【作者】 李圣彦；

【导师】 黄大昉；

【作者基本信息】 东北农业大学 ， 植物学， 2011， 硕士

【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。更多还原

【Abstract】 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.更多还原