【摘要】 低温是种子萌发过程中常见的自然灾害,在我国北方经常发生,这导致种子萌发率较差、降低植株活力等后果。利用cDNA-AFLP技术从低温(4℃)吸胀24h的抗低温吸胀大豆品种中黄22(低温吸胀24h对萌发率无影响的品种)中分离出一个基因片段,命名为GMCHI(GenBank登录号为EU699765),通过RACE方法得到全长为387bp的cDNA序列。在NCBI数据库中的查询表明,GMCHI基因和数据库记录的基因序列同源性较低,因此可以断定GMCHI是在大豆中被发现的新基因。半定量RT-PCR显示GMCHI受ABA和PEG诱导。该基因与PET30A连接后转入原核细胞,经过IPTG诱导,6%SDS-PAGE电泳条带说明,GMCHI在大肠杆菌中能够表达。把诱导表达和非诱导表达的菌落在?20℃下2h后,移至37℃培养20d,发现对照的菌落完全死亡,而诱导表达GMCHI的菌落只有部分死亡,并长出新菌落。RT-PCR检测表明新菌落携带GMCHI基因,证明GMCHI基因的表达提高了大肠杆菌的低温耐性。更多还原

【Abstract】 Low temperature during germination being a natural disaster usually occurs in North of China, which results in low germination rate and low seedling vigor, and even ultimately severe loss in yield. A gene was isolated from soybean Zhonghuang 22 cultivar (tolerant to low temperature in imbibition) for 24 h under 4℃ via cDNA-AFLP, name as GMCHI, GenBank accession No. EU699765. The full-length sequence of GMCHI which was 387 bp in nucleotide was obtained by RACE method. The se- quence of gene shared low similarity with that of genes documented in NCBI. Therefore we inferred that GMCHI was a new gene discovered in soybean. Semi quantitative RT-PCR result revealed that GMCHI was induced by ABA and IPEG.. The gene ligated with Prokaryotic expression vector was transformed into prokaryotic cell, the result of 6% SDS-PAGE induced by IPTG was ex- pressed in Prokaryotes. The E. coli carrying GMCHI survived and showed tolerance to low temperature under the treatment of ?20℃ for 2 h. RT-PCR validated that GMCHI in Prokaryotes improved the adaptation to low temperature.更多还原