StP5CS基因过表达增强高温胁迫下菜用大豆结瘤固氮的能力
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摘要
高温胁迫严重影响菜用大豆的结瘤固氮过程,增强高温胁迫下菜用大豆结瘤固氮的能力对提高菜用大豆产量具有重要意义。采用蛭石栽培的方法,以转StP5CS基因菜用大豆(T6)为试验材料,研究在35℃高温胁迫下StP5CS基因过表达对菜用大豆结瘤固氮的影响。结果显示:转基因株系的株高、干鲜重及根瘤数目、根瘤干鲜重、单株荚数、粒数、种子重均显著高于非转基因株系(WT);转基因株系根瘤中脯氨酸、豆血红蛋白含量及谷氨酰胺合成酶活性均显著高于WT;经qRT-PCR分析,5个结瘤固氮关键基因(GmENOD40-1,GmENOD40-2,GmGS1β1,GmGS1β2及GmLba)在转基因株系中的相对表达量均显著高于WT。结果表明,高温胁迫下StP5CS基因过表达,促进了脯氨酸的积累,显著增强了菜用大豆的耐热性和结瘤固氮能力,进而提高了菜用大豆的产量。
High temperature stress exerts adverse influences on the process of nodulation and nitrogen fixation of vegetable soybean. Enhancing the nitrogen fixation efficiency of vegetable soybean is of significance to increase the yield of vegetable soybean under heat stress. Using the method of vermiculite cultivation, St P5 CS transgenic vegetable soybean(T6) was used as experimental material to study the effects of St P5 CS gene overexpression on nodulation and nitrogen fixation of vegetable soybean under high temperature at 35°C. The results showed that the plant height, dry weight and fresh weight of plants, number of root nodules, dry weight and fresh weight of root nodules, number of pods and number of seeds per plant and seed weight of the transgenic lines were significantly higher than those of the wild type line(WT). Proline, leghemoglobin contents and glutamine synthetase activity in the nodules of the transgenic lines were significantly higher than those of WT. qRT-PCR analysis showed that the relative expression levels of five key genes for nodulation and nitrogen fixation(Gm ENOD40-1, Gm ENOD40-2, Gm GS1β1, Gm GS1β2 and Gm Lba) in the transgenic lines were significantly higher than those of WT. The above results indicate that under high temperature stress, overexpression of St P5 CS gene promotes the accumulation of proline, and significantly enhances the nodulation and nitrogen fixation ability, resulting in the increase of yield in vegetable soybean.
High temperature stress exerts adverse influences on the process of nodulation and nitrogen fixation of vegetable soybean. Enhancing the nitrogen fixation efficiency of vegetable soybean is of significance to increase the yield of vegetable soybean under heat stress. Using the method of vermiculite cultivation, St P5 CS transgenic vegetable soybean(T6) was used as experimental material to study the effects of St P5 CS gene overexpression on nodulation and nitrogen fixation of vegetable soybean under high temperature at 35°C. The results showed that the plant height, dry weight and fresh weight of plants, number of root nodules, dry weight and fresh weight of root nodules, number of pods and number of seeds per plant and seed weight of the transgenic lines were significantly higher than those of the wild type line(WT). Proline, leghemoglobin contents and glutamine synthetase activity in the nodules of the transgenic lines were significantly higher than those of WT. qRT-PCR analysis showed that the relative expression levels of five key genes for nodulation and nitrogen fixation(Gm ENOD40-1, Gm ENOD40-2, Gm GS1β1, Gm GS1β2 and Gm Lba) in the transgenic lines were significantly higher than those of WT. The above results indicate that under high temperature stress, overexpression of St P5 CS gene promotes the accumulation of proline, and significantly enhances the nodulation and nitrogen fixation ability, resulting in the increase of yield in vegetable soybean.
引文
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[20]Kouchi H,Hata S.Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stage of soybean nodule development[J].Molecular and General Genetics,1993,238(1-2):106-119.
[21]赵亚兰,尉亚辉.豆血红蛋白的研究进展[J].西北植物学报,2000,20(4):684-689.
[22]Ott T,Vandongen J T,Gunther C,et al.Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development[J].Current Biology,2005,15(6):531-535.
[23]Seabra A R,Silva L S,Carvalho H G.Novel aspects of glutamine synthetase(GS)regulation revealed by a detailed expression analysis of the entire GSgene family of Medicago truncatula under different physiological conditions[J].BMC Plant Biology,2013,13(1):1-15.
[24]Masalkar P,Wallace I S,Hwang J H,et al.Interaction of cytosolic glutamine synthetase of soybean root nodules with the C-terminal domain of the symbiosome membrane nodulin 26 aquaglyceroporin[J].Biol Chem,2010,285(31):23880-23888.
[25]Cai H M,Xie W B,Lian X M.Comparative analysis of differentially expressed genes in rice under nitrogen and phosphorus starvation stress conditions[J].Plant Mol Biol Rep,2013,31(1):160-173.
[2]Hasanuzzaman M,Nahar K,Alam M M,et al.Physiological,biochemical,and molecular mechanisms of heat stress tolerance in plants[J].Int J Mol Sci,2013,14(5):9643-9684.
[3]Van W S G.Soybean nodule development and senescence:the role of cysteine proteases and their inhibitors[J].Comput Theor Nanos,2015,12(12):1-8.
[4]Ashraf M,Akram N A.Improving salinity tolerance of plants through conventional breeding and genetic engineering:an analytical comparison[J].Biotechnol Adv,2009,27(6):744-752.
[5]Ghanti S K K,Sujata K G,Kumar B M V,et al.Heterologous expression of P5CS gene in chickpea enhances salt tolerance without affecting yield[J].Biol Plantarum,2011,55(4):634-640.
[6]Zhang G C,Zhu W L,Zhu Y L,et al.Enhanced salt tolerance of transgenic vegetable soybeans resulting from overexpression of a novelΔ1-pyrroline-5-carboxylate synthetase gene from Solanum torvum Swartz[J].Hort Environ Biotechnol,2015,56(1):94-104.
[7]Zhu W L,Yang L F,Zhu Y L,et al.Overexpression of rice phosphate transporter gene OsPT2 enhances nitrogen fixation and ammonium assimilation in transgenic soybean under phosphorus deficiency[J].Plant Biol,2016,59(2):172-181.
[8]Mariangela H,Avilio A F.Effects of high temperature on nodulation and nitrogen fixation by Phaseolus vulgaris L[J].Plant and Soil,1993,149(1):95-102.
[9]李永红,魏玉香.鸡冠花耐热性评价方法研究[J].植物研究,2009,29(1):91-95.
[10]职明星,李秀菊.脯氨酸测定方法的改进[J].植物生理学通讯,2005,41(3):355-357.
[11]李合生,孙群,赵氏杰.植物生理生化实验原理与技术[M].北京:高等教育出版社,2000:58-64.
[12]Riley I T,Dilworth M J.Cobalt requirement for nodule development and function in Lupinus-angustifolius L[J].New Phytologist,1985,100(3):347-359.
[13]Kouci H,Hata S.Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development[J].General Genetics,1993,238(1-2):106-119.
[14]She Q X,Sandal N N,Stougaard J,et al.Comparative sequence analysis of cis elements present in Glycine max L.leghemoglobin lba and lbc3 genes[J].Plant Mol Biol,1993,22(5):931-935.
[15]Masalkar P,Wallace I S,Hwang J H,et al.Interaction of cytosolic glutamine synthetase of soybean root nodules with the C-terminal domain of the symbiosome membrane nodulin 26 aquaglyceroporin[J].Biol Chem,2010,285(31):23880-23888.
[16]Libault M,Thibivilliers S,Bilgin D D,et al.Identification of four soybean reference genes for gene expression normalization[J].Plant Genome,2008,1(1):44-45.
[17]Schmittgen T D,Livak K J.Analyzing real-time PCRdata by the comparative CT method[J].Nature Protocols,2008,3(6):1101-1108.
[18]陈慧,邸伟,姚玉波,等.不同大豆品种根瘤固氮酶活性与固氮量差异研究[J].核农学报,2013,27(3):379-383.
[19]Kim G B,Nam Y W.A novelΔ1-pyrroline-5-carboxylate synthetase gene of Medicago truncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation[J].Journal of Plant Physiology,2013,170(3):291-302.
[20]Kouchi H,Hata S.Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stage of soybean nodule development[J].Molecular and General Genetics,1993,238(1-2):106-119.
[21]赵亚兰,尉亚辉.豆血红蛋白的研究进展[J].西北植物学报,2000,20(4):684-689.
[22]Ott T,Vandongen J T,Gunther C,et al.Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development[J].Current Biology,2005,15(6):531-535.
[23]Seabra A R,Silva L S,Carvalho H G.Novel aspects of glutamine synthetase(GS)regulation revealed by a detailed expression analysis of the entire GSgene family of Medicago truncatula under different physiological conditions[J].BMC Plant Biology,2013,13(1):1-15.
[24]Masalkar P,Wallace I S,Hwang J H,et al.Interaction of cytosolic glutamine synthetase of soybean root nodules with the C-terminal domain of the symbiosome membrane nodulin 26 aquaglyceroporin[J].Biol Chem,2010,285(31):23880-23888.
[25]Cai H M,Xie W B,Lian X M.Comparative analysis of differentially expressed genes in rice under nitrogen and phosphorus starvation stress conditions[J].Plant Mol Biol Rep,2013,31(1):160-173.