毛白杨S-腺苷高半胱氨酸水解酶基因PtSAHHA的克隆、表达及SNP关联分析

doi:10.11707/j.1001-7488.20131109

摘要/Abstract

摘要：

组合运用分子生物学及生物信息学方法，首次从毛白杨成熟木质部中分离出编码S-腺苷高半胱氨酸水解酶基因PtSAHHA的cDNA克隆。序列分析表明，毛白杨PtSAHHA cDNA片段全长为1 935 bp，其内部含有大小为1 458 bp的完整开放阅读框架，可编码含有485个氨基酸残基的蛋白质。组织特异性Realtime-PCR结果显示，PtSAHHA在木质部中表达丰度最高，在形成层和老叶中表达丰度中等，在嫩叶中表达丰度最低。在此基础上，组合利用MAGE5.0和DnaSP5软件对毛白杨自然群体中45株基因型个体的PtSAHHA序列进行对比和分析，共检测到166个单核苷酸多态性（SNP）位点，SNP频率为1/15 bp，多样性指数π_T为0.010 58。在外显子区域共检测到88个SNP位点，其中同义突变位点34个，错义突变位点53个及1个无义突变位点；同义突变的核苷酸多样性π_Synonymous= 0.024 91，是非同义突变核苷酸多样性π_{Nonsynonymous}=0.002 33的10倍，推测毛白杨PtSAHHA基因编码区在毛白杨物种演化过程中受到较强的纯化选择压力。对PtSAHHA基因内的SNPs连锁不平衡分析结果表明，随着核苷酸序列长度增加至800 bp，即r² < 0.1，SNPs的连锁不平衡水平已衰退至不明显。在此基础上，在由426株毛白杨基因型个体组成的关联作图群体内，对频率大于10%的42个常见SNP标记位点与6个木材品质性状进行关联分析，检测到3个SNP标记与3个表型性状显著关联。研究结果为基于分子标记的毛白杨优良木材品质性状定向辅助育种提供理论依据。

关键词: 毛白杨, S-腺苷高半胱氨酸水解酶, 单核苷酸多态性, 连锁不平衡, 木材品质性状

Abstract:

In this study, a full-length cDNA clone encoding PtSAHHA was isolated from the cDNA library prepared from mature xylem zone of Populus tomentosa by the bioinformatics and molecular biology. The PtSAHHA cDNA had 1 935 bp length with a corresponding open reading frame (ORF) of 1 458 bp that could encode a protein of 485 amino acids. Tissue-specific expression analysis indicated that the PtSAHHA transcripts were the most abundant mRNA products in stem xylem, intermediate in stem cambium and mature leaf, and the lowest abundant in young leaf. By aligning,comparing and analyzing the genomic sequences of PtSAHHA in 45 random individuals of P.tomentosa with the software of MEGA5.0 and DnaSP5, a total of 166 single nucleotide polymorphisms (SNPs) were detected, with the SNP frequency of 1/15 bp and the diversity (π_T) of 0.010 58. There were 88 SNPs detected in the coding regions of PtSAHHA, of which 34 were synonymous nucleotide substitutions, and 53 were missense mutations and 1 was nonsense mutation. The diversity level of synonymous nucleotide substitutions (π_Synonymous=0.024 91) was tenfold of nonsynonymous nucleotide substitutions (π_{Nonsynonymous}=0.002 33), suggesting that the gene might be evolved under purifying selection at the synonoymous sites of the coding region in P. tomentosa. Linkage disequilibrium test in the PtSAHHA showed that LD declined rapidly within the regions of PtSAHHA with the length increase to 800 bp(r²< 0.1). On this basis, candidate gene-based association analysis of 42 common single-SNPs (frequency > 10%) with six wood traits in an association population of 426 unrelated individuals showed 3 SNPs that were significantly associated with 3 wood traits, respectively. This study provides an important genetic foundation for molecular marker-assisted selection breeding programs with the goals of improving the quality and quantity of wood products of P. tomentosa.

Key words: Populus tomentosa, S-adenosyl-L-homocysteine hydrolase (SAHH), single nucleotide polymorphisms, linkage disequilibrium (LD), wood property

中图分类号:

S718.46

巩琛锐, 王璐, 杜庆章, 张德强. 毛白杨S-腺苷高半胱氨酸水解酶基因PtSAHHA的克隆、表达及SNP关联分析[J]. 林业科学, 2013, 49(11): 67-76.

Gong Chenrui, Wang Lu, Du Qingzhang, Zhang Deqiang. Isolation, Expression, and Association Genetics of the S-Adenosyl-L-Homocysteine Hydrolase Gene A (PtSAHHA) in Populus tomentosa[J]. Scientia Silvae Sinicae, 2013, 49(11): 67-76.

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