【作者】 王艳艳；

【导师】 王相晶；

【作者基本信息】 东北农业大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 土壤盐渍化是影响农业生产和生态环境的主要非生物逆境之一。随着人口的增加和耕地的减少，如何开发利用盐渍化土壤已成为农业生产和环境生态亟待解决的问题。利用生物技术深入开展种质资源的耐盐性研究，挖掘其中优异耐盐相关基因，培育耐盐作物新品种，是改良和利用盐渍化土地，增加粮食产量和维持农业可持续发展最为经济有效的策略之一。本实验以102份小麦地方品种群体为研究材料进行耐盐性鉴定，选出鉴定结果呈中间型的群体进行耐盐基因构成分析；同时，利用RT-PCR技术分离和克隆耐盐相关基因，通过原核表达系统表达目的蛋白，旨在为耐盐小麦种质的利用提供理论依据和应用指导。本研究取得如下研究成果：1．对102份小麦地方品种进行耐盐性鉴定，筛选出22份耐盐性鉴定呈中间型的群体供第四章耐盐基因构成的研究。对盐处理小麦进行形态指标的分析，得出结论：芽期时，相对芽长与盐害指数呈显著的负相关；苗期时，相对根长和盐处理后的根冠比与盐害指数呈显著负相关；小麦种质芽期与苗期的耐盐性没有直接关系。2．对小麦地方品种36号群体进行SSR遗传多样性分析，共检测到26个等位变异，每个SSR位点的变异范围为2～3个，平均每对引物的等位变异为2.36个。每对SSR引物的多态信息含量的范围为0.071～0.512，平均为0.26。可以看出，小麦地方品种群体内部单株之间的遗传变异程度比较小，需要利用一种变异广泛的标记来进行耐盐基因构成的分析。3．利用生物信息学的方法，找到与耐盐相关的基因，设计引物，最后筛选到依据DREB片段设计的引物，通过PCR-SSCP技术对耐盐鉴定呈中间型的22个群体进行基因构成的分析，结果得到了18种带型，共17个单核苷酸突变位点，其中12个位点为转换，3个为颠换，1个为插入，1个为缺失。作物的耐盐性是多基因控制一个较为复杂的数量性状，单从DREB基因的单核苷酸多态性情况还不能完全解释小麦的耐盐性。4．以盐处理的小麦为材料，利用RT-PCR的方法克隆出DREB基因的编码区，与发表的小麦DREB基因有99％的同源性。成功的构建了pET30a+DREB原核表达载体，并利用IPTG诱导了目的蛋白的表达。下一步打算进行DREB在植物中，尤其在小麦中的转基因研究，探讨培育优质耐盐转基因小麦的可能性。更多还原

【Abstract】 Salinization of soil is one of the major abiotic-stresses that influence agricultural productionand environment. With the increasing of world’s population and the decreasing of field, how todevelop and utilize salinized-soil becomes an urgent problem in agriculture production andecological environmental protection. To increase food production for the need of populationgrowing and maintain sustainable development of agriculture, one of the most economic andefficiency strategies to improve and utilize salinized-soil is screening salt-tolerant germplasmresources, discovering and cloning the genes related to salt-tolerance, and breeding new varietiestolerant to salt-stress using biotechniques.102 accessions of wheat landrace groups were studied on the salt-tolerant evaluation, andthe groups which salt-tolerance evaluation results were middle class were selected to study thesalt-tolerance genotypes. Meanwhile, the salt-tolerance related gene were separated and clonedthrough RT-PCR, and the prokaryotic expression system was used to express the target protein.The aim of this study is to provide the theoretical supports and applicable guidances for theutilizations of salt-tolerance wheat germplasm.The study has got following results:1. 102 wheat landrace groups were studied on the salt-tolerance evaluation. 22 groups whichhad middle class evaluation result of salt-tolerance were screened for the analysis of thesalt-tolerance related genotypes. The results of the morphological index analysis of salt-treatedwheat showed that, in the germination stage, the length of relative coleoptile was significantlynegative correlated with the salt injury index; and in seeding stage, the length of relative root andthe ratio of root and shoot after salt treatment were significantly negative correlated with the saltinjury index. The salt tolerance in the germination and seeding stages was different and thecorrelation coefficient was low.2. The genetic diversity of NO.36 wheat landrace group was evaluated. 26 alleles werefound in total, with each SSR locus variation ranging from 2 to 3, and the average allelevariations of each primer was 2.36. The PIC of each primer ranged from 0.071 to 0.512, and theaverage was 0.26. It could be concluded that the variation between the individuals in the samegroup was very small. A broadly varied molecular marker should be needed to analyze thegenotype related to salt-tolerance.3. Salt-tolerance related genes were collected and the primers were designed by the bioinformatics methods. A primer which designed by DREB was screened, and 22 groups whichhad middle class salt-tolerace evaluation results were analyzed for the genotypes related tosalt-tolerance. There were 18 genotypes and 17 SNP loci which included 12 transitions, 3transversions, 1 insert and lindel in 22 wheat landrace groups. The salt-tolerance of crops was acomplex multigene-controlled quantity trait. The SNPs of DREB genes could not describe the salttolerance of wheat completely.4. The RT-PCR technique was used to clone the coding region of DREB in salt-treatedwheat. The cloned fragment was 99％homologous with the published DREB gene. Theprokaryotic expression vector was constructed successfully and the target protein was expressedthrough the inducement of IPTG. The next researth is to study the transgene of DREB in plant,especially in wheat, and discuss the feasibility of the cultivation of high quality and salt-toleranttransgenic wheat.更多还原