杜梨HMGR基因克隆及其转基因烟草种子耐盐性分析

为研究3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)在植物耐盐方面的作用机理,以杜梨(Pyrus betulaefolia Bunge)叶片为材料,通过同源克隆获得开放阅读框为1 815bp的cDNA全长序列,编码604个氨基酸的cDNA全长序列,结构预测显示该蛋白质的N端含有2个保守的跨膜结构域,C端具有催化区,包含HMG-CoA结合结构域和NADP(H)结合结构域。NCBI序列比对发现,其与苹果、沙梨HMGR的氨基酸序列同源性高达97%和93%;MEGA 5.0聚类分析发现该基因属于HMGR1亚类基因,因此,将该基因命名为PbHMGR。RTPCR分析表明,PbHMGR在杜梨韧皮部、木质部、芽、叶片和花中均有表达,在芽中表达量最高。洋葱表皮亚细胞定位发现PbHMGR蛋白在细胞质中呈现点状分布。利用农杆菌侵染叶盘法将PbHMGR转化烟草,转基因T0代种子在添加不同浓度NaCl的培养基上播种,统计萌发率并观察萌发状态,发现转基因烟草种子抗盐胁迫的能力显著高于对照。说明PbHMGR基因可以在一定程度上提高植物种子的耐盐性。

Clone of pear HMGR gene and salt-tolerance analysis of its transgenic tobacco seed

To study the mechanism of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) on salt tolerance in plants, we used birch-leaf pear(Pyrus betulaefolia Bunge.)leaves as materials and obtained a complete cDNA sequence by homology cloning.The open reading frame of this gene was 1815 bp,while it encoded a protein consisting of 604 amino acids.Structure prediction revealed that there were two conserved transmembrane domains in the N-terminal of this protein, and catalytic domains in the C-terminal containing HMG-CoA binding domains and NADP (H) binding domains. Blasting from NCBI,the deduced amino acid sequence showed 97%,93% and 90% of homology rate with apple,flowering peach and sandy pear respectively.From clustering analysis by MEGA 5.0,the results indicated that the obtained gene was a member of HMGR1 subfamily,named as gene PbHMGR.From RT-PCR analysis,the data indicated that PbHMGR expressed widely in phloem,xylem,shoot,leave and flower of birch-leaf pear,but expressed superlatively in shoot.By onion epidermis subcellular positioning analysis,we observed that the PbHMGR protein showed punctate distribution in the cytoplasm.PbHMGR was then transformed into tobacco using the method of agrobacterium infecting leaf discs and sown transgenic T0 seed on medium supplemented with different concentrations of NaCl.The germination rate after 3 days and the germination state was significantly higher in the salt-tolerant transgenic tobacco seed than that of the control.The PbHMGR could thus increase the salt tolerance ability of plant seed.