【作者】 孟强；

【导师】 樊守金；

【作者基本信息】 山东师范大学 ， 植物学， 2017， 硕士

【摘要】 人们在耕作过程中的不合理灌溉、施肥、自然灾害等因素使得全球的土地盐碱化日趋严重。大豆是我国主要的油料作物,但随着育种进程中的不断选择,栽培大豆遗传多样性日趋狭窄。野生大豆是栽培大豆的野生近缘种,并且含有很多优异基因,例如:耐盐、抗旱、耐寒、抗虫、多荚等,尤其在耐盐方面比栽培大豆有着显著的优势。本研究以327份野生大豆种质资源为材料,通过表型性状及SSR分析,筛选并建立野生大豆多样性群体,对该群体进行表型性状及抗逆性多样性分析。并对其中耐盐及盐敏感极端材料进行耐盐生理生化分析、研究耐盐相关基因的表达情况,解析野生大豆耐盐机理。利用大豆发状根体系,通过耐盐相关基因的过表达及CRISPR-cas9技术突变目的基因,研究耐盐相关基因功能,为大豆耐盐育种提供重要信息。主要研究结果如下:1.对327份野生大豆资源的主茎类型、叶形、叶长、叶宽、叶柄长度、荚长、荚宽、花色等农艺性状进行调查,发现这些性状存在丰富的多样性,但有些材料相似性很高。利用课题组以前筛选出的具有较高多态性的10对SSR引物对327份野生大豆进行SSR分析,去除表型及SSR标记一致的材料,最终建立了101份野生大豆的多样性群体。2.对101份野生大豆多样性群体进行遗传多样分析发现,主茎类型、叶长、叶宽、叶柄长度、叶面积、荚长、荚宽、单株籽粒重、苗期株高、干重、耐盐性等方面均表现出了丰富的多样性,变异系数0.18～0.79,多样性指数为1.73～2.05。3.对耐盐材料ST-335和盐敏感材料SS-736进行盐胁迫下生理生化分析,发现盐胁迫条件下,与盐敏感品种SS-736相比,耐盐品种ST-335的叶片相对含水率高,可溶性糖含量高;SOD、POD和CAT活性高;Na+/K+低;且KOR,NHX和NSCC基因表达量低,而SKOR和SOS1基因表达量高。可能是ST-335具有较高的抗离子毒害、渗透胁迫及抗氧化胁迫的能力,且离子转运相关的一些基因通过协同作用控制Na+及K+的吸收和外排,最终决定ST-335具有相对较高的耐盐能力。4.在大豆发状根中过表达SKOR和SOS1基因,提高了大豆复合体的耐盐能力;而过表达NSCC基因,提高了大豆复合体对高盐胁迫的敏感性。在野生大豆发状根中利用CRISPR/Cas9体系成功诱导NSCC基因发生突变,NSCC发生突变的发状根中的Na+/K+比例显著低于野生型。进一步说明这些离子通道相关基因的变化对野生大豆耐盐能力产生影响。该方法也为野生大豆耐盐相关基因功能研究提供重要的理论基础。更多还原

【Abstract】 The global soil salinization has become more and more serious because of all kinds of factors of the cultivation, such as unreasonable irrigation, fertilization,natural disasters .Soybean is the main oil crops in China, but cultivated soybean genetic diversity has become increasingly narrow in the process of the breeding. Wild soybean is wild relatives of cultivated soybean, a lot of excellent genes were stored in the wild species of the soybean, including resistance to salt,drought,cold,insect ,more pod etc, especially, the wild soybean has a good performance in resistance to salt. In this study,we screened and established the wild soybean population with high genetic diversity by analyzing the phenotypic traits and SSR markers of 327germplasm resources of wild soybean. The comparative analysis of physiological and biochemical characters of the salt resistant wild soybean ST-335 and salt sensitive one SS-736 were carried out in this study. we also did a research on expression of salt-tolerant related genes and the salt tolerance mechanism of wild soybean. The effects of SOS1, SKOR and NSCC on soybean salt tolerance were analyzed by gene overexpressing and mutation in soybean hairy roots. The results of this study provided the theoretically basis for deciphering the salt tolerance mechanism and selecting salt tolerant germplasm of wild soybean. The main results were as follows:1.The agronomic traits of 327 wild soybean germplasm resources were investigated, including main stem, leaf shape, leaf length, leaf width, petiole length,pod length, pod width, flower color etc. It was found that the diversity of the wild soybean resources is very high, but similarity also presented in some of them. A group was established which contained 101 wild soybeans and has a rich diversity by combined agronomic traits and SSR analysis which used 10 of SSR primers. The wild soybean which was chosen to consist the group has high polymorphism of SSR and phenotype. The SSR primers were present high diversity.2.It was found that the wild soybean population had a rich diversity of agronomic traits and other aspects t including main stem type, leaf length, leaf width, petiole length, leaf area, pod length, pod width, grain weight per plant, plant height, dry weight, salt tolerance in seedling stage, etc. The variation coefficients range from 0.18 to 0.79 and the diversity index range from 1.73 to 2.05.3.The comparative physiological and biochemical analysis were carried out in ST-335 and SS-736 after 48 hours by 0.9% NaCl treated. The result of this study showed that the relative water and soluble sugar content, the activity of SOD, POD and CAT, the gene expression of SKOR and SOS1 of ST-335 ( salt-tolerant material)were higher than that of SS-736( salt sensitive material ),but Na + / K +and the gene expression of KOR, NHX ,NSCC of ST-335 is lower than that of SS-736. The tolerances to ion poisoning,osmotic stress and oxidative stress may be higher in ST-335 than that of SS-736, and the exchange of Na +, K + in roots may be controlled by the synergy effect of some ion transport genes. It may be the reasons that ST-335 has higher tolerance to salt stress.4.Overexpression of SKOR and SOS1gene in the soybean hairy roots enhanced the salt tolerance ability of soybean complex Overexpression of NSCC gene made the soybean complex became more sensitive to salt stress. Gene mutations were successfully induced by using CRISPR/Cas9 system in wild soybean hairy roots. We found that Na + / K + ratio of NSCC mutations of hairy roots is significantly lower than that of wild type. It was suggested that the salt tolerance ability of wild soybean could be affected by the expression level of ion channel genes. The method used in this study also provide important theoretical basis for searching salt tolerance genes in the wild soybean.更多还原