两种苎麻纤维素合酶基因cDNA序列的克隆及表达

cDNA Cloning and Expression of Two Cellulose Synthase Genes from Boehmeria nivea

从苎麻转录组数据出发,利用 Blast 工具从中分析出与多种植物纤维素合酶高度相似的片段 CL789和Unigene 20360。根据片段信息设计特异性引物,从苎麻[Boehmeria nivea （Linn.） Gaud.]栽培种湘苎3号中克隆纤维素合酶核心片段,并利用5′及3′RACE技术获得2个片段的全长cDNA。两者都具有典型的纤维素合酶特征结构域,表明为2个苎麻纤维素合酶基因 CesA的 cDNA序列,分别命名为 BnCesA2和 BnCesA3。BnCesA2基因编码区全长度3240 bp,编码1079氨基酸多肽；BnCesA3基因编码区全长3120 bp,编码1039氨基酸多肽。对BnCesA2和BnCesA3基因在湘苎1号、湘苎3号、湘潭大叶白和城步青麻苎麻品种木质部和韧皮部荧光定量PCR分析显示,2个基因在不同品种苎麻的木质部及韧皮部都有表达,但表达量存在着一定差异,整体而言 BnCesA2具有更高的表达水平,其木质部和韧皮部的表达都为BnCesA3的2-5倍。推测BnCesA2和BnCesA3均参与了苎麻细胞壁的次生合成。

Two potentially high homologous fragments CL789 and Unigene 20360 were identified as plant cellulose synthase character sequence from the transcriptome data we obtained previously by Blast aligning and homologous screening. The two pairs of specific primers were then designed based on the CL789 and Unigene 20360 sequences information. The intermediate fragments of two cellulose synthase gene cDNA were cloned from ramie variety Xiangzu 3 by RT-PCR. And the whole cDNA was cloned by followed 5′ and 3′ RACE. The full length cDNAs were sequenced and their encoded putative proteins were identified as cellulose synthase by the conserved domain analysis. The‘se two cDNA sequences were named as BnCesA2 and BnCesA3 respectively. The full-length coding sequence of BnCesA2 gene is 3240 bp, and encodes a putative 1079 amino acids. The coding sequence of BnCesA3 gene is 3120 bp, and could be translated into a 1039 amino acids protein. We designed the specific primers based on cDNA sequences of the two genes and their expression levels were tested by quantitative real-time PCR （qRT-PCR）, indicating that BnCesA2 and BnCesA3 were both actively expressed in phloem and xylem in the four different cultivars of ramie. But the level of expression showed significant difference that the BnCesA2 expression was 2 to 5 multiples higher than BnCesA3 in both phloem and xylem. It is speculated that both the BnCesA2 and BnCesA3 participate the primary and secondary cell wall biosynthesis.