狗牙根CdSAP1基因的克隆及耐盐功能解析

发布时间：2019-06-13 20:57

【摘要】：狗牙根(Cynodon dactylon)广泛分布于热带及亚热带地区,是一种耐热,抗旱,耐践踏,坪用性状优良的暖季型草坪草。狗牙根种系里存在一些耐盐性较强的基因型,在盐碱地和盐渍化土壤上有较大的应用潜力。目前关于狗牙根耐盐性的研究主要集中在种质资源耐盐评价和生理机制上,其耐盐分子机制的研究报道尚缺。SAP(Stress Associated Protein)是一种逆境相关的锌指蛋白,具有A20/AN1锌指结构域特征,参与了植物的逆境应答过程,提高了植物的耐盐性,但其调控耐盐性的分子机制尚不清楚。本研究从狗牙根中克隆到SAP家族基因CdSAP1,对其编码的蛋白序列、表达特性及调控耐盐的生理及分子机制进行了分析。主要结果如下:(1)本实验从狗牙根中克隆到CdSAP1基因,其cDNA全长为811bp,ORF全长516bp,编码171个氨基酸。该基因具有典型的A20/AN1锌指蛋白结构域,系统进化树结果表明狗牙根CdSAP1与水稻OsSAP8、OsSAP4和拟南芥AtSAP2相似度最高。荧光定量PCR数据显示,NaCl、ABA、PEG显著诱导了叶片中CdSAP1表达,诱导12 h时表达量最高。(2)将CdSAP1基因异源转化拟南芥,耐盐表型鉴定分析表明,转CdSAP1显著提高了拟南芥的耐盐性。盐胁迫下,转CdSAP1基因拟南芥的种子萌发率显著提高,叶片枯黄率、电解质渗漏率显著下降;转基因拟南芥能保持较高的叶绿素含量、光化学效率、净光合速率和较低的胞间CO2浓度;盐胁迫下转基因拟南芥通过减少Na+积累,维持较低的Na+/K+;转CdSAP1基因促进了脯氨酸及可溶性糖的积累,显著降低了H2O2和O2-的积累。(3)对CdSAP1耐盐相关基因的表达分析显示,盐胁迫下CdSAP1过量表达显著提高了叶片中 AtNHX1、AtP5CS、AtAPX、AtSOD、AtSOS1、和 AtPOD 基因的表达,未影响AtAKTI、AtBADH的表达。CdSAP1过量表达显著提高了根系中AtNHX1、AtAPX、AtSOD、AtPOD 和 AtBADH 的表达,而 AtSOS1、AtAKTI、AtP5CS基因的表达未受影响。
[Abstract]:(Cynodon dactylon) is a kind of warm season turfgrass with heat resistance, drought resistance, trampling resistance and excellent plateau character, which is widely distributed in tropical and subtropical areas. There are some genotypes with strong salt tolerance in dog root lines, which have great application potential in saline-alkali soil and salinized soil. At present, the research on salt tolerance of dog root mainly focuses on the evaluation of salt tolerance and physiological mechanism of germplasm resources, but the molecular mechanism of salt tolerance is still missing. SAP (Stress Associated Protein) is a stress-related zinc finger protein, which has the characteristics of A20/AN1 zinc finger domain. SAP is involved in the process of plant stress response and improves the salt tolerance of plants, but the molecular mechanism of its regulation of salt tolerance is not clear. In this study, the SAP family gene CdSAP1, was cloned from dog root to analyze the protein sequence, expression characteristics and physiological and molecular mechanism of salt tolerance regulation. The main results are as follows: (1) the CdSAP1 gene was cloned from dog tooth roots. The cDNA of ORF was 811bp. the ORF was 516bp. it encodes 171amino acids. The gene has a typical A20/AN1 zinc finger protein domain. The results of phylogenetic tree showed that the similarity between dog root CdSAP1 and rice OsSAP8,OsSAP4 and Arabidopsis AtSAP2 was the highest. Fluorescence quantitative PCR data showed that NaCl,ABA,PEG significantly induced the expression of CdSAP1 in leaves, and the highest expression was at 12 h after induction. (2) the salt tolerance of Arabidopsis thaliana was significantly improved by transgenic CdSAP1. (2) the salt tolerance of Arabidopsis thaliana was significantly improved by transforming CdSAP1 gene into Arabidopsis thaliana. Under salt stress, the seed germination rate of transgenic Arabidopsis thaliana increased significantly, the yellow rate of leaves and electrolyte leakage decreased significantly, the transgenic Arabidopsis thaliana could maintain higher chlorophyll content, photochemical efficiency, net photosynthetic rate and low intracellular CO2 concentration, and the transgenic Arabidopsis thaliana maintained low Na / K by reducing Na accumulation. Transgenic CdSAP1 gene promoted the accumulation of proline and soluble sugar, and significantly decreased the accumulation of H2O2 and O2 -. (3) the expression analysis of CdSAP1 salt tolerance related genes showed that the overexpression of CdSAP1 significantly increased the expression of AtNHX1,AtP5CS,AtAPX,AtSOD,AtSOS1, and AtPOD genes in leaves under salt stress, but did not affect the expression of AtAKTI,AtBADH. CDSAP1 overexpression significantly increased the expression of AtNHX1,AtAPX,AtSOD,AtPOD and AtBADH in roots, while AtSOS1,AtAKTI, increased the expression of AtNHX1,AtAPX,AtSOD,AtPOD and AtBADH in roots. The expression of AtP5CS gene was not affected.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S688.4

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