抗虫基因cry1C~*对早粳稻的转化

【作者】 王薇；

【导师】 李荣田；

【作者基本信息】 黑龙江大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 为了改良黑龙江水稻抗二化螟的遗传特性,采用农杆菌介导外源基因遗传转化的方法,以bar基因为筛选标记,将在ubi启动子调控下、经过密码子优化及DNA序列改造的、人工合成的cry1C^*基因,转入寒区优良水稻品种空育131核基因组中,获得了一批遗传转化苗。在实验过程中,对农杆菌菌株EHA105B（来自中国科学院遗传与发育生物学研究所植物发育研究室的农杆菌菌株EHA105）和EHA105N（来自东北林业大学教育部林木遗传育种与生物技术重点实验室的农杆菌菌株EHA105）的活力进行了比较,还对遗传转化水稻再生苗炼苗方法进行了优化。主要研究结果如下:1.对水稻遗传转化载体pBar-cry1C^*进行酶切和PCR检测验证表明,载体和目的基因均准确无误,将携带目的基因cry1C^*基因的质粒pBar-cry1C^*电转化导入农杆菌,获得了农杆菌转化子EHA105（pBar-cry1C^*）。2.根瘤农杆菌菌株EHA105B的活力比EHA105N强,EHA105B不能用于寒区水稻遗传转化工作,活力较弱的农杆菌菌株EHA105N可以介导外源基因转化北方粳稻。3.黑龙江地区严冬季节产生的遗传转化再生水稻苗采用优化法炼苗,可以确保转基因再生水稻苗的成活率。优化法炼苗在组织培养室炼苗后,将转化苗置于水稻专用营养液中,在光照培养箱中25℃高湿条件下进行无土栽培3-4d,然后再移栽到土壤。4.利用农杆菌介导的遗传转化方法,转化水稻空育131胚性愈伤1800块,共获来自20块独立抗性愈伤的分化再生苗123株,最终成活115株,成活率达到了93.5%。5.对水稻空育131（cry1C^*）T₀代进行PCR分析,123株转化植株中阳性株120株,阳性率97.6%。cry1C^*基因已经整合到黑龙江水稻核基因组中。更多还原

【Abstract】 Stemborers and leaffolders were two groups of Lepidopteran pests that caused severe damage to rice in many areas of the world. Kongyu131, being the most popular rice variety of Heilongjiang province, was highly susceptible to Lepidopteran insects, which greatly reduced the expected yield of Kongyu131. In this study, cry1C^* gene encoding Bacillus thuringiensis（Bt） d-endotoxin was synthesized based on codon optimization as one step toward gene stacking in the resistance management strategy of transgenic rice. The gene was transformed into Kongyu131 （Oryza sativa L.） via Agrobacterium-mediated method. The integration of the cry1C^* gene in rice genome was detected with PCR analyses. Meanwhile, the activity of EHA105B was compared to EHA105N and the optimized hardening method of regenerated plantlets in winter was improved in Heilongjiang province. The main results were as follows:1. The vector pBar-cry1C^* was confirmed in E.coli DH5αwith restriction enzyme digestion and PCR detection.2. The activity of the stain EHA105B of Agrobacterium tumefaciens was more powerful than the strain EHA105N and the EHA105N was suitable for the Agrobacterium-mediated transformation.3. The optimized hardening method of regenerated plantlets elevated the living rate of transgenic plants in winter in Heilongjiang province.4. 123 plants of the rice Kongyu131 transformed with cry1C^* gene from 20 independent resistant calli were regenerated and 115 living transgenic plants were obtained after optimized hardening.5. PCR detection indicated that the cry1C^* gene was integrated into the genome of 120 transgenic plants of T₀ generation of the rice variety Kongyu131.更多还原