马尾松RCA基因的克隆和序列分析  

The Cloning and Sequence Analysis of RCA Genes from Pinus massoniana

作　　者：潘婷[1] 张逢凯 王晓锋[1] 阮倩倩[1] 刘靖[1] 盛璐[1] 季孔庶[1] 

机构地区：[1]林木遗传与生物技术省部共建教育部重点实验室,南京林业大学,南京210037

出　　处：《分子植物育种》2015年第2期391-400,共10页Molecular Plant Breeding

基　　金：国家"十二五"科技支撑项目(2012BAD01B02);林业公益性行业科研专项经费(201104010);江苏高校优势学科建设工程项目(PAPD)共同资助

摘　　要：1,5-二磷酸核酮糖羧化/加氧酶(Rubis CO)活化酶(RCA)是体内活化光合作用限速酶—Rubis CO的重要酶。本研究通过反转录PCR和c DNA末端快速扩增技术,从马尾松中分离得到了Pmrca1(登录号KF420118)和Pmrca2(登录号KF420119)两条RCA基因c DNA序列。两条序列均包含完整的编码区与5'端和3'非翻译区,长度分别为1 816 bp和1 953 bp,编码Pm RCA1和Pm RCA2两个蛋白,长度分别为480和440个氨基酸。序列分析发现两个蛋白质都具有RCA和AAA+蛋白家族(ATPase associated with various cellular activities)特异性结构域和定位于叶绿体的转运肽,Pm RCA1还具有RCA大同工型特有的由两个半胱氨酸(Cys)残基组成的保守结构。多重序列比对显示,这两个蛋白质序列分别与红花槭的RCA大同工型和小同工型的相似性达到78%。将Pm RCA1和Pm RCA2与20种不同物种RCA构建进化树,发现其与地中海松属于同一分支。研究结果为进一步分析马尾松RCA的功能和光合作用机制奠定了基础。1,5-ribulose bisphosphate carboxylase/oxygenase（Rubis CO） activase（RCA） is an important enzyme activating Rubis CO,which is the rate-limiting enzyme of photosynthesis.In this study two full-length c DNA fragments of RCA genes named Pmrca1（accession number KF420118） and Pmrca2（accession number KF420119）were isolated from Pinus massoniana through RT-PCR（Reversed Transcript Polymerase Chain Reaction） and RACE（Rapid-Amplification of c DNA Ends）.Two sequences were 1 816 bp and 1 953 bp in length,respectively,containing the open reading frame and 3＇ and 5＇ untranslated region.Pmrca1 and Pmrca2 encode two proteins named Pm RCA1 and Pm RCA2 whose are 480 and 440 amino acids in length,respectively.Both Pm RCA1 and Pm RCA2 have the specific conversed sequences of AAA＋family and RCA and chloroplast transit peptide.Pm RCA1 has two cysteine residues in the N-terminal which is the feature of conserved sequence of RCA large isoform.The affinity of the RCA large isoform from P.massoniana and Acer rubrum was 78% and the affinity of small isoform from P.massoniana and Acer rubrum was 78% as well.Species phylogenetic tree of Pm RCA1,Pm RCA2 and RCA from 20 species of other plants was constructed,which exhibited that Pm RCA1 and Pm RCA2 were in the same branch with Mediterranean pine.The results of this study gave a foundation for the further analysis of the function of RCA and mechanism of photosynthesis in P.massoniana.

关 键 词：马尾松 RCA RACE c DNA克隆 序列分析 

分 类 号：S791.248[农业科学—林木遗传育种]