苎麻木质素合成酶CAD基因的cDNA克隆及表达分析

cDNA cloning and expression analysis of a lignin synthetase CAD gene in ramie(Boehmeria nivea(L.) Gaud.)

采用PCR结合RACE技术克隆苎麻栽培种湘苎3号CAD基因全长cDNA序列;再运用qRT–PCR技术对木质素含量不同的代表性苎麻品种湘苎1号、湘苎3号、湘潭大叶白和城步青麻的CAD基因在其茎秆韧皮部和木质部的表达进行定量分析;使用1%间苯三酚和25%盐酸对4种苎麻茎横切片进行特异染色,观察品种间木质素染色度。结果表明,获得的苎麻CAD基因cDNA序列全长为1 378 bp(GenBank登录号,KF758396),编码359个氨基酸,推导为苎麻肉桂醇脱氢酶(BnCAD)。该推导蛋白质与已报道的几种植物木质素合成酶CAD的同源性均达90%以上,其中与蒺藜苜蓿的同源性达97%。运用qRT–PCR方法对BnCAD基因表达的定量分析结果显示,苎麻CAD基因在湘苎1号和湘苎3号的韧皮部表达水平明显高于其在木质部的表达水平,而在城步青麻木质部的表达水平高于其在韧皮部的表达水平,该基因在湘潭大叶白的韧皮部和木质部表达量接近。4种苎麻茎横切片的木质素染色结果表明,品种间木质素染色度与CAD基因在其韧皮部的表达量成正相关。

This study is intended to clone CAD from ramie （Boehmeria nivea L. Gaud. cv Xiangzhu 3）, and analyze the expression patterns of CAD in stems from 4 representative ramie cultivar. First, the cDNA sequence of CAD were identified by analyzing the transcriptome data of ramie cultivar Xiangzhu 3 and cloned by RT-PCR combined with RACE method. Secondly, the qRT-PCR was carried out to analyze the expression levels of CAD transcripts in 4 ramie cultivars： Xiangzhu 1, Xiangzhu 3, Xiangtaidayebai and Chengbuqingma. Finally, lignin contents among different cultivars were visually analyzed by histochemical staining of transverse sections of ramie stems using the phloroglucin. The BnCAD cDNA sequence was 1 378 bp in length （GenBank accession number, KF758396） and could be translated into a putative protein with 359 amino acids. Blast and protein structure analysis showed that this cDNA sequence is in high homologous with other plant CAD gene, and with 97% homology compared with Medicago truncatula, which confirmed that this cDNA sequence was the CAD gene. qRT-PCR analysis revealed that the expressions of CAD was much higher in phloem than xylem in Xiangzhu 1 and Xiangzhu 3, while Chengbuqingma the opposite, and no obvious difference was observed in Xiangtandayebai. Histochemical lignin assays showed that the staining degree between different cultivars is directly proportional to the quantitative expression of CAD transcripts, suggesting an important role of CAD during ramie lignin synthesis.