杉木ClLAR基因的克隆及遗传转化拟南芥

为探究杉木种子发育过程中无色花青素还原酶(LAR)基因在类黄酮生物合成途径中对原花青素(PA)合成的分子调控机制,以杉木种子为试验材料,通过RT-PCR结合RACE的方法成功克隆到杉木ClLAR基因全长,该基因的cDNA全长为1625bp,具有1个1242bp的开放阅读框(ORF),编码413个氨基酸。构建pCambia3301-ClLAR植物表达载体,运用冻融法将重组质粒转至农杆菌GV3101,通过花序侵染法获得拟南芥转基因植株,利用Basta溶液和PCR验证筛选出阳性苗,确定目的基因ClLAR已整合至拟南芥基因组中。研究结果为深入分析杉木ClLAR基因调控PA合成的分子机制和ClLAR蛋白功能奠定了基础。

Cloning of ClLAR Gene and Its Transformation in Arabidopsis thaliana

To explore the molecular regulation mechanism of leucocyanidin reductase (LAR) gene in the PA synthesis of the flavonoid biosynthesis pathway during the development of Chinese fir ( Cunninghamia lanceolata ) seeds,Chinese fir seeds were used as experimental materials,the ClLAR gene was cloned by RT-PCR combined with RACE.The results showed that the full length of ClLAR gene cDNA was 1 625 bp,it contained open reading frame (ORF) of 1 242 bp which encoded 413 amino acids.The plant expression vector of pCambia3301- ClLAR was constructed,and the recombinant plasmid was transferred to Agrobacterium tumefaciens GV3101 by freeze-thaw method.The transgenic plants of Arabidopsis thaliana were obtained by inflorescence infection method,and positive seedlings were screened by Basta solution and PCR.The results showed that the target gene had been integrated into the Arabidopsis genome.The results of this study laid the foundation for the further analysis of the molecular mechanism of ClLAR gene regulation of PA synthesis and the function of ClLAR protein in Chinese fir.