两种暖季型草坪草对低温的响应机制及ZjADH基因的克隆与功能分析

发布时间：2017-12-28 03:28

本文关键词：两种暖季型草坪草对低温的响应机制及ZjADH基因的克隆与功能分析　出处：《北京林业大学》2016年博士论文　论文类型：学位论文

【摘要】：日本结缕草及普通狗牙根均为典型的暖季型草坪草,在我国南北地区均有分布。低温伤害是暖季型草坪草在北方地区应用时经常遇到的一种灾害,它不仅会导致草坪草的生长受到损害,甚至会引起植株死亡。研究低温胁迫对暖季型草坪草的伤害机理,探索其抗寒机制具有重要的理论和实际应用意义。ADH基因在植物抵抗逆境胁迫包括低温逆境起着重要的作用,分离并分析日本结缕草ADH基因对于更好的理解结缕草低温响应起着重要的作用。本研究以普通狗牙根和日本结缕草的匍匐茎为材料,研究二者在低温胁迫条件下相关生理指标及分子指标的动态变化,探讨寒冷胁迫对其生理及分子层面的影响；针对抗寒性较强的日本结缕草,设计引物克隆日本结缕草ADH基因全长序列,将其命名为ZjADH,对其进行序列分析和功能分析。研究结果如下：1.低温胁迫下,草坪表观质量下降,两种草坪草的叶绿素含量、类胡萝卜素含量、光化学效率逐渐下降,脯氨酸、可溶性糖及可溶性蛋白逐渐积累,电解质渗透率及丙二醛含量逐渐上升,且低温锻炼能显著降低日本结缕草和普通狗牙根的低温半致死温度。伤害指标评价结果表明日本结缕草抗寒性强于普通狗牙根。2.随着低温胁迫时间的增长,日本结缕草和普通狗牙根的过氧化氢及超氧阴离子含量逐渐增加,SOD和POD活性逐渐升高,APX活性则先升高后降低。CytSOD、 MnSOD、POD、APX基因表达量出现上调,CAT及ChlSOD基因表达量则逐渐下降,FeSOD表达量则先下降再升高。研究表明,低温胁迫下,植株启动抗氧化机制,清除活性氧,降低植物损伤。3.低温胁迫下,两种草坪草的ABA含量呈先升高再降低,CTK含量则逐渐下降。随着胁迫时间的增长,LEA基因表达量逐渐上调,RAB基因表达量先升高再下降。内源激素的变化及低温诱导蛋白的表达对于激活响应机制,提高植株抗寒性具有重要作用。4.设计用于RACE反应的两条引物,进行3'RACE和5'RACE扩增,得到765bp和816bp的片段。通过序列拼接获得该基因的cDNA,序列分析该基因开放阅读框为1140-bp,编码一条含有379个氨基酸的多肽链。将该氨基酸序列进行Blast比较,构建ADH蛋白的系统发育树,发现日本结缕草与沟叶结缕草的ADH亲缘关系最近。克隆的基因为日本结缕草ADH基因,命名为ZjADH, Genbank中登陆号为KT596065。5.通过实时定量RT-PCR方法分析了ZjADH基因在日本结缕草不同组织中转录水平上的表达情况。结果显示,ZjADH基因在根中表达量最高,在叶中表达量最低；ZjADH在低温、干旱和高盐胁迫条件下表达量升高,并且可受到ABA、MeJA和SA信号诱导。6.将含有完整开放性阅读框的ZjADH基因片段插入3302-YFP载体,构建了融合蛋白表达载体ZjADH-YFP,注射法转化烟草叶肉细胞后,利用激光共聚焦显微镜观察发现该蛋白定位于细胞质。7.构建植物表达载体3302-ZjADH,转入到农杆菌GV3101中,通过花序浸泡法,转化野生型拟南芥植株。经草铵膦筛选得到具有草铵膦抗性的幼苗,提取抗性拟南芥叶片DNA,进行PCR检测,证实成功获得转基因植株。提取转基因植株中的总RNA,通过RT-PCR检测,结果显示,转基因拟南芥能够高效表达ZjADH基因。8.对转基因拟南芥进行抗性检测,结果显示：转基因拟南芥植株对低温的抗性明显高于野生型拟南芥；转基因拟南芥中与胁迫相关的基因表达水平明显高于野生型拟南芥植株。对转基因植株进行生理生化分析,结果显示：转基因植物的脯氨酸含量高于野生型拟南芥。本研究表明,低温胁迫下,日本结缕草和普通狗牙根通过渗透调节物质积累、抗氧化代谢、内源激素及低温诱导蛋白的表达,提高植株抗寒性,且日本结缕草抗寒性强于普通狗牙根。以抗寒性较强的日本结缕草为材料,获得了日本结缕草ADH基因,全长1140bp,定位于细胞质,该基因在根中表达量最高,在逆境胁迫条件下,表达量升高显著,成功获得了转基因拟南芥植株,转基因拟南芥对低温抗性与野生型比起来明显提高,初步证明日本结缕草ADH基因与植物逆境响应及抗寒性有关。
[Abstract]:Zoysia japonica and common dog root are typical warm season turfgrass, which are distributed in the north and south of China. Low temperature injury is a common calamity often encountered in the application of warm season turfgrass in the northern area. It will not only cause damage to the growth of turfgrass, but even cause plant death. It is of great theoretical and practical significance to study the damage mechanism of low temperature stress on warm season turfgrass and explore its cold resistance mechanism. ADH gene plays an important role in plant resistance to stress, including low temperature stress. Isolation and analysis of ADH gene from Zoysia japonica plays an important role in better understanding of low temperature response of Zoysia japonica. In this study, Common Bermudagrass and zoysiagrass stolon as materials, study of the two dynamic changes in physiological indexes and molecular indexes under low temperature stress, cold stress to investigate the effects on the physiological and molecular level; the cold resistance of Zoysia japonica strong, primers were designed to clone the full-length sequence of Japan. Zoysia ADH gene, named ZjADH, analyze sequence and function of the. The results are as follows: 1. under low temperature stress, the apparent decline in the quality of the lawn, the chlorophyll content, two kinds of lawn grass carotenoid content, photochemical efficiency decreased, proline, soluble sugar and soluble protein accumulation, membrane permeability and MDA content increased gradually, and the low temperature exercise can significantly reduce the Z.japonica and ordinary Bermuda semilethal temperature. The result of injury index evaluation showed that the cold resistance of Zoysia japonica was stronger than that of common dog root. 2., with the increase of low temperature stress time, the content of hydrogen peroxide and superoxide anion increased gradually, and the activity of SOD and POD increased gradually, while the activity of APX increased first and then decreased. The expression of CytSOD, MnSOD, POD and APX was up-regulated, while the expression of CAT and ChlSOD decreased gradually, while the expression of FeSOD decreased first and then increased. The study showed that under low temperature stress, the plant started antioxidant mechanism, scavenging reactive oxygen species and reducing plant damage. 3. under low temperature stress, the ABA content of two turf grass increased first and then decreased, while the content of CTK decreased gradually. With the increase of stress time, the expression of LEA gene increased gradually, and the expression of RAB gene increased first and then decreased. The changes of endogenous hormones and the expression of low temperature induced proteins play an important role in activating the response mechanism and improving the cold resistance of plants. 4. the two primers used for RACE reaction were designed to amplify 3'RACE and 5'RACE, and the fragments of 765bp and 816bp were obtained. The cDNA of the gene was obtained by sequence splicing, and the sequence analysis of the gene opened the open reading frame to 1140-bp and encodes a polypeptide chain containing 379 amino acids. The phylogenetic tree of the ADH protein was constructed by comparing the amino acid sequence with Blast, and the closest relationship between Zoysia japonica and the ADH of Zoysia japonica was found. The cloned gene is the ADH gene of Zoysia japonica, named ZjADH, and the landfall number in Genbank is KT596065. 5. the expression of ZjADH gene in the transcriptional level of Zoysia japonica was analyzed by real-time quantitative RT-PCR method. The results showed that the expression level of ZjADH gene in roots was the highest, and the expression level in leaves was the lowest. ZjADH expression was increased under low temperature, drought and high salt stress, and it could be induced by ABA, MeJA and SA signals. 6., insert the ZjADH gene fragment containing a complete open reading frame into the 3302-YFP vector, construct the fusion protein expression vector ZjADH-YFP, and transform it into tobacco mesophyll cells by injection, then we observed the localization of the protein in cytoplasm by laser scanning confocal microscopy. 7. the plant expression vector 3302-ZjADH was constructed and transferred into Agrobacterium tumefaciens GV3101 to convert wild type Arabidopsis plants through inflorescence soaking. With glufosinate resistance seedlings by glufosinate screening, extraction resistance of Arabidopsis thaliana leaves DNA, PCR detection, transgenic plants were obtained successfully. The total RNA in transgenic plants was extracted and detected by RT-PCR. The results showed that transgenic Arabidopsis could express the ZjADH gene efficiently. 8., resistance detection of transgenic Arabidopsis thaliana showed that the resistance of transgenic Arabidopsis plants to low temperature was significantly higher than that of wild Arabidopsis thaliana, and the expression level of stress related genes in transgenic Arabidopsis thaliana was significantly higher than that in wild Arabidopsis thaliana plants. The results of physiological and biochemical analysis of transgenic plants showed that the proline content of transgenic plants was higher than that of wild Arabidopsis. This study showed that under low temperature stress, Japanese Zoysia and Common Bermudagrass increased plant cold tolerance through osmotic adjustment substance accumulation, antioxidant metabolism, endogenous hormones and low temperature induced protein expression, and the cold resistance of Zoysia japonica was stronger than that of Common Bermudagrass. The cold resistance of Zoysia japonica as strong material, obtained the Zoysia japonica ADH gene, full-length 1140bp, localized in the cytoplasm, the gene expression level in root was highest in the stress condition, the expression level increased significantly, successfully obtained the transgenic Arabidopsis plants, transgenic Arabidopsis wild and resistance to low temperature type than increased significantly, preliminary evidence of Zoysia japonica ADH gene related to plant stress response and cold resistance.
【学位授予单位】：北京林业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S688.4

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