扁果枸杞表皮蜡质合成相关基因LbCER1的RNAi载体构建

Construction of RNAi expression vector of ECERIFERUM1 gene relating to epidermal wax synthesis in Lycium barbarum ssp. Bianguo

为鉴定醛脱羰基酶(ECERIFERUM1,CER1)基因在旱生植物表皮蜡质烷烃合成过程中的功能,培育适宜干制加工品种,选定扁果枸杞(Lycium barbarum ssp. Bianguo) Lb CER1基因1 469~1 840 bp区段为最佳RNA干扰(RNA interference,RNAi)靶序列,克隆该区段并将其插入p KANNIBAL载体,构建具有发卡状反向重复序列的RNAi中间载体p KANNIBAL-Lb CER1(+)-PDK intron-Lb CER1(-),并将发卡状反向重复序列转入RNAi表达载体p ART27,再用冻融法将p ART27-Lb CER1 (+)-PDK intron-Lb CER1 (-)载体转化根癌农杆菌(Agrobacterium tumefaciens) GV3101,并用限制性内切酶双酶切法和PCR法检测转化的农杆菌菌株。结果表明,RNAi的中间载体和表达载体均结构正确,并且RNAi表达载体p ART27-Lb CER1(+)-PDK intron-Lb CER1(-)成功转化根癌农杆菌GV3101,说明该构建方法正确,将为进一步筛选Lb CER1-RNAi植株,获得适宜干制加工的宁夏枸杞新品种奠定基础。

Aldehyde decarbonylase is a class of enzymes participate in plant alkane biosynthesis.In order to breed new varieties for fruits drying process,ECERIFERUM1( CER1),an aldehyde decarbonylase coding gene was subjected into an RNA interference( RNAi) assay in xerophytic plant Lycium barbarum ssp. Bianguo. The segment between the 1,469 bp to 1,840 bp of LbCER1 coding aldehyde decarbonylase region in Lycium barbarum ssp. Bianguo was chosen as the optimal target of RNAi. An intermediate vector,pKANNIBAL-LbCER1( +)-PDK intron-LbCER1(-),was used to achieve the proper hairpin structure required for RNAi assay. The final hairpin sequence containing reverse complementary fragments of the chosen LbCER1 sequence flanking the PDK intron was inserted into the vector pART27. In addition,the final vector,p ART27-LbCER1( +)-PDK intron-LbCER1(-),was chemically transformed into Agrobacterium tumefaciens GV3101. Restriction enzyme digestion and PCR methods were employed to screen positive intermediate and final vectors. The result showed that A. tumefaciens containing pART27-LbCER1( +)-PDK intron-LbCER1(-) was achieved.Hence,using this method to generate plasmids was correct,which laid the foundation for obtaining new varieties for dry processing through bioengineering of LbCER1-RNAi plants.