亚麻4CL基因克隆及RNAi遗传转化

RACE Clone and RNAi Transformation of Flax 4CL Gene

该实验以前期克隆的亚麻4CL基因片段为靶序列,利用RACE方法克隆其全长cDNA序列(1 957bp),GenBank登录号为KC832864,该序列含有1 650bp完整的开放阅读框。系统进化树分析表明,亚麻4CL基因与盐芥4CL基因亲缘关系较近,而与禾本科的水稻(Oryza sativa)等亲缘关系最远。其编码蛋白预测分析结果显示,亚麻4CL基因由549个氨基酸组成,相对分子量为60kD,等电点为5.3,蛋白质二级结构以无规则卷曲比例最多,其次是α-螺旋。以亚麻4CL基因编码区413bp靶标序列作为干扰区段,扩增其正反向基因片段,构建植物干扰表达载体p1301M-4CL。通过农杆菌介导转入亚麻,DNA及RNA水平上分子检测发现,RNAi结构已经转入亚麻中,并显著降低了其4CL基因的表达量。

In this research,a full-length cDNA sequence of 4CL related to flax lignin synthesis is cloned by RACE-PCR technique on the basis of the segment of this gene cloned freviously. The complete cDNA se- quence of 4CL is 1 957 bp in length,with a complete open reading frame of 1 650 bp. The encoded protein contains 549 amino acids,with a molecular mass of 60 kD and a pI of 5.3. Random coil and u-helix are the main secondary structure of the protein. Phylogenetic analysis indicated that there is high similarity be- tween flax 4CL gene and that of Thellungiella halophila. The full-length cDNA sequence has been regis- tered in GenBank,with an accession number of KC832864. A 413 bp CDS fragment of 4CL is amplified by PCR to construct the RNA interference vector p1301M-4CL,which is transformed to flax by agrobacteri- urn-mediated method. Seven transgenic seedlings selected randomly are detected by GUS and PCR. The results showed that the RNAi vector construction has been integrated into flax genome, which leads to evi- dently depression of the expression of 4CL gene in flax.