【作者】 张岳民；

【导师】 郭兴启； 郑成超；

【作者基本信息】 山东农业大学 ， 生物化学及分子生物学， 2004， 硕士

【摘要】 植物真菌病害一直是影响农作物产量,降低农作物品质的主要原因。在植物抗病基因工程中,通过将抗真菌蛋白基因导入植物来提高植物对真菌病害的抵抗能力已成为一种直接而有效的方法。随着越来越多的抗真菌蛋白被发现,人们试图采用多基因共表达的策略来将多种抗真菌蛋白基因转入植物,以提高植物的抗病能力和抗病谱。但是,由于受到目前可选择的载体数目和载体容量的限制,这个目的很难较好的实现。融合基因技术就是近几年出现的用于解决这一难题的有效途径。通过构建融合基因表达融合蛋白,可以实现多个基因的同步、同水平、同细胞定位表达。正是由于上述优点,融合基因技术正越来越受到人们的关注。本研究选择烟草Ⅰ类几丁质酶(CHI)、小麦硫堇蛋白(THI)和烟草Ⅰ类 β-1,3-葡聚糖酶(GLU)三个已经证明是具有很强抗真菌病害能力的植物抗病蛋白,通过构建融合基因来实现三个蛋白在烟草体内的共表达,以求达到提高烟草对真菌病害的抵抗能力的目的。主要实验结果如下: 1.克隆并修饰了三个抗菌蛋白基因,即烟草Ⅰ类几丁质酶基因(CHI)、小麦硫堇蛋白基因(THI)和烟草Ⅰ类 β-1,3-葡聚糖酶基因(GLU),利用它们构建了两个融合蛋白基因,FG1 和 FG2,其中 FG2 的 3′-端含有一段编码胞内定位信号的序列,而 FG1 的 3′-端则无胞内定位信号序列。 2.构建了含有融合基因的植物表达载体 pBI-FG1、pBI-FG2 和pMR-FG1,同时构建了三个单独基因(CHI、THI 和 GLU)的植物表达载体 pBI-CHI、pBI-THI 和 pBI-GLU,作为对照,用于检测融合基因的抗病能力。 3.利用农杆菌介导法分别获得了转 pBI-FG1、pBI-FG2、pMR-FG1、pBI-CHI、pBI-THI 和 pBI-GLU 六个类群的转基因烟草植株。 4.对 pBI-FG1 表达的融合蛋白的细胞定位进行了检测分析,证明其分泌在细胞间隙,而 pBI-FG2 表达的融合基因定位在细胞内。对融合蛋白各组分蛋白的活性检测表明:三个组分蛋白在融合蛋白中均保持了各自的生物活性。 5.用烟草赤星病原菌(Alternaria alternata)和烟草炭疽病原菌(Colletotrichum corda)对融合蛋白的抗菌能力进行了体外鉴定,发现其 —1—<WP=7>张岳民:三价抗真菌融合基因植物表达载体的构建及转基因烟草的抗病性分析对这两种病原菌的抑制能力大大高于对照蛋白。同时对转融合基因的烟草植株进行了活体接菌实验,发现其抗病能力高于对照植株。 6.对部分 pMR-FG1 和 pBI-FG1 转化植株的 Northern blot 和 Westernblot 分析证明,pMR-FG1 转化植株中融合蛋白的表达在转录水平和翻译水平明显高于 pBI-FG1 转化植株。更多还原

【Abstract】 Plant protection is a major challenge to agriculture worldwide.Significant yield losses due to fungal attack limits crops productivity and canbe very severe local epidemic infections. Transferring antifungal protein genesto plants becomes a most direct and efficient way to obtain fungi resistantplants and has attracted many researchers’ attention because of its highbio-safety and long duration. With more and more antifungal proteins werefound and applied, the problem of multi-gene transforming and expressionarose. Gene fusion, as a novel technology, is a most efficient way to solve thisproblem. In this study, we constructed a fusion gene with tobacco class Ichitinase gene, wheat thionin gene and tobacco class I β-1,3-glucanase gene,and transferred corresponding expression vectors to tobacco, then realized theco-expression of the three antifungal protein genes and enhanced resistanceagainst fungal disease. The main results are as follows: 1. Three antifungal protein genes, tobacco class I chitinase gene, wheatthionin gene and tobacco class I β-1,3-glucanase gene were cloned andmodified slightly used to construct two fusion genes, FG1, FG2, and FG2contains a short carboxyl-terminal propeptide that mediates transport to plantvacuole. 2. Three plant expression vectors containing the fusion genes wereconstructed, and named pBI-FG1, pBI-FG2 and pMR-FG1, respectively. Inaddition, another three vectors containing CHI, THI, GLU, respectively, wereconstructed as control. 3. Six group transgenic tobacco plants were obtained by the standard leaf —3—<WP=9>张岳民:三价抗真菌融合基因植物表达载体的构建及转基因烟草的抗病性分析disc transformation method using kanamycin selection. 4. Expression analysis of transgenic tobacco plants containing FG1 orFG2 constructs showed that the FG1 was secreted extracellularly, while FG2was sorted to the vacuole because it has a targeting information resides in thecarboxyl-terminal propeptide. In addition, they retained their biologicalactivities respectively. 5. The performance of tobacco plants expressing the fusion gene in vitroand in vivo assay revealed significantly enhanced protection against fungalattack when compared with control plants. 6. Northern and Western blot analysis in pMR-FG1 and pBI-FG1transformants showed that pMR-FG1 can improve the gene expression both intranscription level and in translation level.更多还原