油菜基因BnWRI1的克隆及RNAi对种子含油量的影响

Cloning of BnWRI1 Gene and the Effect of RNA Interference on Seed Oil Content in Oilseed Rape

【目的】阐明油菜基因BnWRI1对其种子含油量的影响。【方法】采用RT-PCR技术从油菜中克隆到BnWRI1基因编码区内384bp的片段,将该片段正反向插入干扰载体pGΩ4ARi中,构建成RNAi表达载体pGΩ4ARi-WRI1,通过花粉管通道法将其转入油菜受体品种中双9号中,【结果】获得3棵转基因植株,PCR扩增和基因组Southern杂交证实外源片段已整合在受体油菜基因组中。利用半定量RT-PCR和改进的索氏提取法分别检测转基因T1代植株的BnWRI1基因表达量和种子含油量,结果表明:与非转基因对照相比,转基因植株的BnWRI1基因均受抑制,种子含油量有所降低。【结论】采用RNAi技术可使油菜内源基因BnWRI1沉默,表明油菜基因BnWRI1能有效调控种子含油量。

[Objective] The study was to determine the effect ofBnWRll gene on oil content of oilseed rape seed. [ Method] RT-PCR was employed to isolate a 384 bp fragment from the coding region of BnWRI1, which was then inserted into the RNAi vector of pGΩ4ARi in both forward and reverse directions to form a plant expression vector pGΩ4ARi-WRI1. The vector was introduced into oilseed rape cv. Zhongshuang 9 via pollen-tube pathway method. [ Result] Three transgenic plants were confirmed by PCR and Southern Blot analysis that the foreign fragment integrated into the plant genomes. BnWRI1 transcript levels and seed oil contents were detected by semi-quantitative RT-PCR and improved Soxhlet extraction, respectively. The results showed that BnWRI1 expression was inhibited and seed oil content drastically decreased in each transgenic plant compared with non-transgenic plants. [Conclusion] Silencing endogenous BnWRI1 by RNAi can decrease seed oil content, which suggested BnWRll could effectively regulate seed oil content in oilseed rape.