橡胶树转HbCBF1基因矮化突变体T-DNA插入位点研究

利用经PCR、GUS染色和Southern杂交均证明已成功转入Hb CBF1的、田间表型为矮化的橡胶树植株C1为材料,通过TAIL-PCR的方法获得T-DNA右侧侧翼序列1.6 kb,根据侧翼序列与载体的序列设计引物分析验证该序列真实可靠,并设计引物判断基因的T-DNA插入以杂合位点存在,与此同时,将该侧翼序列与已有的橡胶树基因组测序结果进行比对分析,发现该插入位点处并不存在基因,根据CBF1与Ca MV35s启动子的研究推测,C1植株的矮化表型是由于Ca MV35s启动子驱动抗逆基因导致,而非插入基因造成。

T-DNA Insertion Site of Dwarf Mutant in Hevea brasiliensisTransferred of HbCBF1 Gene

The C1 plant transferred HbCBF1 successfully was used as the material, verified by PCR, GUS staining and Southern blot, the phenotype of C1 in the field was dwarf. Using the method of TAIL-PCR, we got the 1.6 Kb flanking sequence of T-DNA, combing with the sequence of vector pCAMBIA2301, we designed three pairs of primers to validate that the sequence was reliable, and judge that the insertion site of T-DNA was heterozygous. Meanwhile, we compared and analyzed the flanking sequence with the genome sequencing result of rubber, and found that there was no gene in the T-DNA insertion site. According to the study of CBF1 and CaMV35s promoter, we speculated that the dwarf phenotype of C1 was induced by the CBF1 gene driven by CaMV35s promoter, instead of the gene of T-DNA insertion.