稻瘟病菌大片段DNA转化载体的构建

doi:10.3969/j.issn.1001-7216.2015.01.011

摘要/Abstract

摘要：

构建能够转化大片段DNA的载体是进行基因簇功能分析的重要手段。利用cosmid载体和pBHt2载体构建了一个可以在稻瘟病菌中转化大片段DNA的载体,通过农杆菌介导的转化方法,成功地将超过30 kb的大片段DNA转化到稻瘟病菌中。研究结果将促进稻瘟病菌基因簇的功能分析。

关键词: 载体, 转化, 大片段DNA, 稻瘟病菌

Abstract:

It is an important means to study function of gene cluster using special vectors for large DNA fragment transformation. A vector was constructed for large DNA fragment transformation based on cosmid super Cos I vector and pBHt2 vector. More than 30 kb DNA fragment was transferred into the rice blast fungus successfully using Agrobacterium tumefaciens-mediated transformation. The results will promote functional analysis of gene cluster in Magnaporthe oryzae.

Key words: vector, transformation, large DNA fragment, Magnaporthe oryzae

中图分类号:

陈亚平, 施文骁, 王洪凯. 稻瘟病菌大片段DNA转化载体的构建[J]. 中国水稻科学, 2015, 29(1): 91-96.

Ya-ping CHEN, Wen-xiao SHI, Hong-kai WANG. Construction of a Vector for Large DNA Fragment Transformation in Magnaporthe oryzae[J]. Chinese Journal OF Rice Science, 2015, 29(1): 91-96.

图/表 6

图1 Cos-cam融合质粒载体的构建 LB-左边界; RB-右边界。

Fig.1. Construction of Cos-cam vector. LB, Left border; RB, Right border; S, SacⅡ; E, EcoRⅠ; B, BamHⅠ; X, XbaⅠ.

图2 构建的Cos-cam的融合质粒载体的酶切验证结果 M-1 kb+ DNA 梯度标记; 1-完整的Cos-cam载体; 2-酶切后的载体。

Fig. 2. Electrophoresis pattern of Cos-cam vector digested with BamHⅠand XbaⅠ. M, 1 kb+ DNA Ladder marker; 1, Cos-cam vector; 2, Cos-cam vector digested with BamHⅠand XbaⅠ.

图3 用cos-cam载体构建基因组文库时连接效果的检测 M-1 kb DNA梯度标记; 1-连接前的样品; 2-连接后的样品; 3-没有加连接酶的样品。

Fig. 3. Ligation results of Cos-cam vector and genomic DNA. M, 1 kb Ladder DNA marker; 1, Sample before ligation; 2,Ligated sample; 3, Non-ligase sample.

图4 用EcoRⅠ酶切 30个Cos-cam克隆的电泳结果 M1-DL15000 DNA 标记; M2-1 kb+DNA梯度标记; 1~30-30个随机克隆。

Fig. 4. Electrophoresis pattern of Cos-cam clones digested with EcoRⅠ. M1, DL15000 DNA marker; M2, 1 kb+ DNA Ladder marker; 1-30, 30 random clones.

图5 PCR检测转化子的结果泳道1~8-转化子; M-Trans2K +ⅡDNA标记; CK-野生型菌株Guy-11。

Fig. 5. Transformants test by PCR. 1-8, Transformants; M, Trans2K +ⅡDNA marker; CK, Wild type strain Guy-11.

图6 转化子在含有潮酶素的CM平板上的生长情况 CK-野生型菌株Guy-11,其他是转化子。

Fig. 6. Transformants on CM plate containing hygromycin. CK, Wild type strain Guy-11, others are transformants.

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