Journal of Nuclear Agricultural Sciences ›› 2019, Vol. 33 ›› Issue (5): 870-879.DOI: 10.11869/j.issn.100-8551.2019.05.0870

• Induced Mutations for Plant Breeding·Agricultural Biotechnology • Previous Articles     Next Articles

Isolation, Expression and Single Nucleotide Polymorphism Analysis of BlCCoAOMT From Betula luminifera

YU Zicheng, NI Fei, JIANG Cheng, HUANG Huahong*, LIN Erpei, TONG Zaikang   

  1. The State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou, Zhejiang 311300
  • Received:2018-01-05 Online:2019-05-10 Published:2019-03-06

光皮桦BlCCoAOMT基因的克隆、表达及单核苷酸变异分析

俞子承, 倪飞, 江成, 黄华宏*, 林二培, 童再康   

  1. 浙江农林大学亚热带森林培育国家重点实验室,浙江 杭州 311300
  • 通讯作者: *黄华宏,男,教授,主要从事林木遗传育种研究。E-mail:huanghh@zafu.edu.cn
  • 作者简介:俞子承,男,主要从事林木分子遗传研究。E-mail:404398248@qq.com
  • 基金资助:
    国家自然科学基金项目(31470674),浙江省农业新品种选育专项(2016C02056-2)

Abstract: Caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) is a key enzyme involved in lignin biosynthesis, has an important influence on the composition and structure of lignin. To investigate its function in lignin biosynthesis, a BlCCoAOMT gene was cloned from Betula luminifera, and its sequence characteristics and expression patterns were analyzed in this study. The genomic sequences of BlCCoAOMT from 73 individuals were analyzed for single nucleotide polymorphism (SNP) detection. The results showed that the BlCCoAOMT cDNA was 1114 bp in length with an open reading frame (ORF) of 744 bp. The encoding protein was composed of 247 amino acid residues, which contained all eight conserved motifs of plant CCoAOMTs. The expression levels of BlCCoAOMT were relatively high in lignified stem, and increased with the lignification progressed, suggesting that it might participate in lignin biosynthesis of B. luminifera. The gene expression in TW was significantly down-regulated with the process of bending from 6 hours to 7 days which was consistent with the decrease of the lignin content in TW. In total, 119 SNPs were detected, one SNP among which 26 SNPs located in exons of BlCCoAOMT. The frequency of SNP was one SNP per 14 bp on average, indicating that this gene had abundant SNPs among different genotypes. Genetic divergence of BlCCoAOMT was insignificantly different among the populations, and the ratio of nonsynonymous to synonymous mutations was less than 1, suggesting that BlCCoAOMT was mainly subjected to purification selection in the evolution process. The present study laid a foundation for in-depth dissection of lignin biosynthesis mechanism and provided theory basis for further molecular marker assisted breeding in B. luminifera.

Key words: Betula luminifera, CCoAOMT gene, gene cloning, expression pattern, single nucleotide polymorphism

摘要: 咖啡酰辅酶A-O-甲基转移酶(CCoAOMT)是植物木质素合成过程中的一个关键酶,对木质素组成和结构有重要的作用。为探究BlCCoAOMT基因在木质素合成中的作用,本研究从光皮桦中克隆了该基因,并分析其基因结构和表达模式,以及在73个基因型中的单核苷酸多态性(SNP)。结果表明,BlCCoAOMT基因cDNA全长1 114 bp,含有一个744 bp的完整ORF,编码一个由247个氨基酸残基组成的蛋白;该编码蛋白包含高等植物CCoAOMT所有的8个典型保守基序。BlCCoAOMT在木质化茎段中优势表达,且随着木质化程度提高而逐渐增强,表明BlCCoAOMT可能参与光皮桦木质素的生物合成;在拉弯处理的6 h到7 d间,该基因在应拉区发育木质部中的表达显著下调,与应拉木木质素含量的下降相符。共检测到BlCCoAOMT基因有119个SNP位点,平均每14 bp就有1个SNP位点,其中在外显子区域存在26个SNP位点,表明不同基因型中存在丰富SNP变异;在不同群体间BlCCoAOMT基因的分化程度无显著差异,且非同义突变和同义突变的位点的比值均小于1,表明在演化进程中主要受到了纯化选择压力。本研究结果为深入解析光皮桦木质素合成分子机制及后续分子标记辅助育种提供了理论依据。

关键词: 光皮桦, CCoAOMT基因, 基因克隆, 表达模式, 单核苷酸多态性