【作者】 甘德芳；

【导师】 程备久； 朱苏文；

【作者基本信息】 安徽农业大学 ， 生物信息与分子育种， 2006， 硕士

【摘要】 几丁质酶是一种能催化真菌细胞壁的重要成分—几丁质降解的糖苷酶，普遍存在于大白菜、烟草等高等植物中，因在抑制真菌的生长增殖、提高植物的抗病性等方面具有重要作用，现已成为当前科学研究的热点之一。本文通过PCR反应及RACE技术，从大白菜基因组DNA及RNA中分别扩增出大白菜几丁质酶全基因及成熟肽基因，并对其进行了生物信息学分析；构建原核表达载体pET-CHB4，通过IPTG诱导并对该基因表达的蛋白产物进行SDS-PAGE分析、生物活性和功能分析。结果表明：1．根据已知十字花科植物几丁质酶基因的保守序列设计特异性引物，以大白菜叶片基因组DNA为模板，通过PCR反应扩增出长度约为1Kb的特异性DNA片段和几丁质酶N端序列片段（150bp）。选取经1mmol．L^-1水杨酸处理后的大白菜植株叶片提取RNA，利用RACE技术扩增该基因的3′端436bp。2．以大白菜叶片基因组DNA为模板，利用PCR反应扩增出长度为1517 bp的几丁质酶全基因；以水杨酸诱导的大白菜叶片RNA为模板，通过RT-PCR反应扩增出长度为811bp几丁质酶成熟肽基因。3．生物信息学分析表明，该几丁质酶基因全长1517bp，其中内含子序列长度为508bp（DDBJ登录号为AB257452），编码由268个氨基酸残基组成的蛋白质（pI／Mw：8.28／28701.23，www.expasy.org）。与其他物种ClassⅣ类几丁质酶氨基酸序列的多重比较表明（DNAMAN），与油菜ClassⅣ类几丁质酶的同源性达99％，与其他已知高等植物ClassⅣ类几丁质酶的同源性达50％以上。说明所克隆的大白菜几丁质酶基因是编码ClassⅣ类几丁质酶的基因。4．构建了原核表达载体pET-CHB4，利用IPTG诱导表达，SDS-PAGE分析表明，该基因表达的蛋白分子量为-28kD左右，其表达产物主要以包涵体的形式存在。5．通过改变诱导表达的参数发现，25℃的低温并不能改变表达蛋白的可溶性，而在pH9.5的培养基条件下，表达产物的上清液中却有重组蛋白条带出现，表明碱性环境有利于蛋白质的折叠和二硫键的交换。6．以酵母菌为指示菌做抑菌圈试验，结果显示，经不同浓度的IPTG诱导表达的蛋白液处理后，抑菌圈大小变化明显，当IPTG浓度为0.6mmol／L时抑菌效果最佳。7．通过对不同IPTG诱导表达的几丁质酶活力的测定，结果表明，当IPTG浓度为0.6mmol／L时，几丁质酶活力达到最大值2.8U，明显高于原菌液几丁质酶活力1.2U。综上所述，本文成功地从大白菜叶片中克隆出几丁质酶CHB4基因及其成熟肽基因，构建了高效原核表达载体pET-CHB4，优化了该几丁质酶诱导表达的参数，并对该酶进行了功能分析，为该酶的纯化及酶学性质研究提供基本资料，也为进一步转化其他植物，以期获得抗真菌病害的转基因植株提供理论依据。更多还原

【Abstract】 Chitin is the major ingredient of most fungal cell walls. Chitinase, widely existing in higher plant, could catalyze the hydrolysis of chitin and played a very important role in biophylaxis. With PCR and RACE techniques, the full and the mature gene encoding chitinase were isolated from Chinese cabbage genome DNA and cDNA, which was analyzed by bioinformatics method. Prokaryote expression vector pET-CHB4 was constructed and expressed in BL21. The products were analyzed by SDS-PAGE. Biological activity and the function identification were carried out. The results were as follows.1. A specific DNA fragment about 10kb and N-end fragment about 150bp were amplified from genome DNA of Chinese cabbage, and 3’end fragment was amplified by 3’RACE.2. The chitinase gene CHB4 was amplified from genome DNA by PCR and its mature gene was gained by RT-PCR reaction from cDNA.3. Comparing Chinese cabbage chitinase sequence cloned in this experiment with that reported in GenBank composed of 1094bp nucleotide of Brassica napus. It showed that the chitinase sequence of Chinese cabbage is 1517bp in size and contained 1 intron of 508bp (DDBJ accession No. AB257452), encoding a polypeptide of 268 amino acid residues (pI/Mw: 8.28 / 28701.23, www.expasy.org). Analysis of amino acid sequence of several CHIV from different species indicates that it shares 99% identity with brassica napus and over 50% with other higher plants in coding region (DNAMAN). The result suggests that the chitinase gene may function similarly to chitinase of ClassIV.4. Utilizing vector pET-22b(+) to construct expression vector pET-CHB4,through the expression vector induced to express, the result showed that the protein molecular of this expressive gene was 28kDa by analysis of SDS-PAGE and its expression products were mostly composed of inclusion body.5. Through changing parameters of induced expressing, the result showed that solubility of expression products was not change in 25℃,on the other hand, There were recombinant protein bands in lysis supernatant while pH9.5.6. Take Microzyme as demonstrative bacterium to take test on restrain bacterium circle, the result showed that the change of restrain bacterium circles were comparatively rernarkable through dealing with different densities of IPTG induced expression protein liquid ,and the restrain bacterium effect was remarkable in 0.6mmol/L of IPTG density. 7. Through mensurating chitinase activity under induced expression by a series of IPTG, the result showed, at the 0.6mmol/L IPTG , chitinase activity reached maximum 2.8U which was higher than that of original bacterium liquid 1.2U.In sum, the paper had succeeded to clone chitinase gene CHB4 and its mature gene from Chinese cabbage, and construct high-efficient prokaryotic expression vector pET-CHB4, optimize the induced expressive parameter of project bacterium, analyze the function of the chitinase. These results had provided theoretical foundation for the more research of transforming other plants and obtaining anti-disease transgenic plants.更多还原