转抗虫基因Cry1Iem大豆的分子鉴定与功能验证
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摘要
通过转抗虫基因来提高作物的抗虫性是一条有效途径,以此来提高作物的产量和品质。本研究以转抗虫基因Cry1Iem大豆的T2、T3株系作为试验材料,利用常规PCR检测、Southern杂交、荧光定量PCR技术对转抗虫基因Cry1Iem株系进行分子鉴定;同时对40个转基因阳性株系进行室内人工接虫、200个转基因阳性株系室外网室接虫并进行抗虫性鉴定。选取其中10个株系进行常规PCR检测,结果在T2、T3当中都检测到了Cry1Iem、Bar、启动子CaMV35S、Nos目标片段,表明目的基因已被顺利导入受体JN28大豆植株当中并得到了稳定遗传;选取其中5个T2、T3转基因株系进行Southern杂交,结果显示:目的基因以单拷贝的形式整合到了大豆基因组当中;荧光定量PCR检测结果表明,Cry1Iem基因在选取的3个转基因株系中均得到了表达,而在非转化的受体JN28当中未检测到荧光信号。室内抗虫性鉴定结果显示40个转Cry1Iem基因株系豆荚内活虫的成活数量明显低于受体材料JN28大豆豆荚内的活虫数量,表明转基因材料具有显著的抗虫性。室外抗虫性鉴定结果表明:JN28大豆受体植株的虫食率为6.72%,属于感虫品种;而200个转Cry1Iem基因株系的平均虫食率为3.81%,属于抗虫品系。本研究结果为中国转基因抗虫大豆新品种的选育提供部分参考数据。
It is an effective way to improve the insect resistance of crops by transferring insect resistant genes, so to improve crop yield and quality. In this study, T2 and T3 strains of transgenic Cry1 Iem soybean were used as experimental materials, and the molecular identification of transgenic Cry1 Iem strains was carried out by conventional PCR detection, Southern blot and fluorescence quantitative PCR. At the same time, 40 transgenic positive strains were used for indoor artificial inoculation, 200 transgenic positive strains for outdoor inoculation,and the insect resistance was identified. Routine PCR detection was carried out on 10 strains, and the results showed that Cry1 Iem, Bar, promoter CaMV35 S, and terminator Nos target fragment were all detected in T2 and T3,indicating that the target genes had been successfully transferred into receptor JN28 soybean plant and stably inherited. Five T2 and T3 transgenic strains were selected for Southern blot, and the results showed that the target gene was integrated into the soybean genome by single copy. The results of fluorescence quantitative PCR demonstrated that the Cry1 Iem gene was expressed in the three transgenic lines selected, but the fluorescent signal was not detected in the non-transformed receptor JN28. Identification of indoor insect resistance showed that the number of living insects in the pod of 40 transgenic Cry1 Iem strains was significantly less than that of the receptor material JN28, indicating that the transgenic materials had remarkable anti-insect resistance. Identification of outdoor insect resistance showed that the moth-eaten ratio of the JN28 receptor plant was 6.72%, which belonged to susceptible variety. While the average moth-eaten ratio of 200 transgenic Cry1 Iem strains was 3.81%, which belonged to insect-resistant variety. The result of this study could provide some reference data for the breeding of new varieties of transgenic insect-resistant soybean in China.
It is an effective way to improve the insect resistance of crops by transferring insect resistant genes, so to improve crop yield and quality. In this study, T2 and T3 strains of transgenic Cry1 Iem soybean were used as experimental materials, and the molecular identification of transgenic Cry1 Iem strains was carried out by conventional PCR detection, Southern blot and fluorescence quantitative PCR. At the same time, 40 transgenic positive strains were used for indoor artificial inoculation, 200 transgenic positive strains for outdoor inoculation,and the insect resistance was identified. Routine PCR detection was carried out on 10 strains, and the results showed that Cry1 Iem, Bar, promoter CaMV35 S, and terminator Nos target fragment were all detected in T2 and T3,indicating that the target genes had been successfully transferred into receptor JN28 soybean plant and stably inherited. Five T2 and T3 transgenic strains were selected for Southern blot, and the results showed that the target gene was integrated into the soybean genome by single copy. The results of fluorescence quantitative PCR demonstrated that the Cry1 Iem gene was expressed in the three transgenic lines selected, but the fluorescent signal was not detected in the non-transformed receptor JN28. Identification of indoor insect resistance showed that the number of living insects in the pod of 40 transgenic Cry1 Iem strains was significantly less than that of the receptor material JN28, indicating that the transgenic materials had remarkable anti-insect resistance. Identification of outdoor insect resistance showed that the moth-eaten ratio of the JN28 receptor plant was 6.72%, which belonged to susceptible variety. While the average moth-eaten ratio of 200 transgenic Cry1 Iem strains was 3.81%, which belonged to insect-resistant variety. The result of this study could provide some reference data for the breeding of new varieties of transgenic insect-resistant soybean in China.
引文
Chen X.H.,Bai X.,Pan X.,Zhai H.,Ji W.,Li Y.,and Zhu Y.M.,2009,Cultivation of cry1Iem gene transformed soybean and insect resistant assay,Dadou Kexue(Soybean Science),28(6):959-963(陈秀华,柏锡,潘欣,翟红,才华,纪巍,李勇,朱延明,2009,转cry1Iem基因大豆的培育及抗虫性检测,大豆科学,28(6):959-963)
Endo H.,Adegawa S.,Kikuta S.,and Sato R.,2018,The intracellular region of silkworm cadherin-like protein is not necessary to mediate the toxicity of Bacillus thuringiensis Cry1Aa and Cry1Ab toxins,Insect Biochem.Mol.Biol.,94:36-41
Ferrandis M.D.,Juárez-Pérez V.M.,Frutos R.,Bel Y.,and Ferre J.,1999,Distribution of cryl,cryll and cryV genes within Bacillus thuringiensis isolates from spain,Syst.Appl.Microbiol.,22(2):179-185
Gao S.,2016,Identification on insect-resistant gene of cry1Ab13transformation into Glycine max L.Merr,Thesis for M.S.,Jilin Agricultural University,Supervisor:Wang P.W.,pp.20-26(高嵩,2016,抗虫基因cry1Ab13在大豆中的遗传转化及抗虫性鉴定,硕士学位论文,吉林农业大学,导师:王丕武,pp.20-26)
Guo S.D.,1995,Engineering of insect-resistant plant with Bacillus thuringiensis crystal protein genes,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)
Han D.,2016,Transformation and functional analysis of soybean SCTF-1 gene encoding a C2H2-typezinc finger protein,Thesis for M.S.,Jilin Agricultural University,Supervisor:Wang P.W.,pp.8-18(韩丹,2016,大豆C2H2型锌指蛋白基因SCTF-1转化大豆及功能分析,硕士学位论文,吉林农业大学,导师:王丕武,pp.8-18)
He H.,2016,Genetic transformation of soybean with insect-resistant gene cry1Iem,Thesis for M.S.,Jilin Agricultural University,Supervisor:Wang P.W.,pp.18-22(何欢,2016,抗虫基因Cry1Iem转化大豆的研究,硕士学位论文,吉林农业大学,导师:王丕武,pp.18-22)
Hollensteiner J.,Poehlein A.,Sproer C.,Bunk B.,Sheppard A.E.Rosentstiel P.,Schulenburg H.,and Liesegang H.,2017Complete genome sequence of the nematicidal Bacillus thuringiensis MYBT18247,J.Biotechnol.,260(3):48-52
Hou B.K.,and Chen Z.H.,2000,Progress of anti-insect genetic engineering of plants,Zhiwuxue Tongbao(Chinese Bulletin of Botany),17(5):385-393(侯丙凯,陈正华,2000,植物抗虫基因工程研究进展,植物学通报,17(5):385-393)
Hu L.Z.,Guo J.,Qiao L.,Wang H.X.,Hou X.C.,and Zhu Z.X.2016,Identification,expression and evolution analysis of the PDAT genes in soybean(Glycine Max),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(3):677-686(胡利宗,郭婕,乔琳,王红星,侯湘婵,朱自学,2016,大豆PDAT基因的鉴定、表达与进化分析,基因组学与应用生物学,36(3):677-686)
Jia B.H.,Li N.,Yu X.S.,Li H.Z.,Li Y.,Liu Y.T.,Meng F.L.,and Li W.B.,2015,Optimization of soybean embryonic tip regeneration system and transferring cry1C*gene into soybean,Dadou Kexue(Soybean Science),34(5):910-917(贾宝辉,李娜,于希森,李涵哲,李洋,刘玉婷,孟凡立,李文滨,2015,大豆胚尖转化体系优化和cry1C*基因转化大豆的研究,大豆科学,34(5):910-917)
Lan B.X.,Wang L.,Wu Z.K.,and Jiang B.L.,2015,Rapid miniprep extraction of genomic DNA from micro-endosperm maize with modified CTAB method,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):190-194(蓝碧秀,王凛,吴子恺,姜伯乐,2015利用改良CTAB法快速小量提取微胚乳玉米基因组DNA,基因组学与应用生物学,34(1):190-194)
Li H.P.,Li Y.J.,Zhang W.,Xiang P.,and Duan Y.X.,2015,The research of soybean pod borer resistance about foreign and domestic precocious soybean,Dadou Kexue(Soybean Science),34(2):281-288(李红鹏,李艳杰,张武,项鹏,段玉玺,2015,中外早熟大豆对大豆食心虫的抗性研究,大豆科学,34(2):281-288)
Li T.T.,Han L.L.,Zhao K.J.,Chen X.H.,and Zhu Y.M.,2011,Application of disk segmentation method in insect resistance identification of transgenic soybean,Zuowu Zazhi(Crops),4(8):20-22(李婷婷,韩岚岚,赵奎军,陈秀华,朱延明,2011,圆盘分割法在转基因大豆抗虫鉴定上的应用,作物杂志,4(8):20-22)
Liu Y.,2004,Studies on the properties of podborer[Leguminivora glycinivorella(Mats.)]-resistance of soybean and the genetic rule of podborer-resistance,Thesis for M.S.,Northeast Agricultural University,Supervisor:Zhao K.J.,and Wang J.A.,pp.15-17(刘洋,2004,大豆抗食心虫性状及抗虫性遗传规律的研究,硕士学位论文,东北农业大学,导师:赵奎军,王继安,pp.15-17)
Liu Y.J.,2004,Expression of the modified cry1Ac、cry1Ie genes in E.coli,transgenic tobacco plants and transgenic maize plants,Dissertation for Ph.D.,China Agricultural University,Supervisor:Wang G.Y.,pp.73-76(刘允军,2004,人工改造的cry1Ac、cry1Ie基因在大肠杆菌、转基因烟草和玉米中的表达,博士学位论文,中国农业大学,导师:王国英,pp.73-76)
Zhang G.X.,and Ma Z.Q,2003,The technical progress in generating and isolating full-length cDNA,Shengwu Jishu Tongbao(Biotechnology Bulletin),(5):1-5(张光祥,马正强,2003,目的基因全长cDNA合成及克隆技术进展,生物技术通报,(5):1-5)
Zhao X.L.,2004,Methods for controlling soybean moth(Leguminirola glycinioorella),Dadou Kexue(Soybean Science),23(1):77-80(赵晓丽,2004,防治大豆食心虫方法的研究,大豆科学,23(1):77-80)
Endo H.,Adegawa S.,Kikuta S.,and Sato R.,2018,The intracellular region of silkworm cadherin-like protein is not necessary to mediate the toxicity of Bacillus thuringiensis Cry1Aa and Cry1Ab toxins,Insect Biochem.Mol.Biol.,94:36-41
Ferrandis M.D.,Juárez-Pérez V.M.,Frutos R.,Bel Y.,and Ferre J.,1999,Distribution of cryl,cryll and cryV genes within Bacillus thuringiensis isolates from spain,Syst.Appl.Microbiol.,22(2):179-185
Gao S.,2016,Identification on insect-resistant gene of cry1Ab13transformation into Glycine max L.Merr,Thesis for M.S.,Jilin Agricultural University,Supervisor:Wang P.W.,pp.20-26(高嵩,2016,抗虫基因cry1Ab13在大豆中的遗传转化及抗虫性鉴定,硕士学位论文,吉林农业大学,导师:王丕武,pp.20-26)
Guo S.D.,1995,Engineering of insect-resistant plant with Bacillus thuringiensis crystal protein genes,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)
Han D.,2016,Transformation and functional analysis of soybean SCTF-1 gene encoding a C2H2-typezinc finger protein,Thesis for M.S.,Jilin Agricultural University,Supervisor:Wang P.W.,pp.8-18(韩丹,2016,大豆C2H2型锌指蛋白基因SCTF-1转化大豆及功能分析,硕士学位论文,吉林农业大学,导师:王丕武,pp.8-18)
He H.,2016,Genetic transformation of soybean with insect-resistant gene cry1Iem,Thesis for M.S.,Jilin Agricultural University,Supervisor:Wang P.W.,pp.18-22(何欢,2016,抗虫基因Cry1Iem转化大豆的研究,硕士学位论文,吉林农业大学,导师:王丕武,pp.18-22)
Hollensteiner J.,Poehlein A.,Sproer C.,Bunk B.,Sheppard A.E.Rosentstiel P.,Schulenburg H.,and Liesegang H.,2017Complete genome sequence of the nematicidal Bacillus thuringiensis MYBT18247,J.Biotechnol.,260(3):48-52
Hou B.K.,and Chen Z.H.,2000,Progress of anti-insect genetic engineering of plants,Zhiwuxue Tongbao(Chinese Bulletin of Botany),17(5):385-393(侯丙凯,陈正华,2000,植物抗虫基因工程研究进展,植物学通报,17(5):385-393)
Hu L.Z.,Guo J.,Qiao L.,Wang H.X.,Hou X.C.,and Zhu Z.X.2016,Identification,expression and evolution analysis of the PDAT genes in soybean(Glycine Max),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(3):677-686(胡利宗,郭婕,乔琳,王红星,侯湘婵,朱自学,2016,大豆PDAT基因的鉴定、表达与进化分析,基因组学与应用生物学,36(3):677-686)
Jia B.H.,Li N.,Yu X.S.,Li H.Z.,Li Y.,Liu Y.T.,Meng F.L.,and Li W.B.,2015,Optimization of soybean embryonic tip regeneration system and transferring cry1C*gene into soybean,Dadou Kexue(Soybean Science),34(5):910-917(贾宝辉,李娜,于希森,李涵哲,李洋,刘玉婷,孟凡立,李文滨,2015,大豆胚尖转化体系优化和cry1C*基因转化大豆的研究,大豆科学,34(5):910-917)
Lan B.X.,Wang L.,Wu Z.K.,and Jiang B.L.,2015,Rapid miniprep extraction of genomic DNA from micro-endosperm maize with modified CTAB method,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):190-194(蓝碧秀,王凛,吴子恺,姜伯乐,2015利用改良CTAB法快速小量提取微胚乳玉米基因组DNA,基因组学与应用生物学,34(1):190-194)
Li H.P.,Li Y.J.,Zhang W.,Xiang P.,and Duan Y.X.,2015,The research of soybean pod borer resistance about foreign and domestic precocious soybean,Dadou Kexue(Soybean Science),34(2):281-288(李红鹏,李艳杰,张武,项鹏,段玉玺,2015,中外早熟大豆对大豆食心虫的抗性研究,大豆科学,34(2):281-288)
Li T.T.,Han L.L.,Zhao K.J.,Chen X.H.,and Zhu Y.M.,2011,Application of disk segmentation method in insect resistance identification of transgenic soybean,Zuowu Zazhi(Crops),4(8):20-22(李婷婷,韩岚岚,赵奎军,陈秀华,朱延明,2011,圆盘分割法在转基因大豆抗虫鉴定上的应用,作物杂志,4(8):20-22)
Liu Y.,2004,Studies on the properties of podborer[Leguminivora glycinivorella(Mats.)]-resistance of soybean and the genetic rule of podborer-resistance,Thesis for M.S.,Northeast Agricultural University,Supervisor:Zhao K.J.,and Wang J.A.,pp.15-17(刘洋,2004,大豆抗食心虫性状及抗虫性遗传规律的研究,硕士学位论文,东北农业大学,导师:赵奎军,王继安,pp.15-17)
Liu Y.J.,2004,Expression of the modified cry1Ac、cry1Ie genes in E.coli,transgenic tobacco plants and transgenic maize plants,Dissertation for Ph.D.,China Agricultural University,Supervisor:Wang G.Y.,pp.73-76(刘允军,2004,人工改造的cry1Ac、cry1Ie基因在大肠杆菌、转基因烟草和玉米中的表达,博士学位论文,中国农业大学,导师:王国英,pp.73-76)
Zhang G.X.,and Ma Z.Q,2003,The technical progress in generating and isolating full-length cDNA,Shengwu Jishu Tongbao(Biotechnology Bulletin),(5):1-5(张光祥,马正强,2003,目的基因全长cDNA合成及克隆技术进展,生物技术通报,(5):1-5)
Zhao X.L.,2004,Methods for controlling soybean moth(Leguminirola glycinioorella),Dadou Kexue(Soybean Science),23(1):77-80(赵晓丽,2004,防治大豆食心虫方法的研究,大豆科学,23(1):77-80)