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