板栗CmAPs基因的克隆及在芽变短雄花序中的表达分析  

Cloning and Expression Analysis of CmAPs in Mutant Short Catkin of Castanea mollissima

作　　者：段续伟[1] 邓舒[1] 沈元月[1] 冯永庆[1] 秦岭[1] 

机构地区：[1]北京农学院植物科技学院,北京102206

出　　处：《林业科学》2010年第12期49-55,共7页Scientia Silvae Sinicae

基　　金：国家自然科学基金项目(30872039);北京市自然科学基金项目(6082004)

摘　　要：利用RT-PCR与RACE扩增方法,分别从板栗正常雄花序和芽变短雄花序cDNA中分离出编码板栗天冬氨酸蛋白酶cDNA全长的序列,序列分析表明:克隆的cDNA片段总长分别为1 822 bp和1 909 bp,基因内部含有完整的开放阅读框架,大小均为1 539 bp,可编码长度为513个氨基酸残基的蛋白质,所推导的蛋白质氨基酸序列与蓖麻、可可和葡萄的天冬氨酸蛋白酶的蛋白质氨基酸序列相似性分别为83%,80%和75%。正常与芽变短雄花序中天冬氨酸蛋白酶的氨基酸序列存在6个氨基酸的差异,其中有3个位于活性区。后证实分离出的2个天冬氨酸蛋白酶基因在正常雄花序和芽变短雄花序中共同存在,命名为CmAPs1和CmAPs2(GenBank登录号分别为GQ984143和GQ984144)。实时荧光定量PCR结果显示:在雄花序原基形成期、花簇原基形成期和花朵原基形成期,芽变花序中的CmAPs的总表达量均高于正常花序中的总表达量,而在发育的花被原基形成期即程序性死亡发生期,CmAPs在芽变花序中表达量远远低于正常花序中表达量。初步推测CmAPs与短雄花序发育过程中的程序性死亡有关。Two full-length cDNAs encoding aspartic proteinases（APs） were isolated respectively from the normal and mutant short catkin of the Castanea mollissima with RT-PCR and RACE amplification method.The sequence results showed that the full-length cDNA from normal was 1 822 bp,while that from mutant was 1 909 bp.They all contained a 1 539 bp open reading frame（ORF） which encoding a protein of 513 amino acids and had 83%,80% and 75% amino acid sequence similarity with aspartic proteinases in Ricinus communis,Theobroma cacao and Vitis vinifera respectively.The two deduced proteins have six different amino acids,three of which are in the activity areas.Later they were confirmed to be existed both in normal and mutant short catkin,namely CmAPs1 and CmAPs2（accession No.GQ984143 and GQ984144）.The results of Realtime RT-PCR indicated that the expression of CmAPs1 and CmAPs2 gene in mutant catkins was both higher than that in the normal catkins in the primordia formation phase of catkin,floral cluster and flower.However,the expression of CmAPs1 and CmAPs2 gene in mutant catkins was so far lower than that in normal in the stage of perianth primordium formation（programmed cell death happened at the same time）,it is speculated that CmAPs would be related to programmed cell death in the process of short catkin development.

关 键 词：板栗 雄花序 细胞程序性死亡 天冬氨酸蛋白酶 荧光定量PCR 

分 类 号：S718.46[农业科学—林学] S664.2