哈茨木霉T88+perg-22基因的功能研究.pdf

Classified Index: Q933
: 579





Dissertation for the Master Degree in Science



FUNCTIONAL STUDY ON GENE PERG-22 IN
Trichoderma harzianum T88







Candidate: Qu Lianhai
Supervisor: Qian
Academic Degree Applied for: Master of Science
Speciality: Biology
Affiliation: School of Life Science and Technology
Date of Defence: July, 2012
Degree-Conferring-Institution: Harbin Institute of Technology
哈尔滨工业大学理学硕士学位论文
摘要
利用木霉菌作为生物农药来防治各种病原真菌已经在全世界范围内引起研究
者们越来越大的兴趣。本论文以哈茨木霉 T88 为研究对象,采用 RT-PCR 和 PCR
获得了哈茨木霉 T88 perg-22 基因的全部编码序列,该基因编码 616 个氨基酸,理
论分子量为 kD。
利用原始质粒 pSilent-1、pCambia1301 及 perg-22 基因的 EST 序列,构建了该
基因的沉默载体,成功转化了根癌农杆菌 EHA105。通过农杆菌介导的木霉菌遗传
转化体系,获得了哈茨木霉 T88 的转化子。实验中首次采用液体共培养法进行了
木霉菌的诱导转化,并与常用的转膜培养法和复铺培养基法进行了转化效率的比
较,发现液体共培养法的转化效率是后两者的 5-10 倍。
Real Time PCR 检测结果表明转化子中 perg-22 基因的表达量下降了 %,
与能量代谢相关的多条基因表达量也都有不同程度(53%-90%)的下降,表明该
基因与哈茨木霉 T88 的能量代谢有关。检测到转化子中麦角甾醇表达量下降了
%,说明该基因与哈茨木霉 T88 麦角甾醇的合成相关。这一结果,对研究真
菌麦角甾醇的生产以及木霉菌菌剂的工业化有重大的意义。

关键词:哈茨木霉;农杆菌介导;能量代谢;麦角甾醇


- I -
哈尔滨工业大学理学硕士学位论文
Abstract
As a biological pesticide to prevent and treat a variety of pathogenic fungi,
Trichoderma has caused a growing interest of worldwide researchers. This study Select
Trichoderma harzianum T88 as the object, coding sequence of perg-22 has been
obtained by RT-PCR and PCR, this gene encodes 616 amino acids, it’s theoretical
molecular weight is kD.
By using the EST sequence of perg-22 gene and the original plasmid pSilent-1 and
pCambia1301, the gene silencing vector is constructed, and Agrobacterium tumefaciens
EHA105 is essfully transformed. Transformants of T. harzianum T88 were obtained
by Agrobacterium-mediated transformation liquid co-cultured method is
used in the experiment for the first time, and it’s conversion efficiency is about 5-10
times of transfer film culture method plex medium method.
Real Time PCR test results showed