转天山雪莲冷调节蛋白基因烟草的获得及其抗寒性鉴定

从已建立的天山雪莲cDNA文库中随机挑取单克隆,采用通用引物M13扩增出基因序列并测序,然后将该序列在GenBank中进行比对,根据该序列设计特异引物进行PCR,最终获得了冷调节蛋白基因siCOR的完整开放读码框(ORF);构建植物表达载体,通过农杆菌介导法获得转基因烟草,用RT-PCR对转化烟草进行鉴定,收取种子后筛选出纯合的T3株系,并通过冷冻胁迫进行抗寒性分析。结果表明:(1)siCOR大小661bp,与禾本科植物粳稻的冷调节蛋白基因一致性最高(65.04%)。(2)与野生型植株相比,转siCOR基因烟草均叶片浓绿、无分支侧芽,生长快;在冷冻胁迫条件下,转siCOR基因的烟草叶片相对含水量、PSⅡ相对量子产率降低幅度、相对电导率和丙二醛含量的升高幅度均低于野生型烟草植株,而且转siCOR基因烟草植株叶片出现萎蔫症状的时间较迟、程度较轻,受到的伤害也较轻。研究发现,在冷冻胁迫条件下,转siCOR基因烟草植株表现出良好的生理和生长优势,显示出了较强的抗寒特征。

Obtained and Cold Resistance Identification of Tobacco Plants Transformed by siCOR of Saussurea involucrata Kar.et Kir.

Seeking monoclone in established cDNA library randomly for PCR directed with universal M13 primer and sequencing,comparing the sequence in GenBank,designing specific primer according to the sequence,finally we obtained the whole ORF of siCOR.Then plant expression vector pBIN438-siCOR was constructed and NC89 tobacco plants were transformed via Agrobacterium tumefaciens,RT-PCR analysis indicated that siCOR had been integrated into the genome.Homozygous transgenic tobacco plants were used in cold resistance identification.The results show that:(1)The size of siCOR cDNA was 661 bp,the ideutity of amino acid sequence was about 65.04% with Oryza sativa Japonica.(2)siCOR tobacco has deep green leaves,grows faster and has no lateral bud compared with wild type;Under the cold stress,the decrease of relative water content,reduction of relative quantum yield of PSⅡ,the increase of relative conductivity and MDA content of siCOR tobacco are lesser than that of control tobacco evidently.Besides,transgenic plants have slower and lighter symptom.In summary,under the cold stress,siCOR tobacco shows well growth traits and strong cold resistance characteristics.