蝴蝶兰几丁质酶基因的克隆及表达特性分析

Bioinformatics and Expression Analysis of a Chitinase Gene from Phalaenopsis spp.

利用RT-PCR及RACE技术,克隆到蝴蝶兰1个几丁质酶基因PhCHT(GenBank登录号为KT992851),该基因cDNA全长1 210bp,包含37bp的5′-UTR、933bp开放阅读框和240bp 3′-UTR,编码310个氨基酸;该蛋白为糖苷水解酶第19家族成员,兼具有溶菌酶活性;生物信息学分析显示,该蛋白具N-端信号肽和跨膜结构,为胞外分泌蛋白;该蛋白与海枣、谷子、油棕和拟南芥的几丁质酶类似蛋白相近,并且在系统进化树上与甘蔗和陆地棉的Ⅶ类几丁质酶同属一个分支。PhCHT基因的表达分析表明,PhCHT在蝴蝶兰营养器官和生殖器官中均有表达,根中表达量最高;13℃/8℃低温处理3、6、9和15d时该基因的表达被抑制,4℃低温处理1、2和4h表达量升高。研究表明,PhCHT基因能够响应短期的冷胁迫。研究结果为进一步研究蝴蝶兰几丁质酶的系统进化及抗性育种奠定了基础。

Using RT-PCR combined with RACE techniques,we cloned the chitinases gene PhCHT（GenBank accession number：KT992851）from Phalaenopsis.The full length cDNA was 1 210 bp,containing a5′-UTR of 37 bp,a 3′-UTR of 240 bp,and an opening reading frame of 933 bp encoding 310 amino acids;The deduced protein belonged to glycoside hydrolase family 19 and may have lysozyme activity.The bioinformatics analysis indicated that this protein was predicted to be an extracellular secreted protein with a Nterminal signal peptide and a transmembrane structure;It was highly identified with the chitinase-like protein of Phoenix dactylifera,Elaeis guineensis,Setaria italica,and Arabidopsis thaliana.Phylogenetic analysis showed that PhCHTbelongs to a clade with other classⅦ chtinase genes fromGossypium hirsutum and Saccharum spp.The PhCHT gene was expressed in vegetative and reproductive organs with a higher expression level in root than others.The expression of PhCHT was decreased by a low temperature of 13℃/8℃for 3,6,9and 15 d,and increased by 4℃treatment for 1,2and 4h.Our results suggest that the PhCHTplays a crucial role in responding to the short-term cold stress.These results could provide thefoundation for further study the phyletic evolution of chitinase genes and the stress resistance breeding of Phalaenopsis.