【摘要】：狗牙根(Cynodon dactylon)廣泛分布于熱帶及亞熱帶地區(qū),是一種耐熱,抗旱,耐踐踏,坪用性狀優(yōu)良的暖季型草坪草。狗牙根種系里存在一些耐鹽性較強的基因型,在鹽堿地和鹽漬化土壤上有較大的應用潛力。目前關于狗牙根耐鹽性的研究主要集中在種質資源耐鹽評價和生理機制上,其耐鹽分子機制的研究報道尚缺。SAP(Stress Associated Protein)是一種逆境相關的鋅指蛋白,具有A20/AN1鋅指結構域特征,參與了植物的逆境應答過程,提高了植物的耐鹽性,但其調控耐鹽性的分子機制尚不清楚。本研究從狗牙根中克隆到SAP家族基因CdSAP1,對其編碼的蛋白序列、表達特性及調控耐鹽的生理及分子機制進行了分析。主要結果如下:(1)本實驗從狗牙根中克隆到CdSAP1基因,其cDNA全長為811bp,ORF全長516bp,編碼171個氨基酸。該基因具有典型的A20/AN1鋅指蛋白結構域,系統(tǒng)進化樹結果表明狗牙根CdSAP1與水稻OsSAP8、OsSAP4和擬南芥AtSAP2相似度最高。熒光定量PCR數據顯示,NaCl、ABA、PEG顯著誘導了葉片中CdSAP1表達,誘導12 h時表達量最高。(2)將CdSAP1基因異源轉化擬南芥,耐鹽表型鑒定分析表明,轉CdSAP1顯著提高了擬南芥的耐鹽性。鹽脅迫下,轉CdSAP1基因擬南芥的種子萌發(fā)率顯著提高,葉片枯黃率、電解質滲漏率顯著下降;轉基因擬南芥能保持較高的葉綠素含量、光化學效率、凈光合速率和較低的胞間CO2濃度;鹽脅迫下轉基因擬南芥通過減少Na+積累,維持較低的Na+/K+;轉CdSAP1基因促進了脯氨酸及可溶性糖的積累,顯著降低了H2O2和O2-的積累。(3)對CdSAP1耐鹽相關基因的表達分析顯示,鹽脅迫下CdSAP1過量表達顯著提高了 葉片中 AtNHX1、AtP5CS、AtAPX、AtSOD、AtSOS1、和 AtPOD 基因的表達,未影響AtAKTI、AtBADH的表達。CdSAP1過量表達顯著提高了根系中AtNHX1、AtAPX、AtSOD、AtPOD 和 AtBADH 的表達,而 AtSOS1、AtAKTI、AtP5CS基因的表達未受影響。
[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.
【學位授予單位】：南京農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S688.4

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