甘蓝型油菜种子中G3PDH基因特异性表达的ihpRNA载体构建

Construction of a Seed-specific Expression ihpRNA Vector Targeting the G3PDH Gene of Brassica napus

为验证甘蓝型油菜甘油-3-磷酸脱氢酶基因(G3PDH)的功能,采用PCR的方法克隆BnG3PDH557bp的干扰靶序列,将其正向片段插入到中间载体pHurricane的NotI酶切位点、反向重复序列插入到XhoI酶切位点,构建ihpRNA载体反向重复框,BamHI、EcoRI酶切下反向重复框后组装到由种子特异性表达的napin启动子驱动的pCAMBIAl390植物表达载体上。结果表明:PCR扩增产物、限制性内切酶酶切产物与预期片段长度相符。其中,1.7kb的片段包括正向干扰靶片段和部分AtFad2内含子序列,2.3kb大小的片段是干扰反向重复框。种子特异性表达的BnG3PDHihpRNA载体成功构建。

A ihpRNA vector was constructed to study the function of BnG3PDH gene;A 557 bp target BnG3PDH sequence was amplified by PCR,and the positive fragment was transferred into the platform vector pHurricane in enzyme loci of XhoI,and the reverse complementary target DNA fragment was inserted into the enzyme loci of NotI to form the inverse repeat of ihpRNA vector;The inverse repeat fragment was cut off by BamHI、EcoRI and inserted into a modified vector pCAMBIAl390 which under the drive of a rapeseed seed-specific promoter napin.The PCR and restriction enzyme digestion verified the successful construction of the BnG3PDH ihpRNA vector.