甘蔗eEF1A反义表达载体构建及其遗传转化研究

Construction of Plant Expression Vector and Transformation of sugarcane with eEF1A Antisense Gene

延伸因子eEF1A(Eukaryotic elongation factor 1A)是在核糖体催化氨基酸链延伸以及推动、控制蛋白质合成过程中起重要作用的蛋白质因子。利用RT-PCR技术扩增得到甘蔗eEF1A的编码区1420 bp的cDNA序列,反向连接到受体质粒pB I121载体中,构建含GUS基因的甘蔗eEF1A基因反义植物表达载体pB IantiEF。用冻融法将pB IantiEF导入根癌农杆菌菌株EHA105中,通过农杆菌介导法,将eEF1A反义基因转化烟草K346。将获得的43株抗卡那霉素烟草经PCR筛选,得到38株呈阳性的反义转基因植株,阳性植株再经Dot-Southern杂交检测,证明eEF1A基因已整合进其中的34株烟草基因组中,平均转化率为79%。反义转基因烟草植株移栽后表型出现不同程度的变异,叶片变厚,卷曲不能平展,叶背出现明显皱折,并外突成芽,茎、叶的伸长明显受阻,顶芽出现双芽,开花延迟。推测甘蔗延伸因子eEF1A可能与甘蔗茎、叶伸长生长和发育有关。

Eukaryotie elongation factor 1A （eEF1 A）, which broadly exists in the eukaryotic cens, plays a central role in prolonging AA chain,regulating protein synthesis,cell growth and morphology. In this paper,a 1420bp cDNA fragment encoding eEF1A ORF was cloned from sugarcane by RT-PCR amplification ,which was inserted reversely into plasmid pBI121 to construct sugarcane eEF1A antisense plant expression vector pBIantiEF including GUS gene. The constructed vectors were transferred into the Agrobacterium tumefacien EHA105 with the freezing-thawing method,and then the pBIantiEF-EHA105 was transferred into the Nicoliana tabacum variety K346 by agrobacterium-mediated method, and 43 Kan-resistant plants were obtained. After the selection of PCR,38 anti-sense transgentic plants were found positive, and 34 of them were confirmed by Dot-Southern Dlot test that eEFIA gene had been inserted into the genome of Nicoliana tabacum. The antisense transgenic tobacco plants showed different phenotypic variations after transplanting, and their leaves were thicker and curly with puckers in the back, and the growth of stem and leaves was stunted, and there were double buds developed in top of the stems, and flowering was delayed. It was suggested that the eukaryotic elongation factor 1A of sugarcane may be related to the growth and development of stem and leaves of sugarcane.