根据磷酸烯醇丙酮酸羧化酶(PEPC)基因控制油菜籽中蛋白质与脂肪酸合成的原理，构建了PEPC基因片段ihpRNA干扰表达载体，该载体包含了一个PEPC基因片段正反向序列的回文结构．表达RNA干扰的载体结构（ihpRNA），在大肠杆菌体内进行克隆的过程中易被大肠杆菌自身携带的某种酶剪切，剪切位点不确定,可能是一种随机性的剪切．通过反复实验，最终获得了一个与我们设计一致的PEPC基因片段正反向序列结构， RNA干扰表达载体构建获得成功．利用花序浸渍法(floral-dip)转基因将构建的PEPC基因ihpRNA干扰表达载体转移到甘蓝型油菜中，并通过抗性筛选、PCR特异扩增、PCR-Southern杂交和克隆测序，对获得的转基因植株进行了鉴定，转化率达到了069%，说明floral-dip方法有效地实现了ihpRNA干扰表达载体的遗传转化
Abstract
According to the principle that phosphoenolpyruvate carboxylase gene（PEPC）controls rapeseed protein and fatty acid synthesis, we constructed the PEPCgene fragment interference expression vector ihpRNA that contained the palindrome structure of a forward and reverse PEPC gene fragment sequence. The vector structure which expresses RNA interference function (ihpRNA), in the process of cloning in E. coli was easy to cut by restriction enzymes, and splice site is random. Through repeated experiments, we finally got a vector structure which was consistent with our expectations that the forward and reverse sequence of PEPC gene. The construction of interference expression vector ihpRNA was successfully accomplished. The expression vector of PEPCgene was transferred into Brassica napus.L by the transformation method of floral-dip, and then through the resistance screening, PCR-specific amplification, PCR-Southern blot and cloning and sequencing of the obtained transgenic plants were confirmed, and the transformation rate reached 0.69 %. The data will serve to illustrate that the floral-dip method could effectively transform the genetic expression vector ihpRNA.
关键词
花序浸渍法(floral-dip) /
磷酸烯醇丙酮酸羧化酶基因 /
ihpRNA表达载体 /
甘蓝型油菜 /
遗传转化
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Key words
floral-dip method /
phosphoenolpyruvate carboxylase gene（PEPC） /
ihpRNA expression vector /
Brassica napus.L /
genetic transformation
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中图分类号：
S565.4
Q789
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