截形苜蓿液泡膜H+-PPase基因的克隆及功能研究

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	截形苜蓿液泡膜H+-PPase基因的克隆及功能研究
其他题名	Isolation and function analysis of a vacuolar H+-PPase gene from Medicago truncatula
	王建武
学位类型	硕士
导师	王海庆
	2009-05-29
学位授予单位	中国科学院西北高原生物研究所
学位授予地点	西北高原生物研究所
关键词	液泡膜h+-ppase 截形苜蓿耐盐性拟南芥马铃薯
摘要	液泡膜焦磷酸酶（V-H+-PPase）属于除P-、F-和V-类H+-ATPases之外的第4类质子泵。V-H+-PPase水解PPi产生的自由能与H+跨膜转运相偶联，形成质子驱动力，与V-H+-ATPase一起将细胞质中的H+泵入液泡中，可以建立跨液泡膜电化学梯度，为各种溶质分子进出液泡提供驱动力。已有的研究结果表明植物体内V-H+-PPase活性受盐胁迫的调节。从理论上讲，液泡膜上两个H+泵中的任何一个过量表达，都有可能提高H+的利用率，增大质子驱动力，从而使细胞质中更多的盐离子区隔到液泡中以增强植物的耐盐抗旱性。V-H+-PPase由单基因编码，而且在建立跨膜电化学梯度上与由多亚基组成的V-H+-ATPase的作用相当。在拟南芥、烟草、西红柿、水稻、玉米和棉花体内上调V-H+-PPase的表达均提高了这些植物的耐盐性。此外，过表达V-H+-PPase基因还能促进植物生长素的运输，使植物的生物量增大。由此可见，V-H+-PPase基因是一个可用于作物抗逆育种的优良基因。本实验采用EST电子克隆技术，通过RT-PCR克隆了豆科模式植物截形苜蓿(Medicago truncatula)的V-H+-PPase基因MtVP1，进行了生物信息学分析及亚细胞定位，构建了植物表达载体，然后通过根癌农杆菌介导转化了拟南芥和马铃薯，并通过对转基因植株的分子生物学检测及耐盐性实验，鉴定了截形苜蓿V-H+-PPase基因的功能，主要结果如下： 1.截形苜蓿V-H+-PPase基因MtVP1的克隆、生物信息学分析、亚细胞定位及植物表达载体的构建：克隆得到了MtVP1的cDNA序列，该序列包含1个2 298 bp的最大开放读码框。生物信息学分析表明，该基因编码765个氨基酸，其氨基酸序列与来自绿豆、拟南芥等高等植物的Ⅰ类液泡膜H+-PPase的氨基酸序列的一致性高达84%~93%，其蛋白是一个典型的膜蛋白，含有13个跨膜区，含有3个在液泡膜H+-PPase中高度保守的区域(CS1、CS2和CS3)，从结构分析结果推测MtVP1与AVP1在功能上具有相似性。亚细胞定位分析结果显示，MtVP1定位在质膜和液泡膜上。构建了pBI121-MtVP1,2表达载体，用来转化拟南芥和马铃薯。 2.拟南芥转化植株的获得：用花序浸泡法转化拟南芥，在50μg/ml卡那霉素固体MS培养基上筛选阳性苗，然后利用特异性引物进行PCR和RT-PCR鉴定，均扩增出了约1000bp的特异性片段，表明MtVP1已导入拟南芥基因组中。转基因拟南芥的表型观察实验表明，过表达MtVP1能使转基因植株根系更发达，叶片面积增大；但幼苗耐盐性实验表明，转基因株系与对照没有明显差异。 3.马铃薯转化植株的获得：选用马铃薯无菌苗的叶片作为外植体，用农杆菌进行转化，诱导分化成苗后，经PCR鉴定得到了转MtVP1马铃薯植株。耐盐性实验结果表明: 转MtVP1马铃薯植株比对照具有更强的耐盐性。本研究从豆科模式植物克隆到豆科模式植物截形苜蓿(Medicago truncatula)的V-H+-PPase基因MtVP1，并把该基因转到模式植物拟南芥中，确定了该基因具有控制植物生长素运输的功能，过表达该基因能使植物的生物量增大；然后对重要的经济作物马铃薯进行了遗传转化，不仅使转基因马铃薯的叶片面积明显增大，而且提高了转基因马铃薯植株的耐盐性。
其他摘要	Vacuolar H+-pyrophosphatase (V-H+-PPase) belongs to the fourth class of electrogenic proton pump in addition to the P-, F-, and V-type H+-ATPases. The proton pumping reaction couples with the hydrolysis of PPi. V-H+-PPase acidifies vacuoles together with vacuolar V-H+-ATPase in the plant cell, creates a cross-tonoplast electrochemical gradient that drives other solutes in and out of vacuole. It has shown that the level of H+-PPase in plant is regulated under salt stresses. In principle, enhanced expression of either of the vacuolar proton pumps should increase the sequestration of ions in the vacuole by increasing the availability of protons, and increased vacuolar solute accumulation could confer salt and drought tolerance. V-H+-PPase is encoded by a single gene, it can generate a H+ gradient across the vacuolar membrane similar in magnitude to that of the multisubunit vacuolar V-H+-ATPase. Up-regulation of V-H+-PPase in Arabidopsis, tobacco, tomato, rice, maize and cotton results in enhanced salt resistant. Hence, V-H+-PPase gene is a valuable gene for genetic engineering to improve the abiotic resistance of crop plants. In this research, vacuolar H+-PPase cDNA, named MtVP1, was cloned from the legume model plant Medicago truncatula by EST in silico cloning and RT-PCR. Bioinformatics analysis and subcellular localization assay had been completed. The expression vector pBI121-MtVP1,2 which harbored MtVP1 gene was constructed, and had been introduced into Arabidopsis thaliana and potato. The function analysis of MtVP1 transgenic plants have been done by molecular identification and some physiological indexes assay. The key results were as follows: 1. The sequence analysis has revealed that MtVP1 contains a maximum open reading frame of 2 298 bp for 765 amino acids. The deduced amino acid sequence was highly similar to vacuolar H+-PPase from other plants, such as Vigna radiate and Arabidopsis thaliana, with an identity of 84-93%. Computional analysis showed MtVP1 is a typical membrane protein with 13 trans-membrane domains and contained three conservative domains (CS1, CS2 and CS3) in vacuolar H+-PPase. Its sequence conservation and structure similarity with AVP1 implied that MtVP1 had similar function with AVP1. 2. The transgenic plants of Arabidopsis were obtained: The transgenic plants which roote in the Kanamycin medium were examined by PCR and RT-PCR, and 1 000 bp positive signals were obtained, indicated that MtVP1 have been integrated into the genome of Arabidopsis and expressed at transcriptional level. The transgenic plants grow better than wild-type, they have more extensive root systems and more foliage with more bigger leaves. There is no obviously difference between transgenic plants and wild-type under salt tolerance when the plants were plantlet. 3. The transgenic plants of potato were obtained: we use leaves as explants, transformed MtVP1 into potato mediated by Agrobacterium tumefaciens. PCR analysis indicated that MtVP1 had been integrated into the genome of potato. The salt tolerance assay indicated that transgenic plants performed better than that of wild-type. Summarily, MtVP1 was isolated from Medicago truncatula for the first time. First, we transformed MtVP1 into the model plant Arabidopsis thaliana and ascertained the gene’s function that can improve transgenic lines biomass. And then it was introduced into the important crop plant potato and also generated transgenic potato with bigger leaves, and improved salt tolerance of the transgenic lines.
页数	58
语种	中文
文献类型	学位论文
条目标识符	http://210.75.249.4/handle/363003/3246
专题	中国科学院西北高原生物研究所
推荐引用方式 GB/T 7714	王建武. 截形苜蓿液泡膜H+-PPase基因的克隆及功能研究[D]. 西北高原生物研究所. 中国科学院西北高原生物研究所,2009.

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