infection of NDV and VSV at single-cell level.

Isolation, Characterization, and Expression of Goose Avian β-Defensin 10

ZHOU Cai-Yuan, ZHANG Ming-Yue, HAN Zong-Xi, SHAO Yu-Hao, LIU Sheng-Wang, MA De-Ying

Scientia Agricultura Sinica. 2012, 45(5): 999-1009. doi:10.3864/j.issn.0578-1752.2012.05.022

Abstract ( 1052 )

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【Objective】 The objective of this study was to clone avian β-defensin 10 (AvBD10) gene from goose tissues and to characterize the antimicrobial activity of the recombinant GST-AvBD10 fusion protein. 【Method】 The coding sequences of goose AvBD10 was obtained from kidney of goose by RT-PCR, phylogenetic relationships of the goose AvBD10 with those of other avian species and some mammalian were analyzed. In addition, tissue distribution of the gene was detected by real time PCR. AvBD gene coding sequence was inserted into the pGEX-6p-1 vector. The constructs were transformed into competent Escherichia coli BL21 (DE3) cells. Expression of the fusion proteins was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) and the proteins were purified. Furthermore, bioactivities of the recombinant protein were analyzed in vitro. 【Result】 The full length cDNA of goose AvBD10 consisted of 207 nucleotide acids, encoding 68 amino acid residues. Phylogenetic analysis demonstrated that the goose AvBD10 shared the highest amino acid homology with duck AvBD10 (92.7%). The recombinant protein exhibited high antimicrobial activity against 12 bacterial strains investigated. The antimicrobial activity was decreased in high salt concentration and exhibited no hemolytic properties. 【Conclusion】 The goose AvBD10 gene from goose was successfully cloned and expressed in E. coli. The purified recombinant protein showed antimicrobial activity and no hemolytic properties.

Multiplex Tandem PCR Assays for the Detection of Genetically Modified Organisms

WEI Shuang, CHEN Zhen, MA Jun, BAI Wei-Bin, WU Xi-Yang

Scientia Agricultura Sinica. 2012, 45(5): 1010-1016. doi:10.3864/j.issn.0578-1752.2012.05.023

Abstract ( 1026 )

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【Objective】The objective of this study is to develop a multiplex tandem polymerase chain reaction (MT-PCR) gene disk method for the detection of genetically modified organism (GMO). 【Method】 The MT-PCR method was employed to detect NOS terminator, FMV35S promoter, alien genes including NPTⅡ, Cry1Ab, Cry1Ab/Ac, CP4-EPSPS and PAT, which was normally used in transformation, and endogenous genes such as IVR from maize, sad1 from cotton, and PEP from rapeseed meal. Inner and outer primers were specially designed for each gene. Following the first PCR reaction, the multiplexed amplicons were simultaneously amplified for a small number of cycles so as to avoid competition between amplicons. The reaction product was then diluted and analyzed in multiple individual PCRs using primers nested inside the primers used for the multiplexed amplification. As the second PCR used a template enriched in the amplicons of interest, the conditions could be optimized to significantly reduce ‘primer dimer’ formation allowing SYBR Green chemistry to be used for quantification. The final results were achieved by cycling curves and melting curve. 【Result】 The MT-PCR method was rapid (＜2 h), high-throughput (10 genes of each sample), sensitive (＞0.000292 ng), and specific in detecting GMO, such as cotton seeds, maize, rice and rapeseed meal. The method is making very fine distinctions about the three kinds of transgenic species can be made by this method and it fits for a large amount of detection. 【Conclusion】 The MT-PCR method is specific, stable and reliable, and will be an effective tool for the detection of GMO.

Expression of Progestogen Receptor in Inferior Mesenteric Ganglion of Female Goat

WU Wei-Ni, REN Ni, WANG Hong-Mei, XU Yong-Ping, FAN Jie, WANG Zhi-Hao, MING Jia

Scientia Agricultura Sinica. 2012, 45(5): 1017-1022. doi:10.3864/j.issn.0578-1752.2012.05.024

Abstract ( 971 )

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【Objective】 The purpose of the present research was to explore whether inferior mesenteric ganglion (IMG) of female goat has reactivity to the progestogen. 【Method】 Based on immunohistochemical staining and RT-PCR technique, the expression and distribution of progestogen receptor (PR) in the IMG of female goat were detected. 【Result】 The PR immunoreactive products locate widely in the IMG of female goat, they have high staining intensity in the nucleolus, ecumenic staining intensity in the whole cytoplasm and nucleoli. The immunoreactive products also exist in other composition of the IGM.【Conclusion】It was demonstrated that PR existed in the female goat IGM.

Impacts of Technology Training Programs in Farmer Field Schools on the Adoption of Environment-Friendly Technology: Empirical Evidences from the Tomato Production in Beijing

CAI Jin-Yang, HU Rui-Fa, XIAO Chang-Kun, WANG Xiao-Bing

Scientia Agricultura Sinica. 2012, 45(5): 1023-1030. doi:10.3864/j.issn.0578-1752.2012.05.025

Abstract ( 1044 )

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【Objective】The impacts of technology training programs in farmer field schools (FFS) on the adoption of environment-friendly technology was analyzed.【Method】 Through random sampling strategy of the farmers in tomato production in suburbans of Beijing, the econometric method was used to obtain the corresponding results. 【Result】The descriptive statistics indicate that the percentage of farmers who adopt the environment-friendly technology is less than 20%. The empirical analysis shows that under the conditions of other variables are controlled, the proportion of the environment-friendly technology in irrigation adopted by farmers who have attended the technology training programs in FFS is 14.7% higher than that for the farmers who have not attended the training program. In respect to adopting the environment-friendly fertilization technology, the proportion of farmers who have attended the training program in FFS is 8.3% higher than those who have not attended the training program. 【Conclusion】The results show that the training program in FFS significantly enhanced the adoption of environment-friendly technology, especially the irrigation and fertilization technologies in production.

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