摘要
咖啡酰辅酶A-O-甲基转移酶(CCoAOMT,caffeoyl-CoA O-methyltransferase)是木质素合成过程中关键酶之一。研究通过逆转录PCR(RT-PCR)从巨龙竹(Dendrocalamus sinicus L.C.Chia&J.L.Sun)发育早期的竹笋中克隆了CCoAOMT基因(DsCCoAOMT),基因全长cDNA序列为777 bp,编码258个氨基酸,理论分子量28.85 kD,等电点为5.35,蛋白质结构预测的结果显示该蛋白含有丰富的α-螺旋和无规则卷曲。系统进化分析表明,巨龙竹CCoAOMT基因与禾本科植物CCoAOMT基因同源性最高,亲缘关系最近;与非禾本科单子叶植物的亲缘关系次之,然后是裸子植物,与双子叶植物的亲缘关系最远,说明CCoAOMT基因在双子叶植物和单子叶植物之间的差异可能在被子植物进化之前就已经存在。DsCCoAOMT基因密码子偏好性分析的结果显示,该基因选择偏性较强且偏好以G/C结尾;密码子使用频率比较结果发现,DsCCoAOMT基因在微生物中异源表达分析时采用酵母表达系统更为合适,而模式植物拟南芥、烟草、番茄与巨龙竹CCoAOMT密码子使用频率差异较小,尤其烟草、番茄可能是该基因进行转基因功能验证时最为理想的受体。实时荧光定量PCR分析巨龙竹不同组织及竹笋发育过程中DsCCoAOMT基因表达水平的结果显示,该基因在巨龙竹不同组织中的表达具有明显差异,竹笋中表达量最高,茎秆中次之,叶片中最少;随着竹笋的发育,DsCCoAOMT基因的表达量上调并在竹笋发育中期维持较高水平,表明DsCCoAOMT基因可能在巨龙竹快速生长中发挥重要调节作用。本研究的结果为进一步阐明DsCCoAOMT基因的功能和作用机制奠定了基础。
Caffeoyl-CoA O-methyltransferase(CCoAOMT)is one of the key enzymes in lignin biosynthesis.By reversible transcription PCR(RT-PCR)technique,a CCoAOMT gene(DsCCoAOMT)was cloned from the bamboo shoot of Dendrocalamus sinicus L.C.Chia & J.L.Sun.The full-length cDNA of DsCCoAOMT was777 base pairs that encoded a protein of 258 amino acids with predicted molecular mass of 28.85 kD and a basic isoelectric point of 5.35.The deduced protein was rich in helix and coin.The phylogenetic analysis revealed the highest sequence homology of DsCCoAOMT in relative to CCoAOMT from the poaceous plants,followed by the monocotyledon of non-graminaceae and gymnosperms and then some dicotyledons.That suggested a differentiation of CCoAOMT between monocotyledons and dicotyledons before angiosperms evolution.According to the analysis of codon bias,the DsCCoAOMT had high bias toward the synonymous codons with G and C at third codon position.The results of codon usage frequency showed that heterologous expression of DsCCoAOMT might be applicable in the microorganism yeast expression system and in plants Nicotiana tabacum L.and Solanum lycopersicum L..By real-time quantitative PCR,the DsCCoAOMT gene was highly expressed in the shoots,followed by the culms and the leaves at last.As the shoots developed,DsCCoAOMT is up-regulated at the higher level,suggesting that the gene might play an important regulation role in fast-growing D.sinicus L.C.Chia& J.L.Sun.Thus,this study might be helpful for further understanding the function and molecular mechanism of the DsCCoAOMT gene.
引文
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