姜荷花叶绿素降解酶基因PAO的分离及特征

Isolation and Characteristics of Chlorophyll Degrading Gene PAO from Curcuma Alismatifolia

姜荷花是国内新兴的一种热带花卉,其不育苞片尖端带有由叶绿素形成的绿色沉着,严重影响观赏效果.获得叶绿素降解关键酶基因信息有利于后期通过分子育种手段来改良苞片的颜色.为了获得叶绿素降解的关键酶基因PAO信息,本试验在前期通过姜荷花全长转录组测序获得大量转录组信息的基础上,进行筛选分析,获得了2条PAO基因,分别命名为PAO1和PAO2.PAO1基因(GenBank:MT077180),cDNA序列全长1794 bp,开放阅读框1611 bp(10 bp^1620 bp),编码一条536AA的氨基酸序列.PAO2基因(GenBank:MT077181),cDNA序列全长1843 bp,开放阅读框1611 bp(10 bp^1620 bp),编码一条536AA的氨基酸序列.PAO1和PAO2的氨基酸序列,仅存在4个氨基酸残基的差异.采用Blast,Translate tool(ExPASy),Clustal Omega,Find Conserved Domains(NCBI),ProtParam,TMHMM Server,SOPMA,SWISS-MODEL,ClustalX(1.81),MEGA4.1等预测和分析了这2个基因编码蛋白氨基酸的一级结构(包括氨基酸序列的组成、保守区、理化特征等)、二级结构、三级结构及分子系统进化关系.PAO1和PAO2的核苷酸与蛋白氨基酸序列与其他物种的PAO基因都具有很高的同源性,两者都包含PLN02518的保守区.分子系统进化分析表明,姜荷花的PAO1和PAO2组成的小类,与小果野芭蕉PAO(XP_009398041.1)的亲缘关系最为接近,然后与禾本科植物的PAO聚为一大类.本研究为后期通过分子手段改良姜荷花不育苞片的颜色提供基础,并为进一步丰富和探索叶绿素降解代谢理论提供铺垫.

Curcuma Alismatifolia is a new tropical flower in China whose sterile bracts have green spots formed by chlorophyll,leading to its poor viewing effect.Obtaining the key enzyme gene information of chlorophyll degradation is beneficial to the improvement of the bract color through molecular breeding.In order to obtain the key enzyme gene PAO in the process of chlorophyll degradation,on the basis of obtaining a large amount of transcriptome information by sequencing the full-length transcriptome of Curcuma Alismatifolia,we screened and analyzed the transcriptome information and obtained 2 PAO genes named PAO1 and PAO2.The PAO 1 gene(GenBank:MT077180)has a full-length cDNA sequence of 1794 bp and an open reading frame of 1611 bp(10 to 1620 bp),and encodes a 536AA amino acid sequence.The PAO2 gene(GenBank:MT077181)has a full-length cDNA sequence of 1843bp and an open reading frame of 1611bp(10-1620bp),and encodes a 536AA amino acid sequence.The amino acid sequences of PAO1 and PAO 2 differ only by 4 amino acid residues.Using Blast,Translate tool(ExPASy),Clustal Omega,Find Conserved Domains(NCBI),ProtParam,TMHMM Server,SOPMA,SWISS-MODEL,ClustalX(1.81),MEGA4.1,etc.the paper predicted and analyzed their amino acid composition,physical and chemical properties,conserved domains,secondary structure,tertiary crystal structure,and molecular phylogeny.The nucleotide and protein amino acid sequences of PAO1 and PAO2 have high homology with the PAO genes of other species,both of them containing a conserved region PLN02518.Molecular phylogenetic analysis shows that a small cluster of PAO1 and PAO2 are the closest to Musa acuminate PAO(XP_009398041.1),and is therefore aggregated with that of Gramineae plants into a large group.This study provides the basis for improving the color of sterile bracts of Curcuma Alismatifolia by molecular means in the later period,as well as a foundation for further enriching and exploring of the theory of chlorophyll degradation metabolism.