昆虫抗冻蛋白转基因植物表达载体的构建及转基因棉花T1代的检测

摘要

[目的]为了提高转基因质粒的表达效果,基于pCAMBIA2300植物表达载体,改造并构建含有小胸鳖甲抗冻蛋白基因Mpafp149、双CaMV35S启动子、Ω增强子和NOS终止子的高效植物表达载体pCN2300 -Mpafp149.[方法]将抗冻蛋白基因采用不同的注射方法对新疆北疆棉区主栽棉花品种进行了花粉管通道法遗传转化.对获得的转基因棉花T1代植株进行PCR及RT-PCR检测.[结果]对Mpafp149、卡那霉素抗性基因NPTⅡ及CaMV35S进行PCR检测,3个结果的阳性一致率达到了80％.此外,通过磨短进样器针头的长度可大幅提高成铃率,直接注射新陆早42号的成铃率达到了37.59％.[结论]小胸鳖甲抗冻基因Mpafp149已转入T1棉花.通过使用改造的进样器注射可大幅提高成铃率.%[Objective and Method ] To improve the expression effect of the plasmid for plant transformation, a high efficient plant expression vector pCN2300 - Mpafpl49 harboring Mpajpl49 ( an antifreeze protein gene from Microdera punctipennis), two CaMV35S promoters, O enhancer and NOS terminator were constructed. Several cotton varieties cultivated in Xinjiang were transformed with this plasmid through different injection methods with shortened micro - injector. [ Result ] To exclude false PCR result, three PCR detections with specific primers for Mpajp49 ,kanamycin resistance gene NPTII and the CaMV 35S promoter fragment were conducted, respectively. These PCR results show that the positive consistency rate of these three genes reached 80% in T, cotton. Besides, the original length of the needle of the micro - injector is 35.4 mm which is too long to be manipulated properly, so we cut it to the length of 3. 5 mm. The results showed that this change greatly increased boll retention rate of To transgenic cotton. Boll retention rate in Transformed Xinluzao 42 reached 37.59% when cottons were directly injected after removal of chapiter. This boll retention rate was much higher compared to those without micro - injector modification. [ Conclusion ] Mpqfpl49 gene has been transformed into T, cotton plant. RT - PCR detection confirmed this result, though only one transgenic plant showed positive result. Shortened micro - injector can greatly increased boll retention rate of To transgenic cotton.