摘要
水稻是世界的主要粮食作物。非生物逆境伤害是影响水稻产量主要因素之一,耐／抗逆境基因的发掘已成为目前水稻遗传资源与品种改良研究的热点。随着植物分子生物学的发展,一些逆境应答基因已被相继分离并转入水稻中,并获得了提高抗逆性的转基因植株,为抗逆水稻品种的选育开辟新的途径。
植物在生长发育过程中经常遭受逆境胁迫的危害,植物胁迫信号转导途径一直是人们关注的焦点。在逆境胁迫条件下,植物本身能够感知和传导逆境信号,启动或解除胁迫相关基因的表达,进而激活相应的保护代谢途径。
热激蛋白(Heat Shock Protein,HSP)是一类在生物界普遍存在的蛋白质,不仅在应激条件下高效表达,在正常状态的细胞中也广泛存在,参与一些重要的细胞生理活动。HSP90家族是一类分子量为90 kD左右的热激蛋白,它具有帮助蛋白质折叠,保持蛋白质结构完整,调节胞质蛋白亚群表达等功能。为了系统的研究OsHSP90的基因结构、表达特性以及功能等,本研究以OsHSP90为诱饵蛋白,利用酵母双杂交系统筛选其互作蛋白,对筛选出的新基因OSHSPBP进行功能分析,以探究OsHSP90可能的信号传递路径。研究结果简述如下：
1、运用酵母双杂交技术,以OsHSP90为诱饵,利用PEG/LiAc法转化酵母细胞,从水稻低温和干旱cDNA文库中筛选到未知功能蛋白OsHSPBP.
2、通过数据库比对分析OsHSPBP,该基因cDNA全长1321bp,编码区位于184bp-918bp,编码244个氨基酸残基,预测分子量约25.5 kD,等电点约为9.49。通过氨基酸同源性比较发现OsHSPBP与其他植物中的该蛋白具有较高相似性,与玉米(ACG33139相似性最高,为70.40%,且在tify与CCT_2功能域区间相似性明显高于其它部分。
3、运用半定量RT-PCR和实时定量PCR分析OsHSPBP在低温、高温、干旱和高盐等胁迫下以及在水稻不同生理时期各组织中的表达情况,发现其在未经逆境胁迫处理时,有不同程度的表达；高温和高盐逆境下,表达量先升高后减低；低温和干旱逆境下,表达量都升高；在水稻不同生育期的节间、节、叶、叶鞘、枝、颖壳等组织中均有表达,属于非组织特异性表达基因。
4、构建OsHSPBP基因cDNA片段的正、反义表达载体,并以根癌农杆菌介导建立适合水稻的遗传转化体系,从而研究该基因的表达在水稻抗逆应答中的生理功能。同时,构建OsHSPBP-GFP转入1301载体中,对OsHSPBP进行亚细胞定位。
Rice (Oryza sativa L.) is one of the main crops in our country even in the world. Abiotic stress damage is one of the important factors that make its production potential could not be fully exploited. Therefore, the research of adverse resistance is always the important task and finding resistant gene has been the hotspot on both the studying of rice genetic resources and variety improvement researches. With the development of plant molecular biology, some genes coding proteins related to stress have been isolated and transferred to the rice, thereby, the transgenic plants with high stress resistance have been acquired, this has opened up a new era for corp breeding.
Plant is often subjected to biotic stress and abiotic stress under growth and development,the researchers have been focus on stress signal transduction.Under stress conditions, plant itself can perceive and conduct stress signal, start lifting stress-related gene expression, and then activate the appropriate protection of metabolic pathways.
Heat Shock Protein (HSP) exists in the biological universe ubiquitously. HSP not only can perform effectively under stress condition, but also exists in the cells on normal state participating in some important cell activities. HSP90 family is a kind of molecular weight about 90 kD heat shock protein, it has helped protein folding, maintained the integrity of protein structure, and regulated the expression of cytoplasmic proteins. In order to study the genetic structure, expression characteristics and the function of OsHSP90 systematically, this research takes OsHSP90 as bait protein to filter its interact protein by using yeast two-hybrid system, then analyzes the function of the new gene of OsHSPPBP. Results are as follows:
1.According to the approaches provided by Clontech and making use of the yeast two-hybrid tecnology by taking OsHSP90 as bait protein, the unknown function protein was filtered from the low temperature and drought cDNA library of rice by using the PEG/LiAc method to transform yeast cell.
2.Analyzing OsHSPBP by comparing in the data-bases, follows could be found:cDNA of this gene is 1321bp long, its ORF lies in 184bp-918bp encoding 244 amino acid residues. Molecular weight is predicted to be about 25.5 KD and Isoelectric Point is about 9.49. Comparing the amino acid homology, OsHSPBP is found high similarity in the protein to other plants, and maize (ACG33139) similarity is the highest 70.40% and the similarity between the functional areas from tify to CCT_2 are obvious higher than that in other areas.
3. Using the method of semi-quantitative RT-PCR and real-time quantitative PCR to analyze the tissues expression of OsHSPBP in the conditions stressed by low temperature, high temperature, drought and high salt in different physiological stages. It is found to have different expressions in degrees before dealing with adversity stress:the expression is first increased and then reduced under high temperature and high salt; under low temperature and drought, the expression is increased. Meanwhile, it is not tissue specific expressed genes in internodes, nodes, sheaths, branches and glumes in different physiological stages.
4.Constructing expression vector of sense and antisense cDNA fragment of OsHSPBP genes and establishing the genetic transform system for rice mediated by agrobacterium tumefaciens to study the physiological function of the gene expression in resistant response of the rice. At the same time, OsHSPBP-GFP is built into the 1301 vector for subcellular localization of the OsHSPBP.
引文
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