暹罗炭疽菌（Colletotrichum siamense）是一种重要的病原真菌,是我国橡胶树炭疽病（Colletotrichum leaf disease, CLD）的田间优势病原种,也是热带、亚热带地区许多其他农林作物炭疽病的主要病原种。脂滴是细胞的中性脂,存在于绝大多数真核细胞中,是细胞内甘油三酯的主要贮存形式。脂滴在细胞内可以参与脂质代谢、膜转运、蛋白降解和信号传导等。包被蛋白（perilipin）是脂滴表面最丰富的脂质相关蛋白,主要存在于动植物体内,对细胞内脂滴代谢起着重要的调控作用。Cap20是暹罗炭疽菌中包被蛋白的同源蛋白,前期证实Cap20是一个致病相关蛋白,它参与了炭疽菌附着胞膨压形成、脂滴数量和致病性。了解其互作蛋白和互作的生物学意义可能为深入了解脂滴包被蛋白参与的致病分子机制具有重要意义。课题组前期通过酵母双杂交技术在暹罗炭疽菌cDNA文库中筛选到一个与Cap20互作的候选蛋白乙酸激酶,本研究克隆了该乙酸激酶的编码基因,并进行了蛋白结构和同源蛋白聚类分析。结果表明,乙酸激酶编码基因的DNA大小为1312 bp,包括一个内含子,cDNA为1248 bp,编码415个氨基酸,含有一个乙酸激酶结构域,命名为CsAck。然后构建含6×his标签的原核表达载体PET32a-CsAck,利用其和含GST标签的pGEX6p-1-Cap20（先前构建）进行蛋白表达和纯化,获得含有6×His标签的融合蛋白CsAck和含有GST标签的Cap20。通过Pull down验证了Cap20和CsAck蛋白之间的体外互作。最后,构建含有潮霉素转移酶基因HPH的表达载体PXY203-CsAck-S和含有氯嘧磺隆抗性基因ILV1的表达载体pCB1532-GFP-Cap20,将它们共同转化暹罗炭疽菌野生型菌株获得转化子。Co-IP（Co-immunoprecipitation）的结果证实Cap20和CsAck在暹罗炭疽菌中可以相互作用。该结果证实了致病性相关蛋白Cap20和乙酸激酶CsAck在体外和体内均可发生蛋白互作,可为进一步研究Cap20在暹罗炭疽菌中的致病功能和调控机制奠定基础。

Colletotrichum siamense is an important pathogenic fungus, which is the dominant pathogenic species of Colletotrichum leaf disease (CLD) in the field of rubber trees in China. And it is also the main pathogen of anthracnose of many other agricultural and forestry crops in tropical and subtropical regions. Lipid droplets, the neutral lipids of cells, are present in most eukaryotic cells and are the storage form of intracellular triglycerides. Lipid droplets can be involved in lipid metabolism, membrane transport, protein degradation and signaling in the cell. Perilipin is the most abundant lipid-associated protein on the surfaces of lipid droplets, mainly existed in animals and plants, and plays an important role in regulate lipid metabolism. Cap20 is a homologue of perilipin in Colletotrichum genus. It was previously confirmed that Cap20 is a pathogenic associated protein that involved in the formation of turgor pressure of appressorium, the number of lipid droplets and the pathogenicity of Colletotrichum genus. Understanding the interaction proteins of Cap20 and the biological significance of the interactions may be important for gaining insight into how Cap20 is involved in the molecular mechanisms of pathogenesis. In the previous stage, a candidate protein, acetate kinase, which interacts with Cap20, was screened from the cDNA library of C. siamense by yeast two-hybrid technology. In this study, the full length of acetate kinase coding gene was cloned, and the protein structure and phylogenetic analysis were analyzed firstly. The results showed that the acetate kinase coding gene had a DNA size of 1312 bp including an intron, a cDNA of 1248 bp encoding 415 amino acids and containing an acetate kinase structural domain, which was named as CsAck. Then, the prokaryotic expression vector PET32a-CsAck with 6×His tag was constructed, and the fusion protein CsAck with a 6×His tag and Cap20 with a GST tag was obtained by using it and the GST-tag-containing pGEX6p-1-Cap20 (previously constructed) for protein expression and purification. The in vitro interaction between the Cap20 and CsAck proteins was verified by Pull down expriments. Finally, the expression vectors PXY203-CsAck-S containing the hygromycin transferase gene HPH and the pCB1532-GFP-Cap20 with the sulfonylurea resistant gene ILV1 were constructed and co-transformed into C. siamense wild-type strain to obtain transformants. The results of Co-IP (Co-Immunoprecipitation) confirmed that Cap20 and CsAck could interact with each other in C. siamense. The results confirm that the pathogenesis-related protein Cap20 interacts with the acetate kinase CsAck both in vitro and in vivo, which could lay the foundation for further studies on the pathogenic function and regulatory mechanism of Cap20 in C. siamense.