彩叶草羟基丙酮酸还原酶基因CbHPR的克隆及分析

Cloning and Sequence Analysis of A Novel Hydroxypyruvate Reductase Gene,CbHPR,from Coleus blumei

为研究C3植物彩叶草的光呼吸作用,根据获得的HPR EST片段,采用RACE和文库结合的方法,克隆了HPR蛋白的全长cDNA,命名为CbHPR（GenBank accessionNo.EF125078）.序列分析结果表明,该cDNA全长为1393bp,包含一个1161bp的ORF框,编码386个氨基酸;其5′-UTR区含有2个终止子TGA,3′-UTR区具有推测的加尾信号AATAAA;CbHPR蛋白C端具定位于微体的转运信号S-K-L,具有1个保守的2-Hacid_dh_Cdo-main结构域（D-异构体-羟基酸脱氢酶-NAD结合结构域）、5个S磷酸化位点、4个T磷酸化位点和6个Y磷酸化位点.PSORT定位分析显示,CbHPR定位于叶的过氧化物酶体中.多序列比较和进化树分析表明,CbHPR与其他植物HPR一致性高达87%-90%,与拟南芥AtHPR具有相似的功能.CbHPR基因的克隆与分析,为进一步研究该基因在彩叶草的光呼吸作用中的表达调控奠定了基础.

In the attempt to investigate and understand the molecular mechanism of photorespiration ot Coleus blumei ,according to the sequence of an EST fragment with identity to HPR gene,we rapidly cloned a novel HPR gene,denoted Cbhpr（GenBank accession No. EF125078）, using a strategy of RACE combined with eDNA library. The full length of the CbHPR gene was 1 393 bp long and contained a 1 161 bp open reading frame （ORF） encoding a 386 amino acid protein. Two stop codons （TGA） was found in 5＇-UTR and one possible polyadenylation signal, AATAAA, were found in 3＇-UTR. The CbHPR contained a predicted Carboxy-terminal targeting signal （S-K-L） to microbodies, one high conserved 2-Haeid_dh_C domain （D-isomer specific 2-hydroxyacid dehydrogenase,NAD binding domain）, five S, four T and six Y phosphorytation sites. PSORT software predicted the CbHPR was localized in leaf peroxisomes. Multi-sequence comparisons and phylogenetic analysis showed that CbHPR was a novel HPR protein, having high identities from 87 % to 90% with other plant HPRs, and having similar function with Arabidopsis. Cloning and analysis of CbHPR laid the foundation for future researching HPR gene expression and regulation of photorespiration in Coleus blumei.