【作者】 李良；

【导师】 乔代蓉；

【作者基本信息】 四川大学 ， 微生物学， 2005， 硕士

【摘要】 低温会使植物受到不同程度的伤害乃至死亡,同时也是限制农业生产的一种严重的自然灾害。随着植物抗寒机制的研究,抗寒相关基因的克隆以及转基因技术的发展,使利用基因工程培育高效抗寒品种成为一条重要途径。 本文采用十字花科极度耐寒的播娘蒿(Descurainia sophia)作为植物抗寒基因研究的材料。克隆播娘蒿的抗寒基因COR(cold regulated gene),为改造植物耐寒性提供新的材料。该基因的获得对从基因水平上揭示其抗寒机制,提高植物的耐寒性具有十分重要的意义。本实验研究的主要内容包括: 1.播娘蒿COR基因(DsCOR)的克隆。根据拟南芥cor15a基因及油菜的BN115等同源基因的保守区设计同源克隆简并引物。以播娘蒿总cDNA为模板,进行PCR扩增,获得了与拟南芥cor15a基因扩增片段大小相似、序列同源性84.7%的336bp的EST片段,再利用3’RACE和5’RACE技术得到DsCOR的ORF读框和完整的3’端非翻译区序列。该ORF框长417bp,编码139aa的蛋白。 2.生物信息学分析DsCOR与拟南芥cor15a、油菜BN115的序列同源性分别为85.7%和75.4%。使用PHYLIP软件作生物进化分析,油菜中的3个抗寒基因BN115,BN26,BN19被聚为一类,拟南芥中的抗寒基因cor6.6,cor78和苜蓿CAS18被聚为一类。DsCOR同拟南芥的cor15a亲缘关系最近,与拟南芥cor15b也属于一类。由此证明克隆的DsCOR属于COR抗寒基因家族。 3.该蛋白分子量为14.4KD,等电点6.83,为一个酸性蛋白。在pH7.0的环境中它带有-0.13电荷,摩尔消光系数为3840。该蛋白中含有大量的Ala、Lys、Gly、Glu和Ser,含量最高为16.55%,这几种氨基酸占该多肽所有氨基酸的更多还原

【Abstract】 Low temperature may hurt plants variously even make them dead. Cold is an environmental factor that limits the geographical distribution and growing season of many plant species. With the development of mechanism of cold-resist at the gene level, some cold-resist genes have been cloned and technology of transgene, it is possible to odtain excellent cold-resist plant through gene-engineering.We use Cruciferae Descurania sophia(L.) as our cold-resist material, which can tolerance to low temperature. Our laboratory successfully cloned the gene cor (cold regulated gene ) from Descurania sophia(L.) . It is useful to clear up the mechanism of cold-resist at the gene level and advance the ability of cold-tolerance. Our research mainly is : 1. Descurania sophia (L.) cor(DsCOR) gene clone. According to homogene in Arabidopsis thaliana and Barassica , a pair of degenerate primers was designed. A EST sequences was obtained by PCR. We use bioinformatics tools analyzing this EST sequence. The result suggest it was high homologous to Arabidopsis thaliana corl5a. The EST sequence is similar to Arabidopsis thaliana corl5a .Then Using the 3’ RACE and 5’ RACE, we obtain a open reading frame and a complete 3’ non-translation sequence of DsCOR . Trought bioinformatics tools analyzing, sequence in the ORF which is 417bp, can express a protein of 139 aa.2. Bioinformatics tools analyzing DsCOR, the similarity to Arabidopsis thaiiana corlSa and Barassica napus BN115 is 85.7 % and 75.4 %. Through software of PHYLIP, we found our obtained gene DsCOR belongs to COR cold-resist gene family.3. Mw of DsCOR is 14.4 kD, pI is 6.83, short of Pro、 Cys、 Trp. It has the alike second structure with Arabidopsis thaiiana which involves some a—helix second structure. Chlorop prediction results show that it will go to chlorop. signal polypeptide Prediction show that DsCOR has a Precursor polypeptide. And DsCOR has hydrophilic character, then we confer DsCOR may keep soluble in boiling water.4. The characteristic of DsCOR:It has 5 transmembrane regions, which has more transmembrane structure regions than Arabidopsis thaiiana C0R15a. This means DsCOR are more liquid.It can keep membrane steady.Protein site prediction shows that DsCOR has two main site: cAMP- and cGMP-dependent protein kinase phosphorylation site and Protein kinase C phosphorylation site .We infer the two difference between Descurania sophia (L ) and Arabidopsis thaiiana may be the reason why Descurania sophia (L.) have more cold-resisit character than Arabidopsis thaiiana.5. The vector pET32—DsCOR with DsCOR genes was constructed.The gene can well expressed in E. coli BL21 (DS3) . The Mw of fusion protein is 31kD.6. With hydrophilic character, purification the protein in boiling water. A pure protein was obtained.Our experiment is not only important to explain the function of cold resist in Descurania sophia(L.) but also help us to understand phylogenetic analysis in various plants.更多还原