苎麻CCRs基因家族3类成员的生化特性与表达差异

Biochemical characteristics and expression differences of three members of CCRs in ramie(Boehmeria nivea)

肉桂酰辅酶A还原酶(CCR)是木质素生物合成的关键酶,在植物中多以基因家族形式存在。本研究通过苎麻转录组信息,筛选并克隆了4个苎麻CCR基因家族成员(BnCCR、BnCCR-1、BnCCR-2和BnCCR-3)。序列分析结果发现4个BnCCRs可以分为3类,BnCCR属于bona fide CCR类群,BnCCR-1为CCR类似蛋白,而BnCCR-2和BnCCR-3序列在NADPH结合功能域、催化三联体以及CCR底物结合位点上均与bona fide CCR都有差异,形成了苎麻中一个新的CCR类群。跨膜结构分析显示,BnCCR含有1个跨膜螺旋区,其他3个BnCCRse无跨膜螺旋区。结构分析显示,4个BnCCRs的二级和三级结构存在差异,BnCCR-2同源建模模板不同于其他3个BnCCRs。时空表达谱分析发现,BnCCR在快速生长期的木质部具有较高表达水平,而BnCCR-2在快速生长期的韧皮部具有非常高的表达水平。体外酶活测定进一步研究发现,BnCCR具有典型的bona fide CCR催化活性和底物适应性,而BnCCR-2的表现不同于典型CCR的底物适应性,对肉桂酰CoA和芥子酰CoA表现出结合特异性。因此,苎麻中BnCCR参与木质素的生物合成,而BnCCR-2是具有不同于典型CCR结构、组织表达和体外生化功能的一个新类群,BnCCR-2可能不参与或并不仅仅参与木质素的生物合成,这为研究CCR蛋白家族的进化提供了新的参考。

Cinnamoyl-CoA reductases(CCR)is a key enzyme in lignin biosynthesis.CCR mostly exists in the form of gene family in plants.In this study,four family members of CCR(BnCCR,BnCCR-1,BnCCR-2,and BnCCR-3)were screened and cloned by ramie transcriptome information.Sequence analysis revealed that the four BnCCRs can be divided into three categories.BnCCR belonged to bona fide CCR group,BnCCR-1 was a CCR like protein,while BnCCR-2 and BnCCR-3 sequences were different from bona fide CCR in NADPH binding domain,catalytic triad,and CCR substrate binding site,and formed a new group in ramie.Transmembrane structure analysis showed that BnCCR contained two transmembrane regions,and the other three BnCCRs didn’t contain transmembrane regions.Structural analysis indicated that the secondary and tertiary structure of the four BnCCRs were different,and the BnCCR-2 homologous modeling template was different from the other three BnCCRs.The qRT-PCR analysis demonstrated that BnCCR had higher expression levels in xylem at rapid growth stage,BnCCR-2 had the highest expression levels in phloem at rapid growth stage.In vitro enzyme activity analysis further found that BnCCR had typical bona fide CCR catalytic activity and substrate adaptability,while BnCCR-2 was different from the substrate adaptability of typical CCR,and had substrate specificity for cinnamoyl CoA and sinapoyl CoA.Therefore,BnCCR was involved in lignin biosynthesis,while BnCCR-2 was a new group with structure,tissue expression and in vitro biochemical function different from typical CCR.BnCCR-2 may not participate or not only participate in lignin biosynthesis,which provides a new reference for studying the evolution of CCR protein family.