CPB-LAR-GFP表达载体的构建及其在紫花苜蓿愈伤组织中的表达
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摘要
以无色花青素还原酶编码基因LAR为靶序列,构建携带绿色荧光蛋白GFP和抗除草剂bar基因的双标记选择植物表达载体,通过农杆菌介导法对紫花苜蓿进行遗传转化,经PCR检测和PPT筛选出抗性愈伤组织,转基因紫花苜蓿愈伤组织的缩合单宁含量比对照高30.8%。试验证明重组基因已转化至紫花苜蓿愈伤组织中并发挥生理功能,为研究缩合单宁合成与代谢的分子调控机制和培育具有抗除草剂和抗臌胀病的牧草新品种奠定基础。
Leucoanthocyanidin reductase is a key enzyme in plant condensed tannins synthesis.The target was Leucoanthocyanidin reductase encoded by LAR gene,the expression vector was constructed with the double-marker expressing green fluoresce protein GFP gene and herbicide resistance bar gene.Transgenic alfalfa callus were obtained by Agrobacterium tumefaciens mediated transformation,and the resistant callus were screened by PPT.The condensed tannins content in transgenic alfalfa callus was 30.8% higher than that of control.The recombinant gene had been transformed to alfalfa callus and played a physiological function.These research work will provide the fundamental information and the method for studying the regulation mechanism of condensed tannins synthesis and further alfalfa breeding through transgenic technology with character of resistance to herbicide and bloat disease.
Leucoanthocyanidin reductase is a key enzyme in plant condensed tannins synthesis.The target was Leucoanthocyanidin reductase encoded by LAR gene,the expression vector was constructed with the double-marker expressing green fluoresce protein GFP gene and herbicide resistance bar gene.Transgenic alfalfa callus were obtained by Agrobacterium tumefaciens mediated transformation,and the resistant callus were screened by PPT.The condensed tannins content in transgenic alfalfa callus was 30.8% higher than that of control.The recombinant gene had been transformed to alfalfa callus and played a physiological function.These research work will provide the fundamental information and the method for studying the regulation mechanism of condensed tannins synthesis and further alfalfa breeding through transgenic technology with character of resistance to herbicide and bloat disease.
引文
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[3]Paolocci F,Bovone T,Tosti N,et al.Light and an exogenous transcription factor qualitatively and quantitatively affect the biosynthetic pathway of condensed tannins in Lotus cornicuental latus leaves[J].Journal of Experim Botany,2005,56:1093-1103.
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[5]Hichri I,Barrieu F,Bogs J,et al.Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway[J].Journal of Experimental Botany,2011,62:2465-2483.
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[9]王艳,李建龙,潘永年,等.草坪草转基因育种研究进展[J].草原与草坪,2007(6):13-17.
[10]顾垒,毕玉芬.转基因苜蓿研究的现状和前景[J].草原与草坪,2004(1):17-21.
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[12]王旺田,张金文,王蒂,等.马铃薯块茎糖基转移酶基因的克隆及其RNAi载体的构建[J].作物学报,2011,37(11):1926-1934.
[13]陈春艳,马晖玲,董文科.甘肃红豆草无色花青素还原酶LAR基因的克隆和表达分析[J].草业学报,2015,24(6):177-187.
[14]Tanner G J,Francki K T,Abrahams S,et al.Proanthocyanidin biosynthesis in plants:purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA[J].Journal of Biology Chemistry,2003,278:31647-31656.
[15]Bogs J,Downey M O,Harvey J S,et al.Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves[J].Plant Physiology,2005,139(2):652-663.
[16]Paolocci F,Robbins M P,Madeo L,et al.Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosyn thesis:structure,expression analysis,and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus[J].Plant physiology,2007,143(1):504-516.
[17]王关林,方宏筠.植物基因工程原理与技术[M].北京:科学出版社,1998:547-548.
[18]王关林,方宏筠.植物基因工程(第二版)[M].北京:科学出版社,2002:742-744.
[19]陈春艳,马晖玲,董文科.‘甘肃红豆草’肌动蛋白基因片段的克隆及序列分析[J].甘肃农业大学学报,2014,49(6):97-102.
[20]Terrill T H,Row An A M,Douglas G B,et al.Determination of extractable and bound condensed tannin concentrations in forage plans protein concentrate meals and cereal grains[J].Journal of the science of food and agriculture,1992,58:321-329.
[21]Chalfie M,Tu Y,Euskirchen G,et al.Green fluorescent protein as a maker for gene expression[J].Science,1994,263(5148):802-805.
[22]Jim H,Brad A.GFP in plant[J].Technical Focus,1995,8(11):10-11.
[23]陈玉香,周道玮.转基因牧草研究进展[J].中国草地,2002,24(3):59-63.
[24]安惠惠,马晖玲,李坚,等.农杆菌介导的Lyz-GFP基因对匍匐翦股颖Penn A-1转化和表达的研究[J].草业学报,2012,21(2):141-148.
[25]D′Halluin K,Boteerman J,de Greef W.Engineering of herbicide-resistant alfalfa and evaluation under field conditions[J].Crop Science,1990,30:866-871.
[26]刘艳芝,王玉民,刘莉,等.Bar基因转化草原1号苜蓿的研究[J].草地学报,2004,12(4):273-280.
[2]Koupai-Abyazani M R,McCallum J,Muir A D,et al.Purification and characterization of a proanthocyanidin polymer from seed of alfalfa(Medicago sativa cv.Beaver)[J].Journal of Agricultural and Food Chemistry,1993,41:565-569.
[3]Paolocci F,Bovone T,Tosti N,et al.Light and an exogenous transcription factor qualitatively and quantitatively affect the biosynthetic pathway of condensed tannins in Lotus cornicuental latus leaves[J].Journal of Experim Botany,2005,56:1093-1103.
[4]Zhao J,Pang Y,Dixon R A.The mysteries of proanthocyanidin transport and polymerization[J].Plant Physiology,2010,153:437-443.
[5]Hichri I,Barrieu F,Bogs J,et al.Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway[J].Journal of Experimental Botany,2011,62:2465-2483.
[6]Verdier J,Zhao J,Torres-Jerez I,et al.MtPAR MYB transcription factor acts as an on switch for proanthocyanidin biosynthesis in Medicago truncatula[J].Proceedings of the National Academy of Sciences USA,2012,109:1766-1771.
[7]杨茁萌,陈颖,李林,等.红豆草浓缩单宁生物合成相关蛋白的研究[J].草原家畜(增刊),1996(9):47-54.
[8]俞金蓉,玉永雄,陈丽梅.生物技术在紫花苜蓿中的应用[J].草原与草坪,2005(5):65-69.
[9]王艳,李建龙,潘永年,等.草坪草转基因育种研究进展[J].草原与草坪,2007(6):13-17.
[10]顾垒,毕玉芬.转基因苜蓿研究的现状和前景[J].草原与草坪,2004(1):17-21.
[11]Smith A K,Frank M,Phil M,et al.Reducing post-harvest losses of forage protein[J].Iger Innovationa,2004(8):30-33.
[12]王旺田,张金文,王蒂,等.马铃薯块茎糖基转移酶基因的克隆及其RNAi载体的构建[J].作物学报,2011,37(11):1926-1934.
[13]陈春艳,马晖玲,董文科.甘肃红豆草无色花青素还原酶LAR基因的克隆和表达分析[J].草业学报,2015,24(6):177-187.
[14]Tanner G J,Francki K T,Abrahams S,et al.Proanthocyanidin biosynthesis in plants:purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA[J].Journal of Biology Chemistry,2003,278:31647-31656.
[15]Bogs J,Downey M O,Harvey J S,et al.Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves[J].Plant Physiology,2005,139(2):652-663.
[16]Paolocci F,Robbins M P,Madeo L,et al.Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosyn thesis:structure,expression analysis,and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus[J].Plant physiology,2007,143(1):504-516.
[17]王关林,方宏筠.植物基因工程原理与技术[M].北京:科学出版社,1998:547-548.
[18]王关林,方宏筠.植物基因工程(第二版)[M].北京:科学出版社,2002:742-744.
[19]陈春艳,马晖玲,董文科.‘甘肃红豆草’肌动蛋白基因片段的克隆及序列分析[J].甘肃农业大学学报,2014,49(6):97-102.
[20]Terrill T H,Row An A M,Douglas G B,et al.Determination of extractable and bound condensed tannin concentrations in forage plans protein concentrate meals and cereal grains[J].Journal of the science of food and agriculture,1992,58:321-329.
[21]Chalfie M,Tu Y,Euskirchen G,et al.Green fluorescent protein as a maker for gene expression[J].Science,1994,263(5148):802-805.
[22]Jim H,Brad A.GFP in plant[J].Technical Focus,1995,8(11):10-11.
[23]陈玉香,周道玮.转基因牧草研究进展[J].中国草地,2002,24(3):59-63.
[24]安惠惠,马晖玲,李坚,等.农杆菌介导的Lyz-GFP基因对匍匐翦股颖Penn A-1转化和表达的研究[J].草业学报,2012,21(2):141-148.
[25]D′Halluin K,Boteerman J,de Greef W.Engineering of herbicide-resistant alfalfa and evaluation under field conditions[J].Crop Science,1990,30:866-871.
[26]刘艳芝,王玉民,刘莉,等.Bar基因转化草原1号苜蓿的研究[J].草地学报,2004,12(4):273-280.