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2025年4月6日 星期日
  2018, Vol. 26 Issue (10): 1678-1687    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
二穗短柄草Bra1基因的克隆及表达分析
刘瑶瑶1,武艳芳2,高璐2,李霞2,王永丽2,孙建中2,蒋建雄1
1. 江苏大学
2.
Cloning and Expression Analysis of Bra1 Gene in Brachypodium distachyon
1, 1, 1, 1, 1, 1, 2
1.
2. Jiangsu University
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摘要 植物阿魏酰转移酶(feruloyl transferase)是负责将阿魏酸从阿魏酰CoA转移至阿拉伯木聚糖分子上的关键酶之一,在阿拉伯木聚糖与木质素的连接上起到关键作用,因此与细胞壁抗降解屏障的形成密切相关。本研究以禾本科模式植物二穗短柄草(Brachypodium distachyon)为材料,通过qRT-PCR技术克隆得到了Bra1(PlantGDB No.: 5g14720)基因的cDNA序列,其片段大小为1 369 bp。该基因的ORF编码443个氨基酸,蛋白质的理论分子质量为48.45 kD;基因原核表达以及蛋白质谱分析确定该基因的开放阅读框能正确编码蛋白,分子量大小与理论值基本一致。生物信息学分析显示该Bra1蛋白的氨基酸序列中包含有BAHD酰基转移酶家族特有的HXXXD功能区以及DFGWG保守结构域,说明其是BAHD酰基转移酶家族的一个成员。对二穗短柄草基因组中89个BAHD酰基转移酶氨基酸序列的系统进化树分析发现,这89个酰基转移酶大致划分为4个大的亚族,每个大亚族又包含几个小的亚族,Bra1蛋白(属于第I亚族)与他人研究过的与香豆酸(p-coumalic acid, p-CA)代谢相关的阿魏酰基转移酶(属于第Ⅲ亚族)不属于同一亚族,其蛋白功能可能存在差异;同时,从89个蛋白的编码基因中选取了第Ⅲ亚族另外一个小亚族中的几个基因及与第Ⅲ亚族亲缘关系较远的第Ⅰ亚族的几个基因做了表达分析。qRT-PCR分析表明Bra1基因与其他几个基因相比较,表达量高并且稳定,并且在二穗短柄草成熟期的茎、叶和幼穗等组织器官中的表达量约为其幼苗期的2倍,与二穗短柄草体内阿魏酸的积累特征基本一致,表明这个基因可能参与调控植物细胞壁中的阿魏酸(ferulic acid, FA)介导的交联反应。本研究的结果为该基因的生物学功能研究提供了资料,为禾本科能源植物开发利用和农作物秸秆的再生利用提供了新思路。
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刘瑶瑶
武艳芳
高璐
李霞
王永丽
孙建中
蒋建雄
关键词 二穗短柄草阿魏酰基转移酶基因克隆表达特征    
Abstract:Feruloyl transferase is one of the key enzymes responsible for transferring ferulic acid from ferulic acid CoA to arabinolxylan molecules, which plays a key role in the connection between arabinoxylan and lignin molecules. Therefore, ferulolyl transferase is closely related to the formation of anti-degradation barrier in plant cell wall. In this study, the cDNA sequence of Bra1 (PlantGDB No.: 5g14720) gene in Brachypodium distachyon was cloned by qRT-PCR technique, and the length of which was up to 1 369 base pairs. The open reading frame (ORF) of Bra1 gene encoded a deduced protein with 443 amino acid residues, with a theoretical molecular weight of 48.45 kD. The prokaryotic expression and mass spectrometry analyses further confirmed that the gene can encode the protein correctly, and the molecular weight was consistent with the theoretical value. Bioinformatics analysis indicated that the amino acid sequence of Bra1 protein contained a HXXXD domain and a DFGWG conserved domain that were unique to the BAHD acyltransferase family, as indicated that Bra1 protein was a member of BAHD acyltransferase family. The phylogenetic analysis of 89 BAHD acyltransferases in Brachypodium distachyon suggested that these acyltransferases could roughly be divided into four subfamilies, each of them containing several small groups, and the Bra1 protein (subfamily I) did not belong to the same subfamily as the other previously characterized BAHD acyltransferases (subfamily Ⅲ) whose functions were mainly related to the metabolism of p-coumalic acid (p-CA), and thus, Bra1 protein may be different in function from those BAHD acyltransferases. In addition, the expression profiles of 10 BAHD acyltransferase genes in the subfamily Ⅲ and the subfamily I which was far related to the subfamily Ⅲ were analyzed by the qRT-PCR, and the results showed that the Bra1 gene was with a higher and more stable expression compared with other genes, and furthermore the expression of Bra1 gene in the mature stem, leaf and spike were almost twice as high as that in young tissues, and its expression character was consistent with the accumulation of ferulic acid (FA) in Brachypodium distachyon suggesting that the Bra1 gene may be involved in regulating the cross-linked reaction mediated by ferulic acid in plant cell walls. This study provides important data for the identification of biological function of Bra1 gene as well as for the development and utilization of grass energy crops and the recycle utilization of crop straw biomass.
Key wordsBrachypodium distachyon    Feruloyl transferase    Gene cloning    Expression characteristic
收稿日期: 2018-03-08      出版日期: 2018-09-26
基金资助:极耐热性酶的高效表达提高水稻秸秆生物质转化的机制研究;外源嗜热FAE酶表达促进芒草生物质降解转化的过程与机制
通讯作者: 蒋建雄     E-mail: jxjiang2002@163.com
引用本文:   
刘瑶瑶 武艳芳 高璐 李霞 王永丽 孙建中 蒋建雄. 二穗短柄草Bra1基因的克隆及表达分析[J]. , 2018, 26(10): 1678-1687.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2018/V26/I10/1678
 
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