海藻糖-6-磷酸合成酶基因的序列优化与表达研究（英文）

Sequence Optimization and Expression Analysis of Trehalose-6-phosphate Synthase Gene

为了获得一个优良的玉米抗旱育种基因资源,借助于生物信息学手段和分子生物学实验操作技术,从木棉植物中获得了一个编码海藻糖-6-磷酸合成酶的抗旱基因序列。根据玉米对密码子使用的偏爱性,合成了1个可用于玉米转化并提高其耐旱性的新的海藻糖-6-磷酸合成酶基因,进一步通过DNA分子的酶切和连接等操作将该基因构建成表达质粒,再导入大肠杆菌和酵母细胞以鉴定其是否能在原核和真核细胞中表达。最后该基因被构建到植物表达载体上,并转入农杆菌细胞。结果表明,新合成基因全长为2 586 bp,包含完整的开放阅读框,编码861个氨基酸;构建出的原核和真核表达质粒与预期的结果相符,该基因可用于细胞转化试验;该新合成基因能在大肠杆菌和酵母细胞中有效表达出海藻糖-6-磷酸合成酶;该基因被成功构建到植物表达载体p CAMBIA-2300上,并转入了农杆菌细胞LBA4404工程菌株。综合上述结果认为,获得了1个新的海藻糖-6-磷酸合成酶基因,该基因能在原核和真核细胞中有效表达出海藻糖-6-磷酸合成酶,构建的植物基因表达质粒可以直接用于玉米抗旱转基因育种。

To provide one elite drought-resistance gene resource for maize breeding program,a trehalose-6-phosphate synthase(TPS)gene sequence was obtained from Gossypium arboretum by bioinformatics analysis and molecular experimental operation.The gene was optimized and synthesized based on maize preferred codons.Afterwards,the gene was cloned into expression plasmids by multiple DNA restriction enzyme digestion and molecular ligation operations,and further transformed into E.coli and yeast cells to identify its expression status.Finally,the new TPS gene was cloned into plant expression vector.The results showed that the new synthesized gene had 2 586 bp in length,it contained a whole open reading frame and encoded 861 amino acids;constructed prokaryotic and eukaryotic expression plasmids can agree with our predicted results,this suggested that the gene could be used for cell transformation experiment;the synthesized TPS gene could effectively express trehalose-6-phosphate synthase in E.coli and yeast cells;the TPS gene was successfully ligated into plant expression vector pCAMBIA-2300,and further transformed into engineering strain LBA4404 of Agrobacterium.Comprehensively,it was concluded that a new TPS gene was obtained in this experiment,which could express trehalose-6-phosphate synthase in prokaryotic and eukaryotic cells.Therefore,the constructed plant expression plasmid can be directly applied in maize drought-resistance breeding program.