【作者】 蔡伦；

【导师】 张富春；

【作者基本信息】 新疆大学 ， 动物学， 2005， 硕士

【摘要】 土壤盐分是制约作物产量的主要因素,土壤盐渍化严重影响着农业的产量,尤其是使灌溉农业作物减产,同时盐分过高还可造成盐碱地,限制土壤的利用。随着分子遗传学和植物转基因技术的发展,利用生物技术提高作物的耐盐性,使作物在盐胁迫环境中能正常生长并提高作物的产量,正受到越来越多的关注并取得了一定的成果。作物可以通过多种途径达到对盐胁迫的耐受,如操纵生物合成途径,在作物体内表达耐盐相关基因,改变基因的胁迫反应特性等。目前,已经证实SOS(Salt Overly Sensitive)盐胁迫信号途径在植物耐盐方面起着关键的调控作用,同时也控制着离子的自身稳定(homeostasis)。信号通路中Na~+/H~+逆向转运蛋白基因(NHX1)倍受人们关注,NHX1是液泡膜中的一种Na~+/H~+反向运输体,可促进钠离子在液泡中的区隔化效应,跨液泡膜的pH为其提供能量。避免细胞质中的高Na~+盐毒害,维持高的K~+/Na~+比,达到离子平衡和渗透平衡,钠离子和氯离子区隔化在液泡中,不仅可作为有效的渗透调节剂,还可减小细胞毒性,从而提高植物的耐盐能力,解决植物生长受抑制等问题。本研究通过RT-PCR(Reverse Transcription-Polymerase Chain Reaction)的方法,它是基于同源序列克隆候选基因家族成员的快捷途径,鉴定和克隆新疆野生耐盐碱植物盐角草、盐爪爪、盐穗木NHX1基因、花花柴、盐爪爪、盐桦钙调蛋白(CaM)基因,并进行序列分析。研究工作首先根据GenBank 上已发表的各种物种的NHX1 基因序列,分析其保守性并根据保守区设计了7 条引物(上游4 条,下游3 条),分别组合经RT-PCR 扩增出新疆野生耐盐碱植物盐角草、盐爪爪、盐穗木NHX1 基因的核心片段,克隆于pMD18-T 载体,经DNA 序列测定后,结果与GenBank 中NHX1 基因序列进行同源性比较,确定克隆出的核心片段为NHX1基因的核心更多还原

【Abstract】 Salt stress is the major factor that limits and decreases the output of crops in many parts of the world, particularly for the irrigated land. Overly high soil salinity can cause salina and limit the utilization of soil. Advances in molecular genetics and plant transformation in recent years have made it possible to employ biotechnological strategies in crops of salt-tolerant characters, keeping normal growth and without loss of production in the saline environment. Crops are able to tolerate salt stress by many ways, such as regulation of the pathways of biological synthesis, expression salt-related genes, altering genes’reaction characters regarding to the stress, etc.. So far, it has been proved that the SOS(Salt Overly Sensitive) pathway plays key role in plant salt tolerance and the ionic homeostasis. In this process, the Na+/H+ antiporter gene (NHX1) has been noticed widely, this antiporter energized by the ?pH across the tonoplast, facilitates vacuolar compartmentalization of the cation. As a fundamental mechanism in salt tolerance, an active antiporter would function to sequester Na+ into the vacuole, which results in avoidance of cytoplasmic Na+ toxicity and maintenance of a high cytoplasmic K+/Na+ ratio. In parallel, vacuolar Na+ would serve as an osmoticum necessary for cellular H2O homeostasis. Based on previous research, we employed local halophytes in Xinjiang as materials, performed the following research work: cloned NHX1 gene from Salicornia europaea、Kalidium foliadum and Halostachys caspica by RT-PCR and RACE; cloned calidium genes from Karelinia caspica, Kalidium foliadum and Betula halophila, carried out sequence analysis and preliminary function assay. At first, the conservative regions of various NHX1 gene sequences published in Genbank were analyzed, and seven primers (4 upstream, 3 downstream) were designed for amplification of core fragment of NHX1. Total RNA was isolated from plant tissue and cDNAs were synthesized by reverse transcription. Different combinations of these primers were used to amplify the core fragment of NHX1. The core fragments were cloned into pMD18-T cloning vector, then sequence analysis was conducted with positive clones. Sequencing results showed that the core fragments from Salicornia europaea、Kalidium foliadum and Halostachys caspica were similar to the AtNHX1 (abbreviation of NHX1 for Arabidopsis thaliana) sequence (GI: 30690553). With a series of primers designed according to the sequences of the cloned core fragments, the 3’ends of the cDNAs were obtained by 3’rapid amplification of cDNA ends (RACE), respectively. For the 5’unknown regions of these genes, the up-stream primers at the start-codon region was designed, the down-stream primers are the primers used to cloning the core cDNA. Combining sequences of the 3’ends, 5’ends and core cDNA, the full-length sequences of the NHX1 cDNA were assembled. To obtain open reading frame (ORF) sequence of NHX1 gene, three pairs of primers were designed according to the assembled cDNA sequence. PCR products were cloned into pMD18-T cloning vector and sequenced. Results confirmed that ORF of the NHX1 gene were completely involved in the full-length sequence. Three genes shared high homology. Comparing with the other plants, especially with the plants in Chenopodiaceae, NHX1 gene were highly conservative. The results may have important implications for the elucidation of the different abilities of halophytes and glytophytes for salt tolerance, and may present a feasible method to utilize much more salina area in Xinjiang. In addition, plant expression vector harboring with NHX1 gene of sarlicornia was constructed, and transformation of explants of brassica was carried out to investigate the function of this gene further more. Specific primers for calidium (CaM) gene were designed according to published sequences. RT-PCR were conducted to amplify calidium genes from Karelinia caspica、Kalidium foliadum and Betula halophila. Sequence analysis revealed that the cloned fragments contained entire calmodulin ORF. Sequence similarity was performed by BLAST network service. The results showed that high similarity (about 85%) among three calmodulin genes in terms of evolution status was observed. The homology of calmodulin genes between these three plants and the other plants was up to 80%. It indicated that calmodulin gene was relatively stable in plant evolution. Our research work no doubts has promoted the utilization of salt-resistant genes isolated from local halophytes in improving the ability of plant in salt tolerance and in the near future developing salt-resistant crops, and we believe , by genetic engineering, more and more salt-resistant plants will be developed and in application.更多还原