铁皮石斛RCA2基因克隆与生物信息学分析

Cloning and bioinformatics analysis of RCA2 gene in Dendrobium officinale

核酮糖1,5-二磷酸羧化酶/加氧酶是光合碳同化作用的关键酶,它对调节铁皮石斛Rubisco活性和光合速率具有直接的作用,对其进行基因等方面的研究,会对改变植物光合速率打下良好基础。该研究以一年生铁皮石斛叶片为材料,采用RT-PCR和RACE技术成功克隆了Rubisco活化酶(RCA)基因,并对其进行生物信息学分析。结果表明:RCA基因全长1 730 bp,命名为RCA2(Gen Bank登录号KT205842),其中5'-UTR 81 bp、3'-UTR 326 bp,开放阅读框1 323 bp,编码440个氨基酸,分子质量为48.53 k Da,等电点为6.19,包含P-loop NTPase超家族基因结构。氨基酸多重序列比对发现RCA2核苷酸序列与蝴蝶兰的相似性高达87%。RCA2编码蛋白为亲水性蛋白;亚细胞定位于叶绿体基质;蛋白质二级结构分析,α螺旋占30.68%,延伸链占25.45%,不规则折叠占43.86%。RCA2编码蛋白质的功能预测发现,RCA2在中间代谢起到了一个非常重要的角色。该研究发现对铁皮石斛光合作用关键酶Rubisco的分析可为其光合作用特性的发掘提供理论基础,并为提高温室大棚栽培效率提供理论依据。

Ribulose-1,5-bisphosphosphate carboxylase/oxygenase was a key enzyme of photosynthetic carbon assimilation,it had direct adjustive effects Rubisco activity and photosynthetic rate of in Dendrobium officinale,and laid a good foundation for changing plant photosynthetic rate by studying the gene. RCA gene was cloned from leaf of annual D. officinale using RT-PCR and RACE,and was analyzed by bioinformatics. The results showed that the full length c DNA of RCA was 1 730 bp length,the cloned RCA gene was named RCA2（ Gen Bank accession No. KT 205842）. It was contained a 5＇-untranslated region（ 5＇-UTR）（ 81 bp）,3＇-UTR（ 326 bp）,and an opening reading frame（ ORF）（ 1 323bp）,which could be translated into a 410-amino-acid putative peptides with a molecular weight of 48.53 k Da and a theoretical p I of 6.19,with a domain of P-loop_NTPase super-family. Multiple sequence alignment results showed that the homology of nucleotide sequence between the RCA2 and the reported Phalaenopsis genes was 87%. RCA2 protein was belong to hydrophilic protein,sub-cellular localization was in the chloroplast. The RCA2 protein secondary structure components showed that a- helix（ h）,extended strand（ e）,and random coil（ c） accounted for 30. 68%,25. 45% and43.86%. RCA2 encoding protein function prediction showed that RCA2 played a very important role in intermediary metabolism. This study of the key enzyme-photosynthesis Rubisco in Dendrobium officinale provides the information for the research of the photosynthetic characteristics,and the basis for improving the efficiency of greenhouse cultivation.