|
|||||
|
|
| 哈茨木霉(Trichoderma harzianum)多种胞壁降解酶基因表达载体的构建及转化水稻 | |
| 引用本文： | 刘梅,孙宗修,徐同.哈茨木霉(Trichoderma harzianum)多种胞壁降解酶基因表达载体的构建及转化水稻[J].浙江大学学报(农业与生命科学版),2004,30(6):596-602. |
| 作者姓名： | 刘梅 孙宗修 徐同 |
| 作者单位： | 1. 浙江大学,植保系,浙江,杭州,310029;中国水稻研究所,农业部水稻生物学重点实验室,浙江,杭州,310006 2. 中国水稻研究所,农业部水稻生物学重点实验室,浙江,杭州,310006 3. 浙江大学,植保系,浙江,杭州,310029 |
| 基金项目： | 国家自然科学基金，中-意合作项目，浙江省科技厅资助项目，云南大学校科研和教改项目 |
| 摘 要： | 为提高水稻的抗病性,利用哈茨木霉(Trichoderma hazianum)P1菌株的3个胞壁降解酶基因ech42、nag70与gluc78构建了7个植物表达载体,每个基因受独立的Act1启动子调控.构建的7个载体不仅包含3个外源基因的所有组合(A,B,C,A+B,A+C,B+C,A+B+C),而且具有双元载体本身携带的HPT基因与Gus基因,为研究不同T-DNA长度、不同基因组合与不同基因排列方向对植物遗传转化效率以及外源基因在转基因植株中表达的影响提供了一套比较完整的材料.利用本实验室的农杆菌高效转化体系,将所有组合的7个载体分别转入粳稻品种石狩白毛(Oryza sativa L ssp. Japonica cv. Ishikari-shiroge)中,共获得再生植株1800余株.对部分再生植株进行了PCR检测,证明96%的植株至少携带有外源基因中的一个,80%以上的植株整合有完整的外源基因片断. |
| 关 键 词： | 哈茨木霉(Trichoderma hazianum) 胞壁降解酶 植物表达载体 农杆菌介导法转化 水稻 |
| 文章编号： | 1008-9209(2004)06-0596-07 |
| 修稿时间： | 2003年9月15日 |
Construction of several plant expression vectors using multiple cell wall degrading enzyme genes from Trichoderma harzianum and rice transformation |
|
| LIU Mei.Construction of several plant expression vectors using multiple cell wall degrading enzyme genes from Trichoderma harzianum and rice transformation[J].Journal of Zhejiang University(Agriculture & Life Sciences),2004,30(6):596-602. | |
| Authors: | LIU Mei |
| Abstract: | In order to enhance rice resistance against fungal pathogens by applying the synergistic interaction of different cell wall degrading enzymes from Trichoderma, three different cell wall degrading enzyme genes from Trichoderma harzianum P1 were placed respectively downstream of Act1 promoter. Based on this work, 7 different plant expression vectors were constructed by cloning each gene alone or either two genes in combination or three genes together into binary vector pCAMBIA1305.2. Three kinds of methods was used to solve different problems in the process of vector construction: (1) Restriction solution was used directly to ligation reaction for recovered DNA fragment usually influenced ligation frequency; (2) Using one cohensive end and one blunted end to carry out the ligation reaction was more effective than two blunted ends when restriction ends must be modified; (3) Choosing appropriate mediated plasmid to introduce new restriction sites around certain DNA fragment and avoid linear ends modification. The constructed vectors also carried hpt gene and Gus gene which was already placed in two sides of the multiple cloning sites of binary vector pCAMBIA1305.2, that provided a suit of systematic materials for further study on the influence of T-DNA size, gene combination and gene direction on transformation frequency and gene expression. Seven combinations of CWDE genes were transformed into Oryza sativa L ssp. Japonica cv. Ishikari-shiroge respectively mediated by Agrobacterium tumefaciens. More than 1,800 independent regenerated plantlets of seven populations were obtained. PCR analysis of part of transgenic plants revealed that about 96 percent of them integrated with at least one out of three exogenous genes, more than 80 percent had intact exogenous fragment. |
| Keywords: | Trichoderma harzianum cell wall degrading enzymes plant expression vector agrobacterium-mediated transformation rice |
| 本文献已被 CNKI 维普 万方数据 等数据库收录！ | |
| 点击此处可从《浙江大学学报(农业与生命科学版)》浏览原始摘要信息 | |
| 点击此处可从《浙江大学学报(农业与生命科学版)》下载免费的PDF全文 | |