捕光叶绿素a/b结合蛋白是植物光系统I(photosystem I,PSI)中与色素分子结合的膜蛋白,由Lhca基因家族编码,主要参与光合作用中光能的捕获与传递。本研究对甘蔗叶片cDNA文库测序,获得甘蔗PSI中Lhca3基因的cDNA序列,命名为ScLhca3(Gen Bank登录号为KU215669)。生物信息学分析表明,ScLhca3的开放读码框(opening reading frame,ORF)长度为804 bp,编码267个氨基酸,分子量为28.91 k D,等电点为8.96;ScLhca3被定位于叶绿体,无信号肽,存在3个明显的跨膜区域,含有典型的捕光叶绿素a/b结合蛋白功能域(chlorophyll a/b binding domain),为亲水性非分泌蛋白。多序列比对和进化分析表明,ScLhca3在不同物种间具有较强的保守性,具有种属特性。构建原核表达载体p GEX-6P-1-ScLhca3,通过IPTG诱导表明,ScLhca3蛋白与预测大小一致。亚细胞定位试验显示,ScLhca3与报告基因GFP的融合蛋白定位于叶绿体中。实时定量PCR分析表明,ScLhca3在成熟叶片中的相对表达量最高,根中几乎不表达,具有明显的组织特异性;在Cd Cl2、ABA和H2O2外源胁迫下,ScLhca3均上调表达;在黑暗、Na Cl和PEG胁迫下则下调表达。
The Lhca gene family in green plants encodes several light-harvesting chlorophyll a/b-binding proteins that harvest and transfer light energy to the reaction center of photosystem I(PSI) in photosynthesis. The cDNA sequence of Lhca3 gene was firstly obtained from sugarcane leaf full-length cDNA library through sequencing and validated by homology comparison. It was designated ScLhca3 and submitted to the Gen Bank(accession number: KU215669). ScLhca3 contains an 804 bp open reading frame(ORF) and encodes a deduced protein of 267 amino acids, with a molecular weight and p I of 28.91 k D and 8.96, respectively. Bioinformatics analysis showed that ScLhca3 is a hydrophilic non-secretory protein with three transmembrane domains and a chlorophyll a/b binding domain. Sequence multi-alignment and phylogenetic analysis demonstrated that the ScLhca3 protein sequence shared a high identity with Lhca3 from other plants, with the specificity of species. The protokaryotic expression vector p GEX-6P-1-ScLhca3 was constructed and expressed in E. coli cells under the induction of IPTG. The subcellular localization experiment showed that the ScLhca3 fused with GFP, a reporter protein, was located on chloroplast. Real-time quantitative PCR analysis showed the expression of ScLhca3 had a clear tissue specificity, and was upregulated by Cd Cl2, ABA, and H2O2, but downregulated by darkness, Na Cl, and PEG.