云南松PyCADs基因的克隆与表达分析

Cloning and expression analysis of PyCAD genes in Pinus yunnanensis

【目的】研究云南松肉桂醇脱氢酶的表达特性及其在木质素生物合成过程中的作用。【方法】以一年生云南松苗木为材料,通过测定嫩茎、嫩芽和针叶中18种木质素通路小分子的相对含量,并结合转录组数据联合分析,对云南松CAD基因进行筛选、克隆和生物信息学分析,同时利用RT-qPCR技术分析其在不同组织中的表达模式。【结果】在检测的18种木质素通路小分子中,嫩芽以5-O-咖啡酰莽草酸、5-O-对香豆酰莽草酸和咖啡酸3种小分子的相对含量较高,嫩茎以3-O-对香豆酰奎宁酸、4-香豆酸、阿魏酸、松柏醛、L-苯丙氨酸、松柏醇、对-香豆醇、L-酪氨酸和芥子醛9种小分子的相对含量较高,而在针叶中则以松柏苷、香豆醛、咖啡醛和紫丁香苷4种小分子的相对含量较高。云南松转录组中筛选出4个CAD家族成员,通过系统进化分析发现云南松PyCAD-3与AtCAD1同源,PyCAD-1、PyCAD-2和PyCAD-4与松属类CAD、拟南芥AtCAD4、AtCAD5亲缘关系最近。相关性分析表明PyCAD-1和PyCAD-2与上下游木质素通路小分子相关性较强。PyCAD-1和PyCAD-2开放阅读框全长分别为570和1083 bp,编码蛋白分子量分别为20.38和39.30 kDa。实时荧光定量PCR结果分析显示,PyCAD-1基因的相对表达量在嫩芽、嫩茎中最高;PyCAD-2基因的相对表达量在嫩茎中最高。【结论】PyCAD-2可能直接参与云南松木质素的生物合成,而PyCAD-1则可能存在一定的功能冗余。研究结果为云南松CAD基因家族及木质素代谢途径相关基因研究提供参考。

【Objective】The paper aimed to comprehend the expression characteristics of cinnamyl alcohol dehydrogenase from Pinus yunnanensis and its involvement in lignin biosynthesis.【Method】The relative concentrations of 18 specific small molecules within the lignin pathway were assessed in the stems,buds,and needles of one-year-old P.yunnanensis.Additionally,the CAD genes of P.yunnanensis were identified,cloned and bioinformatics analyzed by comprehensive analysis of transcriptome data.RT-qPCR was employed to examine their expression patterns across various tissues.【Result】The findings revealed that among the 18 lignin pathway molecules,the relative content of 5-O-caffeoylshikimic acid,5-O-p-coumaroyl shikimic acid,and caffeic acid were higher in the buds,while the relative content of 3-O-p-coumaroyl quinic acid,p-coumaric acid,ferulic acid,coniferylaldehyde,L-phenylalanine,coniferyl alcohol,p-coumaryl alcohol,L-(-)-tyrosine,sinapinaldehyde were higher in the stems.In addition,the relative content of coniferin,p-coumaraldehyde,caffeic aldehyde,and syringin were higher in the needles.Four members of the CAD family were screened from the transcriptome of P.yunnanensis,and phylogenetic analysis showed that PyCAD-3 of P.yunnanensis was homologous to AtCAD1,and PyCAD-1,PyCAD-2 and PyCAD-4 were closely related to CAD of Pinus,AtCAD4 and AtCAD5.The results of correlation analysis showed that the PyCAD-1 and PyCAD-2 were closely related to small molecules in the lignin pathway upstream and downstream.The open reading frame of PyCAD-1 and PyCAD-2 was 570 and 1083 bp,respectively,and the molecular weights of PyCAD-1 and PyCAD-2 were 20.38 and 39.30 kDa,respectively.The results of RT-qPCR showed that the relative expression of PyCAD-1 was the highest in bud and tender stem,and PyCAD-2 was the highest in tender stem.【Conclusion】It is speculated that PyCAD-2 may be directly involved in lignin biosynthesis,while PyCAD-1 may have some functional redundancy.The study provides a theoretical basis for studying CAD genes family and genes related to the lignin metabolism pathway of P.yunnanensis.