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作物学报 ›› 2008, Vol. 34 ›› Issue (01): 76-83.doi: 10.3724/SP.J.1006.2008.00076

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

苎麻纤维素合成酶基因cDNA的克隆及表达分析

田志坚1;易蓉1;陈建荣2;郭清泉3;张学文1,*   

  1. 1湖南农业大学生物科学技术学院,湖南长沙 410128; 2中国农业科学研究院麻类研究所, 湖南长沙 410205; 3湖南农业大学苎麻研究所, 湖南长沙 410128
  • 收稿日期:2007-04-23 修回日期:1900-01-01 出版日期:2008-01-12 发布日期:2008-01-12
  • 通讯作者: 张学文

Cloning and Expression of Cellulose Synthase Gene in Ramie [Boehme- ria nivea (Linn.) Gaud.]

TIAN Zhi-Jian1,Yi Rong1,CHEN Jian-Rong2,GUO Qing-Quan3,ZHANG Xue-Wen1,*   

  1. 1 College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, Hunan; 2 Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, Hunan; 3 Institution of Ramie, Hunan Agricultural University, Changsha 410128, Hunan, China

  • Received:2007-04-23 Revised:1900-01-01 Online:2008-01-12 Published:2008-01-12
  • Contact: ZHANG Xue-Wen

摘要:

以苎麻 [Boehmeria nivea (Linn.) Gaud.] 栽培种湘苎3号为材料, 通过简并引物RT-PCR结合RACE技术首次成功克隆苎麻纤维素合成酶基因BnCesA1 5′端450 bp序列以外的全部cDNA 序列, 序列长3 276 bp, 编码一段938个氨基酸的蛋白质。经基因比对及蛋白质结构分析确证是苎麻纤维素合成酶基因。半定量RT-PCR分析显示:BnCesA1在苎麻根、茎、叶和芽组织中均有表达, 其表达量为茎>叶>芽>根, 相对表达量依次为 0.791、0.381、0.319和0.183。从其表达模式可以推测该基因同时参与了苎麻细胞初生和次生细胞壁纤维素的合成。

关键词:

苎麻, 纤维素合成酶, cDNA克隆, 表达分析

Abstract:

In this study, total RNA was extracted from ramie, and then cDNA was obtained through reverse transcription. Degenerate primer was designed to amplify a fragment of cellulose synthase gene so as to obtain the gene using RACE. The fragment contained the whole 3′-end with ploy(A) and most 5′-end excluding 450 bp of the 5′-end of the expected cDNA. There sequences were sequenced respectively and spliced into a cDNA sequence which was 3 276 bp in length, and could be translated into protein with 938 amino acids in the reading frame. BLAST and protein structure analysis confirmed that this cDNA sequence was the Ramie cellulose synthase gene. According to the cellulose synthase gene denomination regulation, it was designated as BnCesA1 and submitted to GenBank l. Its accession number is DQ077190. In order to investigate the expression and regulation mechanism of BnCesA1 in different tissues of Ramie, the nonconservative sequence at 3′-end of BnCesA1 was amplified by semi-quantitative RT-PCR respectively at the same time. Taking 18S rRNA gene as an inner control, the integrate optic density (IOD) of BnCesA1 and 18S rRNA gene bands were detected with gel analysis software and defined the ratio of IOD as relative expressive quantity. The results indicated the expression of BnCesA1 could be found in leaf, stem, root and bud in Ramie. The expression level in tissues from high to low is stem, leaf, bud and root, with the relative content of 0.791, 0.381, 0.319, and 0.183 respectively. The deduced BnCesA1 protein shows a high concordance and homology to Arabidopsis thaliana AtCesA1 (87/93%). Thus BnCesA1 maybe serve a dual role as a CesA involved in both primary and secondary cell wall biosynthesis. Further studies are currently in progress to substantiate this hypothesis.

Key words:

Rmaie (Boehmeria nivea), Cellulose synthase, cDNA cloning, Expression analysis

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