甘蔗细胞壁转化酶基因(SoCIN1)克隆及其在转基因烟草中的表达特性分析

【目的】克隆甘蔗细胞壁转化酶基因(So CIN1)并分析其在转基因烟草植株中的表达特性,为研究CIN1基因功能提供参考。【方法】克隆So CIN1基因,利用基因重组技术构建植物正义表达载体p BI121-So CIN1,采用叶盘法经农杆菌EHA105介导将其转化烟草品种K346。经抗性筛选和PCR扩增验证后获得转So CIN1基因烟草植株,采用半定量PCR检测叶片So CIN1基因的表达量,并测定叶片可溶性糖含量、CIN活性及株高(以野生型烟草为对照)。【结果】克隆获得的So CIN1基因长度为1731 bp,与Gen Bank已公布的So CIN1基因(JQ312298)核苷酸序列同源性达100%,并通过构建其植物正义表达载体转化烟草。PCR扩增鉴定结果表明,So CIN1基因已整合至烟草基因组DNA。对F0和F1代进行转基因烟草连续筛选,获得4个转So CIN1基因烟草株系(T-1、T-2、T-3和T-4)。在4个株系的叶片中均检测到So CIN1基因的表达,其中以在T-1株系叶片中So CIN1基因表达量最高,其次是T-3株系,最低的是T-4株系。4个株系叶片的可溶性糖含量、CIN活性及株高均高于野生型烟草,其中CIN活性显著高于野生型烟草(P0.05,下同),且T-1株系CIN活性高于其他3株系;T-1、T-2和T-3株系叶片可溶性糖含量分别显著高于野生型烟草39.68%、19.95%和31.15%;T-1、T-2、T-3和T-4株系的株高较野生型烟草分别提高了15.57%、2.90%、5.74%和5.22%,但仅T-1株系与野生型烟草存在极显著差异(P0.01)。【结论】转So CIN1基因烟草叶片过表达So CIN1基因,能提高烟草的CIN活性和可溶性糖含量,促进植株生长,由此推测该基因在甘蔗生长发育和蔗糖积累过程发挥作用。

Cloning of cell wall invertase gene(SoCIN1)in sugarcane and its expression characteristics in transgenic tobacco

Objective]In the present experiment,cell wall invertase gene(SoCIN1)of sugarcane was cloned and its expression characteristics in genetically modified tobacco plants were studied to provide reference for function research of gene SoCIN1.Method]Coding region sequence of gene SoCIN1 was cloned. The sense expression vector pBI121-So-CIN1 was constructed by gene recombination technology,and it was transformed into tobacco variety K346 by leaf discs method through Agrobacterium tumefaciens EHA105. SoCIN1 genetically modified tobacco was obtained after resistance screening and PCR amplification. Expression of gene SoCIN1 in leaf,soluble sugar content in leaf,CIN activity and plant height were measured by semi-quantitative PCR(wild tobacco as CK).Result]The coding region sequence of gene SoCIN1 cloned was 1731 bp,whose nucleotide sequence homology was 100%with that of gene SoCIN1(JQ312298)pub-lished by GenBank. Plant sense expression vector was constructed to transform tobacco. The results of PCR amplification showed that gene SoCIN1 had been integrated into tobacco genome DNA . Transgenic tobacco was screened continuously through F0 and F1 generation,and four SoCIN1 genetically modified tobacco lines(T-1,T-2,T-3 and T-4)were obtained. Expression of gene SoCIN1 could be detected in leaves of four transgenic tobacco lines,among which the highest was found in T-1,followed by T-3 ,and the lowest in T-4. Compared with wild tobacco,soluble sugar content,CIN activity in leaves and plant height of four transgenic lines improved obviously. CIN activities in four transgenic lines were signifi-cantly higher than that of wild type(P<0.05,the same below),and CIN activity of T-1 was higher than those of other three transgenic tobacco lines. Soluble sugar contents in leaves of T-1,T-2 and T-3 were significantly higher than that of wild type tobacco 39.68%,19.95%and 31.15%respectively. Plant height of T-1,T-2,T-3 and T-4 increased by 15.57%, 2.90%,5.74%and 5.22%respectively compared with wild type,but only T-1 was significantly higher than that of wild tobacco.Conclusion]Overexpression of gene SoCIN1 in SoCIN1 genetically modified tobacco leaves can raise CIN activity and increase soluble sugar content of tobacco leaves and promote plant growth. It is speculated that the gene SoCIN1 may play a role in sugarcane growth and sucrose accumulation.