【作者】 李新玲；

【导师】 杨传平； 徐香玲；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2007， 博士

【摘要】 逆境胁迫（如干旱、高盐、低温等）是影响植物生长发育的主要因素，严重影响农作物的产量和品质，并日益恶化生态环境。如何提高植物的抗非生物胁迫能力来满足日益增长的粮食需求，已经成为现代育种工作急需解决的关键问题之一。应用基因工程手段开发耐逆资源，克隆耐逆基因，培育耐逆农作物品种已成为现代农业研究的一个重要内容，也是维持农业可持续发展的有效策略。为了进一步探讨植物抗逆反应机理，本文以羊草、烟草和拟南芥作为主要实验材料，开展了植物耐逆分子机制和相关基因工程研究，主要结果如下。测定了灰绿型羊草的耐盐相关生理指标，并与黄绿型羊草进行了比较，各种生理生化指标的变化趋势充分反应了灰绿型羊草是一个高抗盐性材料，其中一定含有众多的耐盐基因。利用RT-PCR技术从灰绿型羊草RNA中扩增到了3个通读的且具有醛脱氢酶基因家族保守结构域的序列LC-1、LC-2和LC-3。结构上的同源性比较表明，LC-1为一种乙醛脱氢酶基因：LC-2为甜菜碱醛脱氢酶基因；LC-3为一个未知的全新的醛脱氢酶基因。这三个序列在GenBank上的注册号分别为DQ458294，DQ497618，DQ458293．以LC-1为靶序列，采用RACE技术克隆到了全长1712bp的羊草乙醛脱氢酶基因LC-ALDH，该基因包括1503bp的ORF，编码501个氨基酸，含有66bp的5’-UTR和144bp的3’-UTR，在终止密码子下游72bp处有单一的加尾信号AATAAA，在GenBank上的登陆号为EF492045。经生物信息学分析，LC-ALDH具有醛脱氢酶基因家族绝对保守的谷氨酸活性位点、半胱氨酸残基和组氨酸残基。与水稻的OsALDHla基因的同源性最高，氨基酸的一致性达到87％，系统发生学分析表明LC-ALDH属于细胞质的醛脱氢酶基因。Real Time RT-PCR结果表明，LC-ALDH基因受高盐、干旱及低温等逆境胁迫的诱导，并在这些诱导条件下其表达量显著升高。推测该基因在抑制细胞膜脂过氧化反应、抵御氧化胁迫的作用中起重要的作用。利用PCR技术从拟南芥基因组中克隆了CBF2基因和rd20A启动子，并构建了由rd29A启动子调控的CBF2基因的植物表达载体pBI-RD-CBF₂，通过农杆菌介导法转化烟草品种龙江851。转基因植株后代遗传学分析表明，外源基因以单拷贝的形式插入到烟草基因组中并遵循孟德尔3∶1的遗传分离规律；耐盐性鉴定表明，转基因烟草在150mM NaCl胁迫下尚可正常生长，而野生型烟草在100mM NaCl胁迫下盐害症状明显；电导率测定也表明，在盐胁迫下转基因烟草叶片细胞质膜的损伤程度低于对照株。说明CBF2基因的过量表达有利于提高植物对盐胁迫的耐受性。但有部分T₀代转基因烟草在形态上发生了明显改变，如植株矮化、叶片皱缩、花色变淡、顶端优势减弱、节间缩短等，推测与CBF2基因所含的AP2保守结构域有关。更多还原

【Abstract】 Adversity stresses such as drought, high salt and freezing is a major factor that influences plant development. It has a serious impact on the output and quality of the crop, even on the environment. How to improve the ability to resist abiotic stress and to meet the increasingly demands of provision has become one of the key problems in the modern breeding works. It has became an important content in modern agriculture research to cultivating the new crop variety which can bear the stress and developing the tolerant resources and cloning the genes which are relevant to help plant to withstand the adversity. It is also an efficient strategy to keeping agricultural sustainable development. In this research, for further study the mechanism of stress tolerance in plant, Arabidopsis thaliana, tobacco and Leymus Chinensis were used as the main experimental materials to study the molecular mechanism of stress tolerance in plant and do some researches on the gene engineering. The main results are as follow:By measuring the relevant physiological index of grey green type Leymus Chinensis under the salt stress and comparing with yellow green type Leymus Chinensis, the variety trends of physiological indexes show that grey green type Leymus Chinensis is a high salt tolerant material and there are many genes related to salt tolerance in it. We get three read-through sequence LC-1、LC-2 and LC-3 with a conservation domain which belong to the aldehyde dehydrogenase family by RT-PCR method from Leymus Chinensis. Homologous comparison with their structure shows that LC-1 is a aldehyde dehydrogenase gene, LC-2 is a betaine aldehyde dehydrogenase gene and LC-3 is an unknown aldehyde dehydrogenase gene. These sequences are all enrolled in GeneBank and the number is DQ458294, DQ497618 and DQ458293, respectively.We clone the full-length aldehyde dehydrogenase gene which contain from Leymus Chinensis by RACE which use LC-1 as the target sequence. This gene contains 1503bp ORF including 66bp 5’-UTR and 144bp 3’-UTR, and codes 501 amino acid. There is a tailing signal AATAAA at the terminator downstairs 72bp and the registered number is EF492045 at GeneBank. LC-ALDH has an absolute conservative glutamic acid active site, cysteine residue and histidine residue of aldehyde dehydrogenase by bioinformatics analysis. There is 87% homology between the LC-ALDH and the OsALDHla gene in rice. Analysis of phylogenesis shows that LC-ALDH belongs to the aldehyde dehydrogenase gene in cytoplasm. The result of Real time RT-PCR shows that LC-ALDH is induced by drought, high salt and freezing and its expression increased prominently under these conditions. We confer that this gene plays an important role in restraining membrane lipid peroxidation and resisting the oxidation stress.After isolating the CBF2 gene and the rd29A promoter from Arabidopsis and constructing a plant expressive vector pBI-RD-CBF2 in which the CBF2 gene regulated by rd29A promoter, we transformed this vector to the tobacco Longjiang 851 by Agrobactorium. By the genetic analysis of the transgenic offspring, it shows that the exogenous gene inserts tobacco genome as one copy form and follows the Mendel 3:1 heredity separate law. The analysis of salt tolerance shows that transgenic tobacco can grow normally under the 150mM NaCI while the wild type tobacco represent harmfully under the 100ram NaCI. The result of the relative conductance shows that the damage extent of the cytoplasmic membrane in transgenic tobacco is lower than in the wild type tobacco. The experiment account for that the over expression of CBF2 gene is propitious to improve the plant salt tolerance. But the form of the TO generation of some transgenic tobaccos are obviously changed, including the plant stunting, leaf crimpling, color of the flower thinning, apical dominance weakening and node shortening, which was conferred that the changing is correlate to the AP2 conservative domain of CBF2 gene.更多还原