摘 要:Cl-毒害是盐害的一个主要因素，氯离子通道蛋白基因（CLC）可能是植物调控Cl-平衡的一个关键基因。本研究对抗盐型草坪草‘阳江’狗牙根进行CdCLCc基因全长克隆，并进行生物信息学和荧光定量表达分析。结果表明，此基因全长2205 bp，编码734个氨基酸，与AtCLCc和OsCLCc蛋白分别有66%和78。5%相似性，表明CdCLCc和已知植物的CLCc基因有非常高的同源性。通过蛋白的三维结构模拟，发现其与AtCLCc和OsCLCc蛋白存在较大的差异，提示其功能上可能存在较大的不同。荧光定量PCR分析证明盐胁迫下CdCLCc在抗盐型狗牙根中表达量较高。对狗牙根盐胁迫下植株Cl-分析表明，抗盐型材料具有较强的把Cl-隔离在根系的能力，这可能与此基因具有较强的把Cl-隔离在根系细胞液泡中的能力有关。76756

毕业论文关键词：狗牙根，氯离子通道，克隆，生物信息学

Abstract: The chloride ion toxicity is one of the main factors of salt injury, and chloride channel (CLC) is a key gene for the regulation of chloride ion homeostasis in plants。 In this study, we cloned a CdCLCc gene from the salt-tolerant bermudagrass cultivar, ‘Yangjiang’, and then the bioinformatics and fluorescence quantitative expression of this gene was analyzed。 The result indicated the CdCLCc is 2205 bp in length and encoded 734 amino acids。 The protein of CdCLCc had 66% and 78。5% similarity with AtCLCc and OsCLCc proteins, respectively, which showed that CdCLCc had the very high homology with known plant CLCc genes。 Through simulated the three-dimensional structure of the protein, we found there were many differences between CdCLCc and other two proteins, prompted they may differ in larger on its function。 Fluorescence quantitative PCR analysis showed that CdCLCc in salt-tolerant bermudagrass had higher expression under salt stress。 Through analyzed the Cl- contents in bermudagrass under salt stress, we found salt-tolerant bermudagrass had stronger ability of isolating Cl- in the root, which may be related to the CdCLCc gene had stronger ability of pumping Cl- into the vacuole of root cell。

Key words: Cynodon dactylon, chloride channel, cloning, bioinformatics

目 录

1  前言 5

2  材料和方法 7

2。1  植物材料 7

2。2  植物材料RNA提取 7

2。3  RNA质量检测 8

2。4  mRNA反转录合成cDNA 8

2。5  狗牙根CLC（氯离子通道蛋白）基因的克隆 9

2。5。1  中间片段获取 9

2。5。2  5’端RACE 9

2。6  CdCLCc的生物信息学分析 10

2。7  CdCLCc的荧光定量PCR（qRT-PCR）分析 10

2。8  盐胁迫下狗牙根体内氯离子的含量测定 11

2。9  数据分析 11

3  实验结果及分析 11

3。1  CdCLCc基因的全长克隆 11

3。2  CdCLCc基因的生物信息学分析 12

3。2。1  CdCLCc蛋白的氨基酸序列比对 12

3。2。2  CdCLCc蛋白的三维结构分析 13

3。2。3  CdCLCc基因的系统进化树分析