【作者】 王轶；

【导师】 李旭锋； 杨毅；

【作者基本信息】 四川大学 ， 遗传学， 2005， 硕士

【摘要】 植物防御素是一类广泛分布于植物种子的阳离子、低分子量的短肽。其能强烈抑制病原微生物的入侵,是植物先天性非特异免疫的重要组成部分。尤其是被认为能通过真菌细胞膜上特异受体而强烈抑制丝状真菌的生长。研究表明,其保守的含有8个Cys,而形成4个二硫键。因其与动物防御素同源而得名。 本实验根据GenBank数据库中登记的十字花科植物防御素基因序列,运用Primer5.0软件设计了两个引物5’-GCGGAATTCATGGCTAAGTTTGCTTCC-‘3和5’-CGCTCTAGATTAACATGGGAAATAACAGATAC-‘3,并为后插入到改造自Pharmacia公司的pGEX—2T克隆表达载体GTK中而加入了EcoRⅠ和XbaⅠ的酶切位点(粗体部分)。从正在萌发的甘蓝型油菜“蜀杂九号”的种子提取总RNA,反转录为cDNA。并以此为模板进行PCR扩增,成功地从甘蓝型油菜中克隆出长243bp编码80个氨基酸的防御素基因的cDNA序列。 将所得甘蓝型油菜防御素基因的cDNA片段插入到pGEX—2T表达载体GTK的EcoRⅠ和XbaⅠ多克隆位点之间。将重组原核表达质粒转化到E.coli BL21的感受态细胞中,获得了表达甘蓝型油菜防御素基因的重组子。在2×YTA培养基中,通过0.1mM的IPTG诱导,18℃培养2h,经SDS-PAGE分析,获得了与理论值相符合的34KD的融合蛋白。而且经Western-blot进一步检测,证明了融合蛋白的表达。 在融合蛋白的初步纯化中发现:不论是改变IPTG浓度还是降低培养温度,融合蛋白均以不溶性的包涵体形式存在。所以选择对细胞毒副作用小的0.1mM更多还原

【Abstract】 Plant defensins, a kind of low MW cationic peptides widely distributed in many plant seeds, have strong ability to inhibit pathogen. As multifunctional effetor molecules of non-specificity innate immunity, defensins are the key components in plant immunity system. Plant defensins are thought to interact with specific receptor harboring in the cell membrane and inhibit the growth of fungi, especially filamentous fungi. Plant defensins have conserva(?)e eight Cys residues and form four intramolecular cysteine disulfide bonds.According to the known sequences of Cruciferae plant defensin genes from Gen-Bank, two primers, 5’ -GCGGAATTCAT GGCTA AGTT TGCT T CC- ’3 and 5’-CGC TCTAGATTA ACATGG GAA A TAACAGATAC- ’3, were designed. The total RNA was extracted from Brassica Napus(Shuza No, 9) seeds and reverted into cDNA, then amplifyied plant defensin gene by using PCR. The plant defensin cDNA sequence, with the length of 243bp coding 80 amino acids, was successfully cloned from Brassica Napus.The target DNA was inserted into the expression vector, GTK, which is derived from the pGEX—2T, then transformed to E. coli BL21. Having been cultivated in 2 X YTA medium and induced by 0. lmM IPTG at 18 °C for 2 hours, the E. coli BL21 effectively expressed the prospective 34kD fussion protein which could be detected by SDS-PAGE. Moreover, the result of Western-blot proved this conclusion.In the course of the fussion protein purification , the fussion protein was expressed as the form of inclusion bodies in E. coli either varying concentration of IPTG or adjusting the cultivated temperature. While IPTG is toxicity to cells and expression system has relative higher expression efficiency induced at 18°C, we selected 0.1 mM IPTG and induction at 18°C as the condition of the fussion protein purification. Then a series of purification steps were done, including supersonic breaking cell, inclusion bodies washing, inclusion bodies soluting in a buffer containing 8M urea, 50mM Tris- HCl(PH8.0) and 0.4% ME. Generally, this work gave the prerequisite conditions to further purification of Brassica napus defensins and took the first step of more advanced research aimed at regulators of plant defensin genes.更多还原