农杆菌介导转Bt cry1Ah基因抗虫玉米的研究
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摘要
目前在植物基因工程中常常需要转化来源于微生物的基因。这种原核生物的基因的编码区序列使用的密码子多为植物使用频率很低的稀有密码子,这就可能导致基因在植物中的表达量很低。而通过密码子优化可以显著地提高外源基因在植物中的表达。
Bt cry1Ah基因是本实验室从苏云金芽胞杆菌(Bacillus thuringiensis,Bt)菌株BT8中克隆的一种新型杀虫蛋白基因,其编码蛋白对鳞翅目害虫的杀虫活性强于目前使用的cry1A类基因,在实验室前期工作中已根据植物密码子偏好性对cry1Ah基因进行过一次优化。
本研究首先利用一次优化的cry1Ah基因构建植物表达载体pAhmG,该载体以2mG2-epsps为筛选标记基因,以草甘膦异丙胺盐作为筛选剂。利用农杆菌介导法转化玉米自交系Z31幼胚,得到40株T0代转化植株,其中14株经分子检测为阳性植株,对T1代植株进行了田间生物活性检测。通过PCR、RT-PCR、Western blot、ELISA等分子检测结果表明cry1Ah基因可以在玉米中表达,并稳定遗传。但是研究中发现得到的转基因事件Cry1Ah蛋白的表达量比较低(最高仅为0.008%)并且在玉米中有降解的现象,这会影响到抗虫效果。
随后,对cry1Ah基因根据玉米基因密码子偏好性进行了二次改造,优化后的cry1Ah基因与一次改造的基因序列相似性为75.95%。构建了两个分别含有crym1Ah单基因(pmAhb)、及crym1Ah/1Ie双基因(pmAhIeb)的植物表达载体,以草胺膦乙酰转移酶基因(phosphinothricin acetyltransferase,bar)为筛选标记基因。利用农杆菌介导法转化玉米自交系Z31幼胚,结果得到33株T0代crym1Ah基因转化植株,其中14株经PCR及RT-PCR检测为阳性植株,并对T0代再生植株进行Western blot和ELISA检测。检测结果显示Cry1Ah蛋白在玉米中正确的表达,最高达到0.045%;表达量较pAhmG转基因植株提高5-10倍左右。
本研究通过密码子优化方法提高cry1Ah基因的表达,并获得了对玉米螟有抗性的转基因植株,对今后外源基因的高效利用、抗虫转基因玉米的研发奠定了基础。
At present some transgenes for plant genetic engineering were from microbiology. The codon of microbiology genes′coding sequence was mostly plant rare codons, which could lead to genes’expression very low in plants. Codon optimization could significantly improve foreign genes’expression in plants.
Bt cry1Ah gene was a novel insecticidal protein gene cloned from a Bacillus thuringiensis isolate BT8 in China, and its encoding product′s insecticidal activity to lepidopterous pests was higher than the currently used cry1Ac or cry1Ab. Previous work had done that the cry1Ah gene was modified according to the plant codon bias in our laboratory.
In this study, the first modification of cry1Ah gene and glyphosate-toleranct 2mG2-epsps gene were used to construct an insect-resistant/glyphosate-tolerance bivalent plant expression vector pAhmG, glyphosate isopropylamine salt as a screening agent, 2mG2-epsps gene as a selectable maker gene, the vector was transfer into maize inbred Z31 immature zygotic embryos by Agrobacterium-mediated. Forty transformed plants of T0 generation were obtained, fourteen of which were positive events through molecular detection. To further analyze the T1 generation plants, the transgenic plants′molecular detection and insect-resistant bioassay were done. The transgenic plants′molecular detection including PCR、RT-PCR、Western blot、ELISA revealed that Bt cry1Ah gene was expressed and inherited to next generation stably. But in this study, the expression of Cry1Ah protein in transgenic maize was found very low (the highest is 0.008%) and degradation that reduced the effect of insect resistance.
Then, according to the maize coden bias, the cry1Ah gene was modified again. The second modified cry1Ah gene′s nucleotide similarity to the first modified cry1Ah gene was 75.95%. The two plant expression vectors pmAhb and pmAhIeb were constructed, bar gene as a selectable maker gene. The vectors were transfered into maize inbred Z31 immature zygotic embryos by Agrobacterium-mediated transformation, obtaining thirty-three transformed plants of T0 generation. Through PCR and RT-PCR detections, fourteen positive transgenic events were obtained. Western blot and ELISA detections were done. The Cry1Ah protein was correctly expressed in transgenic maize and the highest expression was up to 0.045% of maize soluable proteins, which were 5-10 times higher than the expression in transgenic plants harboring pAhmG vector.
In this study, the expression of cry1Ah gene was improved through codon optimization, and the transgenic maizes with Asian corn borer resistance were obtained. It laid the foundation for the efficient use of foreign genes and the development of insect-resistant transgenic maize in the future.
Bt cry1Ah基因是本实验室从苏云金芽胞杆菌(Bacillus thuringiensis,Bt)菌株BT8中克隆的一种新型杀虫蛋白基因,其编码蛋白对鳞翅目害虫的杀虫活性强于目前使用的cry1A类基因,在实验室前期工作中已根据植物密码子偏好性对cry1Ah基因进行过一次优化。
本研究首先利用一次优化的cry1Ah基因构建植物表达载体pAhmG,该载体以2mG2-epsps为筛选标记基因,以草甘膦异丙胺盐作为筛选剂。利用农杆菌介导法转化玉米自交系Z31幼胚,得到40株T0代转化植株,其中14株经分子检测为阳性植株,对T1代植株进行了田间生物活性检测。通过PCR、RT-PCR、Western blot、ELISA等分子检测结果表明cry1Ah基因可以在玉米中表达,并稳定遗传。但是研究中发现得到的转基因事件Cry1Ah蛋白的表达量比较低(最高仅为0.008%)并且在玉米中有降解的现象,这会影响到抗虫效果。
随后,对cry1Ah基因根据玉米基因密码子偏好性进行了二次改造,优化后的cry1Ah基因与一次改造的基因序列相似性为75.95%。构建了两个分别含有crym1Ah单基因(pmAhb)、及crym1Ah/1Ie双基因(pmAhIeb)的植物表达载体,以草胺膦乙酰转移酶基因(phosphinothricin acetyltransferase,bar)为筛选标记基因。利用农杆菌介导法转化玉米自交系Z31幼胚,结果得到33株T0代crym1Ah基因转化植株,其中14株经PCR及RT-PCR检测为阳性植株,并对T0代再生植株进行Western blot和ELISA检测。检测结果显示Cry1Ah蛋白在玉米中正确的表达,最高达到0.045%;表达量较pAhmG转基因植株提高5-10倍左右。
本研究通过密码子优化方法提高cry1Ah基因的表达,并获得了对玉米螟有抗性的转基因植株,对今后外源基因的高效利用、抗虫转基因玉米的研发奠定了基础。
At present some transgenes for plant genetic engineering were from microbiology. The codon of microbiology genes′coding sequence was mostly plant rare codons, which could lead to genes’expression very low in plants. Codon optimization could significantly improve foreign genes’expression in plants.
Bt cry1Ah gene was a novel insecticidal protein gene cloned from a Bacillus thuringiensis isolate BT8 in China, and its encoding product′s insecticidal activity to lepidopterous pests was higher than the currently used cry1Ac or cry1Ab. Previous work had done that the cry1Ah gene was modified according to the plant codon bias in our laboratory.
In this study, the first modification of cry1Ah gene and glyphosate-toleranct 2mG2-epsps gene were used to construct an insect-resistant/glyphosate-tolerance bivalent plant expression vector pAhmG, glyphosate isopropylamine salt as a screening agent, 2mG2-epsps gene as a selectable maker gene, the vector was transfer into maize inbred Z31 immature zygotic embryos by Agrobacterium-mediated. Forty transformed plants of T0 generation were obtained, fourteen of which were positive events through molecular detection. To further analyze the T1 generation plants, the transgenic plants′molecular detection and insect-resistant bioassay were done. The transgenic plants′molecular detection including PCR、RT-PCR、Western blot、ELISA revealed that Bt cry1Ah gene was expressed and inherited to next generation stably. But in this study, the expression of Cry1Ah protein in transgenic maize was found very low (the highest is 0.008%) and degradation that reduced the effect of insect resistance.
Then, according to the maize coden bias, the cry1Ah gene was modified again. The second modified cry1Ah gene′s nucleotide similarity to the first modified cry1Ah gene was 75.95%. The two plant expression vectors pmAhb and pmAhIeb were constructed, bar gene as a selectable maker gene. The vectors were transfered into maize inbred Z31 immature zygotic embryos by Agrobacterium-mediated transformation, obtaining thirty-three transformed plants of T0 generation. Through PCR and RT-PCR detections, fourteen positive transgenic events were obtained. Western blot and ELISA detections were done. The Cry1Ah protein was correctly expressed in transgenic maize and the highest expression was up to 0.045% of maize soluable proteins, which were 5-10 times higher than the expression in transgenic plants harboring pAhmG vector.
In this study, the expression of cry1Ah gene was improved through codon optimization, and the transgenic maizes with Asian corn borer resistance were obtained. It laid the foundation for the efficient use of foreign genes and the development of insect-resistant transgenic maize in the future.
引文
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练云,贾志伟,何康来等. 2008.人工改造的Cry1Ac和Cry1Ie基因在烟草中共表达对棉铃虫有更好的杀虫活性.科学通报. 53 (6):658-663
梁雪莲,郭平毅,孙毅等. 2005.玉米3种非组培转基因方法转化外源bar基因研究.作物学报. 31 (12):1648-1653
刘桂玲,陈举林,李平海. 2004.转基因玉米的研究进展与展望.农业生物技术科学. 20 (4):36-38
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欧阳立明,吴宏文,喻子牛等. 2001.害虫对转Bt基因植物抗性的治理策略.植物保护学报. 28 (2):183-188
渠柏艳,于海清,韩兆雪等. 2004.可去除选择标记的转Bt基因抗虫玉米研究.分子植物育种. 2 (5):649-653
沈世华,张秀君,郭奕明等. 2001.玉米基因转化的离体子房注射及其转基因植株的鉴定. 植物学报. 43 (10):1055-1057
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伍晓丽,朱祯,李晚忱等. 2004.农杆菌介导豇豆胰蛋白酶抑制剂基因(cpti)在玉米中的遗传转化.作物学报. 30 (3):297-298
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朱常香,宋云枝,张杰道等. 2002.抗虫、抗除草剂转基因玉米的获得及遗传研究.山东农业大学学报(自然科学版). 33 (2):120-125
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黄璐,卫志明. 1999.农杆菌介导的玉米遗传转化.实验生物学报. 32 (4):381-387
李葱葱,刘娜,康岭生等. 2006.转基因抗虫玉米BT蛋白表达量的研究.玉米科学. 14 (31): 40-41
李慧芬,刘翔,曲强等. 2002.转抗虫融合基因(cry1Ac3-cpti)玉米(Zea mays L.)植株的获得及其抗虫性分析.自然科学进展. 12 (1):37-40
练云,贾志伟,何康来等. 2008.人工改造的Cry1Ac和Cry1Ie基因在烟草中共表达对棉铃虫有更好的杀虫活性.科学通报. 53 (6):658-663
梁雪莲,郭平毅,孙毅等. 2005.玉米3种非组培转基因方法转化外源bar基因研究.作物学报. 31 (12):1648-1653
刘桂玲,陈举林,李平海. 2004.转基因玉米的研究进展与展望.农业生物技术科学. 20 (4):36-38
刘谦,朱鑫全. 2001.生物安全.科学出版社. 25-27
刘源霞,兰进好,杜景平等. 2007.山东农业科学. 6:21-24
欧阳立明,吴宏文,喻子牛等. 2001.害虫对转Bt基因植物抗性的治理策略.植物保护学报. 28 (2):183-188
渠柏艳,于海清,韩兆雪等. 2004.可去除选择标记的转Bt基因抗虫玉米研究.分子植物育种. 2 (5):649-653
沈世华,张秀君,郭奕明等. 2001.玉米基因转化的离体子房注射及其转基因植株的鉴定. 植物学报. 43 (10):1055-1057
王关林,方宏筠. 2002.植物基因工程.第二版.科学出版社
王国英,杜天冰,张宏等. 1995.用基因枪将Bt毒蛋白基因转入玉米及转基因植株再生.中国科学(B辑). 25 (1):71-76
王振营,鲁新,何康来等. 2000.我国研究亚洲玉米螟历史、现状与展望.沈阳农业大学学报. 31 (5):402-412
伍晓丽,朱祯,李晚忱等. 2004.农杆菌介导豇豆胰蛋白酶抑制剂基因(cpti)在玉米中的遗传转化.作物学报. 30 (3):297-298
岳同卿. 2009.转Bt cry1Ah基因抗虫玉米的研究.中国农业科学院.博士学位论文
张荣,王国英,张晓红等. 2001.根癌农杆菌介导的玉米遗传转化体系的建立.农业生物技术学报. 9 (1):45-48
朱常香,宋云枝,张杰道等. 2002.抗虫、抗除草剂转基因玉米的获得及遗传研究.山东农业大学学报(自然科学版). 33 (2):120-125
Cambell C.H., Gowri G., 1990. Codon usage in higher plants, green algae, and cyanobacteria. Plant Physiol. 92: 1–11
Chu C.C., Wang C.C., Sun C.S., et al., 1975. Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen source. Scientia Sinica. 18: 659-668
Dean C., Tamaki S., Dunsmuir P., et al., 1986. mRNA transcripts of several plant genes are polyadenylated at multiple sites in vivo. Nucleic Acids Research. 14: 2229–2240
Denolf P., Hendrickx K., Van D. J., et al. 1997. Cloning and characterization of Manduca sexta and Plutella xylostella midgut aminopeptidase N enzymes related to Bacillus thuringiensis toxin-binding proteins. European Journal of Biochemistry. 248(3):748-761
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