哈茨木霉caf类似基因ThCon1的克隆及功能初探  

Cloning and Functional Research of a caf-like Gene ThCon1 in Trichoderma harzianum TH-33

作　　者：牛静[1,2] 李梅[1,2] 范亮波[1,2] 刘卫德[1,2] 冀颖[1,2] 刘增亮[1,2] 蒋细良[1,2] 陈书华[1,2] 

机构地区：[1]中国农业科学院植物保护研究所/农业部作物有害生物综合治理重点实验室,北京100081 [2]国家生物农药质量监督检验中心,延吉133000

出　　处：《中国生物防治学报》2013年第1期89-96,共8页Chinese Journal of Biological Control

基　　金：国家自然科学基金项目(30671400;31071728);国家高技术研究发展计划(2011AA10A205);农业部"948"项目(2011-G4);中央级公益性科研院所基本科研业务费专项(1610142012005);植物病害生物学国家重点实验室开放基金(SKL20110P13)

摘　　要：采用染色体步移方法从产孢量显著减少的哈茨木霉Trichoderma harzianum T-DNA插入突变株1413中克隆出caf(染色体组装蛋白基因)类似基因。序列分析显示该基因全长3418 bp,共有5个外显子,4个内含子,开放阅读框长为2904 bp(GenBank登录号:JN802132),编码967个氨基酸,有两个保守的功能结构域WAC_Acf1_DNA_bd和DDT domain,该氨基酸序列与血红丛赤壳Nectriahaematococca mpVI 77-13-4的核染色质组装蛋白(chromatin assembly protein)有69%相似性,推测该基因编码的蛋白为一个核染色质组装蛋白,命名为ThCon1。构建了ThCon1基因置换载体pDHt/SKThCon1∷hph,采用ATMT方法转入野生型哈茨木霉TH-33菌株,筛选到1株表型发生明显差异的突变株,PCR证实该突变株中ThCon1基因已被置换为潮霉素抗性基因。对突变株进行表型分析,结果显示:与野生菌株相比,突变株生长速率下降,菌落稀薄,产孢量减少。表明ThCon1基因影响哈茨木霉的生长和产孢。A caf-like gene of full length DNA, named ThConl, was obtained by genome walking from the ATMT mutation strain Trichoderma harzianum 1413 whose conidial yield was significantly decreased. ThConl gene encoded a homolog of chromatin assembly protein with 967 amino acid residues, containing two conserved domains WAC Acfl DNA bd and DDT. It showed a high resemblance to that of Nectria haematococca mpVI 77-13-4 with 69% identity. To clarify its role in T. harzianum TH-33, we constructed a pDHt/SK ThConl：：hph vector to knockout the ThConl from wild T. harzianum TH-33 by Agrobacterium tumefaciens-mediated transformation method, and obtained a ThConl negative mutant. Analysis on the differences in mycelial growth and conidial yield between wild type and ThConl negative mutants indicated that ThConl negative mutant showed slower mycelial growth, decreased conidial yield. These results suggest that the gene ThConl may plays a role in mycelial growth and conidiation in T. harzianum.

关 键 词：哈茨木霉 ThCon1 核染色质组装蛋白 基因置换 

分 类 号：Q785[生物学—分子生物学] S482.292[农业科学—农药学]