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旨在模式生物拟南芥中获得玉米漆酶ZmLac5基因过表达转化体,以研究该基因功能。分离玉米自交系掖478根部总RNA,反转录PCR法获得玉米漆酶基因ZmLac5全长,测序与序列分析表明,ZmLac5 cDNA的开放阅读框为1 758 bp,编码一个由586个氨基酸组成的蛋白质。同源性比对和进化树分析显示该基因与高粱Sb03g039570.1和谷子Si000813m在进化上亲缘关系较近。采用In-Fusion试剂盒构建了过表达载体pCUB-Ubi::ZmLac5,通过电击法将过表达载体导入根癌农杆菌LBA4404中,并成功转化拟南芥(Columbia ecotype),获得PCR阳性T1代植株62株,转化率为1.28%,T2代阳性植株60株。本研究为基于模式生物体过表达分析该基因生物学功能奠定了分析基础。
In order to study the function of this gene, an attempt was made to obtain a corn laccase ZmLac5 overexpressing transformant in a model organism Arabidopsis thaliana. Total RNA was isolated from maize inbred line Ye 478 and reverse transcribed PCR was used to obtain the full length of maize laccase gene ZmLac5. Sequencing and sequence analysis showed that the open reading frame of ZmLac5 cDNA was 1 758 bp encoding a 586 amino acid protein. Homology comparison and phylogenetic tree analysis showed that the gene was phylogenetically closer to Sb03g039570.1 and millet Si000813m. The overexpression vector pCUB-Ubi :: ZmLac5 was constructed by In-Fusion kit. The overexpression vector was introduced into Agrobacterium tumefaciens LBA4404 by electroporation and transformed into Columbia ecotype successfully. PCR-positive T1 plants were obtained 62 strains, the conversion rate was 1.28%, 60 strains of T2 generation positive plants. This study laid the foundation for the analysis of the biological function of this gene based on the over expression of model organism.