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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2015, Vol. 35 Issue (12): 58-64    DOI: 10.13523/j.cb.20151209
技术与方法     
改良ATMT转化技术在深绿木霉基因敲除中的应用
周于聪, 谢秋瑾, 宋凯, 杨朝晖, 陈捷, 李雅乾
上海交通大学 农业与生物学院 农业部都市南方重点试验室 上海 200240
Optimal Agrobacterium tumefaciens-Mediated Transformation Technology Applied in Gene Knockout for Trichoderma atroviride
ZHOU Yu-cong, XIE Qiu-jin, SONG Kai, YANG Zhao-hui, CHEN Jie, LI Ya-qian
School of Agriculture and Biology, Shanghai Jiao Tong University, Key Laboratory of Urban Agriculture(South)Ministry of Agriculture, Shanghai 200240, China
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摘要：

木霉菌是环境中具有重要经济价值的丝状真菌之一。高效率的基因敲除技术是深入研究木霉菌功能的必要手段。研究改进了传统的农杆菌介导的遗传转化技术(ATMT),成功构建深绿木霉T23中碳代谢抑制因子 cre1基因敲除突变株。首先查找深绿木霉全基因组序列,比对并扩增 cre1基因侧翼序列,以改造后的p1300qh质粒为骨架构建 cre1敲除载体pC1300qh: cre1-up∷hyg∷ cre1-down,转化到农杆菌AGL-1。通过优化ATMT转化中木霉菌分生孢子浓度,改良培养方式和延长诱导转化时间等参数,获得最佳转化条件:木霉菌分生孢子浓度为8×106,筛选培养基改为IM培养基,诱导转化时间延长,成功筛选到可能的转化子10个。最后,经鉴定有1个转化子为 cre1敲除转化子,9个为T-DNA随机插入。因此,为深绿木霉菌基因功能研究提供了可借鉴的高效便捷的遗传转化方法。
关键词： 筛选鉴定;  深绿木霉T23;  碳代谢抑制因子CRE1;  优化;  农杆菌介导的遗传转化方法ATMT    
Abstract:

Trichoderma.spp was one of most economic filament fungi in various environments. And the efficient gene knock out technologies are key ways to do intensive researches of gene functions in Trichoderma.spp. The aim is to optimize the Agrobacterium tumefaciens-mediated transformation (ATMT), and successfully construct the cre1 gene knock out strain of Trichoderma atroviride. Firstly, the strain of T.atroviride 23 is adopted as original strain, analysis of genome sequence and confirmation DNA sequence of cre1 and its flanking sequence by combination of bioinformatics and molecular genetics operation strategy. Secondly, the flanking sequences of the upstream and downstream for cre1 are cloned and purified and then litigated into the plasmid p1300qh in sequence. Then, the knock-out vector p1300qhsilent-cre1 is transform into Agrobacterium tumefaciens AGL-1. The spore concentration, culture mode and inducing period were optimizing. The optimal condition of ATMT is as follows:concentration of conidia attains 8×106, IM medium the screening medium and increase of the period of induction. As a result, 8 tranformants which was much more than that by traditional methods were got. After verification, there were one cre1 gene knock out strain needed and seven tranformants of T-DNA random insertion. This contributes to an efficient, reliable and convenient method of ATMT for gene functional analysis in Trichoderma atroviride.
Key words: Agrobacterium tumefaciens-mediated transformation    Identification    Trichoderma atroviride 23    Carbon catabolite repressor CRE1    Optimization
收稿日期: 2015-04-13 出版日期: 2015-12-22
ZTFLH:  Q819  
基金资助:

国家自然科学基金(31201557)、上海市自然科学基金(12ZR1414100)、高等学校博士学科点新教师专项科研基金(20120073120070)资助项目
通讯作者: 李雅乾     E-mail: lauren@sjtu.edu.cn
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引用本文:

周于聪, 谢秋瑾, 宋凯, 杨朝晖, 陈捷, 李雅乾. 改良ATMT转化技术在深绿木霉基因敲除中的应用[J]. 中国生物工程杂志, 2015, 35(12): 58-64.

ZHOU Yu-cong, XIE Qiu-jin, SONG Kai, YANG Zhao-hui, CHEN Jie, LI Ya-qian. Optimal Agrobacterium tumefaciens-Mediated Transformation Technology Applied in Gene Knockout for Trichoderma atroviride. China Biotechnology, 2015, 35(12): 58-64.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20151209    或    https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I12/58

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