农杆菌介导转Bt基因海岛棉的获得及抗棉铃虫效果分析
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摘要
本研究采用农杆菌介导法,对海岛棉胚性愈伤组织进行遗传转化。经抗性培养基筛选得到抗性胚性愈伤组织,进而嫁接得到抗性植株。通过对抗性植株进行PCR以及Southern blot检测,确定了Bt基因已经整合至抗性植株的基因组中。通过对这些转基因植株进行室内抗虫性鉴定,调查棉铃虫幼虫的死亡情况、幼虫对转基因植株叶片的取食情况以及幼虫的生长状况为指标来综合评价其抗性程度,结果表明,6棵转基因植株对棉铃虫均具有一定的抗性,5号及6号植株抗性级别达到了抗。
In this study,Agrobacterium tumefaciens-mediated method was used to genetically transform embryogenic calli of island cotton.The resistant embryogenic callus was screened by resistance medium and grafted to obtain resistant plants.The Bt gene was integrated into the genome of the resistant plant by PCR and Southern blot detection of the resistant plants.The results showed that 6 transgenic plants were identified for indoor resistance to evaluate the resistance of these transgenic plants to investigate the mortality of Helicoverpa armigera larvae,the larvae feeding on transgenic plants and the growth status of larvae.The plants had a certain resistance to cotton bollworm,and the resistance levels of plants 5 and 6 reached resistance.
In this study,Agrobacterium tumefaciens-mediated method was used to genetically transform embryogenic calli of island cotton.The resistant embryogenic callus was screened by resistance medium and grafted to obtain resistant plants.The Bt gene was integrated into the genome of the resistant plant by PCR and Southern blot detection of the resistant plants.The results showed that 6 transgenic plants were identified for indoor resistance to evaluate the resistance of these transgenic plants to investigate the mortality of Helicoverpa armigera larvae,the larvae feeding on transgenic plants and the growth status of larvae.The plants had a certain resistance to cotton bollworm,and the resistance levels of plants 5 and 6 reached resistance.
引文
Ding X.L.,2015,Study on the Agrobacterium tmefaciens-mediated transformation of Bt gene into sea island cotton(Gossypium Barbadense L.),Thesis for M.S.,Xinjiang Agricultural University,Supervisor:Chen Q.J.,and Qu Y.Y.,pp.1-55(丁喜莲,2015,农杆菌介导的海岛棉转Bt基因遗传转化研究,硕士学位论文,新疆农业大学,导师:陈全家,曲延英,pp1-55)
Guo S.D.,1995,Advances in plant Bt insect resistance gene engineering,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),28(5):8-13(郭三堆,1995,植物Bt抗虫基因工程研究进展,中国农业科学,28(5):8-13)
Guo S.D.,Wang Y.,Sun G.Q.,Jin S.Q.,Zhou T.,Meng Z.G.,and Zhang R.,2015,Twenty years of research and application of transgenic cotton in China,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),48(17):3372-3387(郭三堆,王远,孙国清,金石桥,周焘,孟志刚,张锐,2015,中国转基因棉花研发应用二十年,中国农业科学,48(17):3372-3387)
He Y.T.,Qu Y.Y.,Kong Q.P.,Yan Y.L.,Shang S.,Zheng L.Y.,and Chen Q.J.,2008,Primary studies on the effects of callus induction and differentiation in Gossypium barbadense,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),6(3):597-602(贺雅婷,曲延英,孔庆平,严勇亮,尚爽,郑良玉,陈全家2008,海岛棉愈伤组织诱导及分化的影响因素初探,分子植物育种,6(3):597-602)
Kong Q.P.,2002,Production situation and comparative advantage analysis of island cotton in China,Zhongguo Mianhua(China Cotton),29(12):19-22(孔庆平,2002,我国海岛棉生产概况及比较优势分析,中国棉花,29(12):19-22)
Li R.H.,Xia Y.S.,Liu S.Z.,Sun L.L.,Guo P.G.,Miao S.Y.,and Chen J.H.,2009,CTAB-improved method of DNA extractionin plant,Shiyanshi Yanjiu Yu Tansuo(Laboratory Research and Exploration),28(9):14-16(李荣华,夏岩石,刘顺枝,孙莉丽,郭培国,缪绅裕,陈健辉,2009,改进的CTAB提取植物DNA方法,实验室研究与探索,28(9):14-16)
Sakhanokho H.F.,Zipf A.,Rajasekaran K.,Saha S.,and Sharma G.C.,2001,Induction of highly embryogenic calli and plant regeneration in upland(Gossypium hirsutum L.)and pima(Gossypium barbadense L.)cottons,Cell Biology&Molecular Genetics,41(4):1235-1240
Su Y.M.,2015,Research progress of transgenic breeding of Xinjiang island cotton,Nanfang Nongye(South China Agriculture),9(24):252-256(苏豫梅,2015,新疆海岛棉转基因育种的研究进展,南方农业,9(24):252-256)
Wei L.M.,1995,Study on callus induction and differentiation of sea island cotton,Shengwu Jishu(Biotechnology),5(5):26-29(魏良民,1995,海岛棉愈伤组织诱导和分化研究,生物技术,5(5):26-29)
Wei Y.H.,Ma L.L.,He L.L.,Chai M.L.,Zhu H.G.,Sun J.,and Zhang W.,2013,Agrobacterium infecting flower stigma for sea-island cotton(Gossypium barbadense L.)genetic transformation,Xinjiang Nongye Kexue(Xinjiang Agricultural Sciences),50(1):15-21(魏延宏,马玲玲,何兰兰,柴蒙亮,朱华国,孙杰,张薇,2013,利用农杆菌侵染花柱头法进行海岛棉遗传转化,新疆农业科学,50(1):15-21)
Wang Y.X.,Wang X.F.,and Ma Z.Y.,2007,A new method of cotton grafting and its application,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),40(2):264-270(王彦霞,王省芬,马峙英,2007,棉花高效嫁接新方法及其应用,中国农业科学,40(2):264-270)
Weng Q.,2007,Establishment of stem-tip regeneration system of island cotton and Agrobacterium-mediated transformation of insect-resistant genes,Thesis for M.S.,Xinjiang Agricultural University,Supervisor:Qu Y.Y.,pp.1-68(翁琴,2007,海岛棉茎尖再生体系的建立及农杆菌介导抗虫基因转化研究,硕士学位论文,新疆农业大学,导师:曲延英,pp.1-68)
Yao H.S.,Zhang Y.L.,Yi X.P.,Xue J.,Luo Y.,Luo H.H.,and Zhang W.F.,2015,Study on differences in comparative canopy structure characteristics and photosynthetic carbon assimilation of field-grown pima cotton(Gossypium barbadense)and upland cotton(G.hirsutum),Zhongguo Nongye Kexue(Scientia Agricultura Sinica),48(2):251-261(姚贺盛,张亚黎,易小平,薛军,罗毅,罗宏海,张旺锋,2015,海岛棉和陆地棉叶片光合特性,冠层结构及物质生产的差异,中国农业科学,48(2):251-261)
Zhang H.,Li R.,Liu L.,Xia Q.Z.,and Zhang M.J.,2011,Study on efficient cotton graftting,Hubei Nongye Kexue(Hubei Agricultural Sciences),50(24):5060-5062(张浩,李锐,刘亮,夏启中,张明菊,2011,棉花高效嫁接技术研究,湖北农业科学,50(24):5060-5062)
Zhang J.L.,2009,Study on identification method of insect resistance of transgenic insect resistant cotton,Thesis for M.S.,Huazhong Agricultural University,Supervisor:Guo X.P.,pp.1-51(张金龙,2009,转亡基因抗虫棉抗虫性鉴定方法研究,硕士学位论文,华中农业大学,导师:郭小平,pp.1-51)
Zhou Y.,Xie H.W.,Liu C.A.,Kuang Q.,Pan J.T.,Chen Q.S.,Xu W.B.,Yu C.,and Peng X.J.,2016,Cloning of lectin Le4gene from leguminosaeand the aphid resistance effect of its protein production,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3474-3480(周英,谢红卫,刘长爱,况琼,潘境涛,陈秋生,徐伟标,余潮,彭晓珏,2016,豆科凝集素基因Le4的克隆及其表达产物对蚜虫的抗性,基因组学与应用生物学,35(12):3474-3480)
Guo S.D.,1995,Advances in plant Bt insect resistance gene engineering,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),28(5):8-13(郭三堆,1995,植物Bt抗虫基因工程研究进展,中国农业科学,28(5):8-13)
Guo S.D.,Wang Y.,Sun G.Q.,Jin S.Q.,Zhou T.,Meng Z.G.,and Zhang R.,2015,Twenty years of research and application of transgenic cotton in China,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),48(17):3372-3387(郭三堆,王远,孙国清,金石桥,周焘,孟志刚,张锐,2015,中国转基因棉花研发应用二十年,中国农业科学,48(17):3372-3387)
He Y.T.,Qu Y.Y.,Kong Q.P.,Yan Y.L.,Shang S.,Zheng L.Y.,and Chen Q.J.,2008,Primary studies on the effects of callus induction and differentiation in Gossypium barbadense,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),6(3):597-602(贺雅婷,曲延英,孔庆平,严勇亮,尚爽,郑良玉,陈全家2008,海岛棉愈伤组织诱导及分化的影响因素初探,分子植物育种,6(3):597-602)
Kong Q.P.,2002,Production situation and comparative advantage analysis of island cotton in China,Zhongguo Mianhua(China Cotton),29(12):19-22(孔庆平,2002,我国海岛棉生产概况及比较优势分析,中国棉花,29(12):19-22)
Li R.H.,Xia Y.S.,Liu S.Z.,Sun L.L.,Guo P.G.,Miao S.Y.,and Chen J.H.,2009,CTAB-improved method of DNA extractionin plant,Shiyanshi Yanjiu Yu Tansuo(Laboratory Research and Exploration),28(9):14-16(李荣华,夏岩石,刘顺枝,孙莉丽,郭培国,缪绅裕,陈健辉,2009,改进的CTAB提取植物DNA方法,实验室研究与探索,28(9):14-16)
Sakhanokho H.F.,Zipf A.,Rajasekaran K.,Saha S.,and Sharma G.C.,2001,Induction of highly embryogenic calli and plant regeneration in upland(Gossypium hirsutum L.)and pima(Gossypium barbadense L.)cottons,Cell Biology&Molecular Genetics,41(4):1235-1240
Su Y.M.,2015,Research progress of transgenic breeding of Xinjiang island cotton,Nanfang Nongye(South China Agriculture),9(24):252-256(苏豫梅,2015,新疆海岛棉转基因育种的研究进展,南方农业,9(24):252-256)
Wei L.M.,1995,Study on callus induction and differentiation of sea island cotton,Shengwu Jishu(Biotechnology),5(5):26-29(魏良民,1995,海岛棉愈伤组织诱导和分化研究,生物技术,5(5):26-29)
Wei Y.H.,Ma L.L.,He L.L.,Chai M.L.,Zhu H.G.,Sun J.,and Zhang W.,2013,Agrobacterium infecting flower stigma for sea-island cotton(Gossypium barbadense L.)genetic transformation,Xinjiang Nongye Kexue(Xinjiang Agricultural Sciences),50(1):15-21(魏延宏,马玲玲,何兰兰,柴蒙亮,朱华国,孙杰,张薇,2013,利用农杆菌侵染花柱头法进行海岛棉遗传转化,新疆农业科学,50(1):15-21)
Wang Y.X.,Wang X.F.,and Ma Z.Y.,2007,A new method of cotton grafting and its application,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),40(2):264-270(王彦霞,王省芬,马峙英,2007,棉花高效嫁接新方法及其应用,中国农业科学,40(2):264-270)
Weng Q.,2007,Establishment of stem-tip regeneration system of island cotton and Agrobacterium-mediated transformation of insect-resistant genes,Thesis for M.S.,Xinjiang Agricultural University,Supervisor:Qu Y.Y.,pp.1-68(翁琴,2007,海岛棉茎尖再生体系的建立及农杆菌介导抗虫基因转化研究,硕士学位论文,新疆农业大学,导师:曲延英,pp.1-68)
Yao H.S.,Zhang Y.L.,Yi X.P.,Xue J.,Luo Y.,Luo H.H.,and Zhang W.F.,2015,Study on differences in comparative canopy structure characteristics and photosynthetic carbon assimilation of field-grown pima cotton(Gossypium barbadense)and upland cotton(G.hirsutum),Zhongguo Nongye Kexue(Scientia Agricultura Sinica),48(2):251-261(姚贺盛,张亚黎,易小平,薛军,罗毅,罗宏海,张旺锋,2015,海岛棉和陆地棉叶片光合特性,冠层结构及物质生产的差异,中国农业科学,48(2):251-261)
Zhang H.,Li R.,Liu L.,Xia Q.Z.,and Zhang M.J.,2011,Study on efficient cotton graftting,Hubei Nongye Kexue(Hubei Agricultural Sciences),50(24):5060-5062(张浩,李锐,刘亮,夏启中,张明菊,2011,棉花高效嫁接技术研究,湖北农业科学,50(24):5060-5062)
Zhang J.L.,2009,Study on identification method of insect resistance of transgenic insect resistant cotton,Thesis for M.S.,Huazhong Agricultural University,Supervisor:Guo X.P.,pp.1-51(张金龙,2009,转亡基因抗虫棉抗虫性鉴定方法研究,硕士学位论文,华中农业大学,导师:郭小平,pp.1-51)
Zhou Y.,Xie H.W.,Liu C.A.,Kuang Q.,Pan J.T.,Chen Q.S.,Xu W.B.,Yu C.,and Peng X.J.,2016,Cloning of lectin Le4gene from leguminosaeand the aphid resistance effect of its protein production,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3474-3480(周英,谢红卫,刘长爱,况琼,潘境涛,陈秋生,徐伟标,余潮,彭晓珏,2016,豆科凝集素基因Le4的克隆及其表达产物对蚜虫的抗性,基因组学与应用生物学,35(12):3474-3480)