苎麻肉桂酰辅酶A还原酶基因cDNA序列的克隆与分析

cDNA Cloning and Analysis of Cinnamoyl-CoA Reductase Gene from Boehmeria nivea

根据苎麻转录组测序信息,利用RACE法克隆出2个肉桂酰辅酶A还原酶基因全长cDNA序列,对其进行生物信息学分析,并利用荧光定量技术对苎麻快速生长期、成熟期和成熟后期该基因在木质部和韧皮部的表达模式进行研究。结果表明,BnCCR1全长1056 bp,编码277个氨基酸,Blast比对BnCCR1基因与白桦、蓖麻CCR基因核苷酸序列相似性均为70%,推测的氨基酸与蓖麻CCR基因氨基酸序列相似度为77%,蛋白质预测显示其N端存在一个3 Beta-HSD/Epimerase/NAD-binding-10的保守域;BnCCR2基因全长1291 bp,编码248个氨基酸,Blast比对BnCCR2基因与毛果杨CCR基因核苷酸序列相似度为74%,推测的氨基酸与蓖麻、毛果杨CCR基因氨基酸序列相似度均为81%,蛋白质预测显示其N端存在一个3 Beta-HSD/Epimerase/NAD-binding-4的保守域;BnCCR1和BnCCR2基因编码蛋白三维模型与矮牵牛CCR基因相似度分别达30.68%、44.77%,建模结果可靠;荧光定量结果显示BnCCR1和BnCCR2基因具有时期表达差异性,不具有组织表达差异,但不同组织表达量具有差异。推测BnCCR1和BnCCR2基因是存在于苎麻木质素代谢中的两种CCR基因。

Two cDNA sequences of cinnamoyl-CoA reductase genes were cloned by RACE technology base on transcriptome sequencing data of Boehmeria nivea, and their bioinformatics were analyzed. Their expression levels in tissues of phloem and xylem were also tested respectively in rapid-growth stage, maturation stage and late maturity stage by quantitative Real-time PCR. The results showed that the BnCCR1 was 1056 bp in length and encoding 277 amino acids. Blast and protein structure analysis showed that its eDNA sequence shared a homology of 70% compared with Betula platyphylla and Ricinus communis CCR, and its amino acids did a homology of 77% compared with that of Ricinus communis. Protein prediction showed its protein N terminal with a conservative field of 3 Beta-HSD/Epimerase/NAD-binding-10. The coding sequence of BnCCR2 gene was 1291 bp, and could be translated into a 248 amino acids. Blast and protein structure analysis showed that its cDNA sequence had a homology of 74% compared with Populus trichocarpa CCR, and its amino acids had a homology of 81% compared with these of Ricinus communis and Populus trichocarpa. Protein prediction showed its protein N terminal with a conservative field of 3 Beta-HSD/ Epimerase/NAD-binding-4. Three-dimensional structures of BnCCR1 and BnCCR2 had relatively high similarity with CCR genes in petunias, and the similarity was 30.68% and 44.77% respectively. The result of qRT-PCR indicated that the expression of both BnCCR1 and BnCCR2 was different in different periods, while not in different tissues, but the levels of expression in different tissues showed significant differences. We speculated that BnCCR1 and BnCCR2 are different CCR genes in lignin metabolism of Boehmeria nivea, and the result of this study provide a theoretical basis for further exploration of their function in the ramie lignin biosynthesis and regulation.