盐角草胆碱单加氧酶基因的克隆及其在烟草中的表达研究

【作者】 葛庆燕；

【导师】 安利佳；

【作者基本信息】 大连理工大学 ， 生物化工， 2005， 硕士

【摘要】 本研究利用RT-PCR和RLM-RACE技术获得了盐角草的CMO cDNA全序列。CMO cDNA全长1844bp,5’端非编码区154bp,3’端非编码区361bp,含有1个可能的加polyA信号:AATAA,开放阅读框1329个核苷酸,编码一个由442个氨基酸构成的多肽,与辽宁碱篷、滨藜、菠菜、甜菜CMO的氨基酸序列同源性分别为84%、79%、79%、78%,具备Rieske-type(2Fe-S)蛋白的保守Cys-His对——“CTH”和“CPYH”,包括保守的多铁原子核结合域“HVPYAH”。同时根据RLM-RACE的原理和5’端非编码区序列的分析,推测出目的基因上游的转录起始位点可能为g。 克隆了CMO基因的编码区,构建了其植物表达载体pBI121-CMO,冻融法将质粒导入根癌农杆菌(Agrobacterium tumefaciens)GV3101,获得工程菌GV3101-pBI121-CMO。 农杆菌介导法将CMO基因导入烟草(Nictiana tabacum L.cv.89),获得卡那霉素抗性植株。PCR初步证明外源CMO基因已转入烟草中,转化烟草膜的相对电导率明显低于对照,说明盐胁迫下转基因烟草的膜结构所受损伤小于对照。更多还原

【Abstract】 The full length CMO cDNA was cloned from Salicornia europaea by RT-PCR and RLM-RACE. CMO cDNA (1844bp) included a 154bp 5’ UTR, a 361bp 3’ UTR, a putative polyadenylylation signal and a 1329bp open reading frame encoding a 442-amino-acid polypeptide which was 84%, 79%, 79% and 78% to CMO sequences of Suaeda liaotungensis, A.hortensis, A.tricolor and B. vulgaris in amino acid homology respectively. The deduced amino acid sequence included the conserved mononuclear Fe-binding motif "CTH" and "CPYH" and the conserved Rieske-type iron-sulfur cluster-binding region "HVPYAH". G was considered as the possible transcription initiation site based on the principle of RLM-RACE and the analysis of CMO 5’ UTR sequences.The CMO ORF was cloned and the plant expression vector pBI121-CMO was constructed. The resultant plasmids pBI121-CMO was transferred into Agrobacterium tumefaciens (GV3101) by the liquid nitrogen freezing thaw method.The CMO gene was transferred into tobacco (Nictiana tabacum L. cv. 89) via Agrobacterium mediation. PCR analysis showed that the CMO gene was transferred into tobacco. Relative electronic conductivity demonstrated less membrane damage in transgenic plants than in the wild type.更多还原