元宝枫KCS基因家族的鉴定及时空表达分析

Identification and spatio-temporal expression profiles of the KCS gene family in Acer truncatum

【目的】为深入研究元宝枫KCS基因与神经酸生物合成中功能的关联性及高神经酸元宝枫分子育种提供参考。【方法】采用改良的CTAB法提取元宝枫根、茎、叶、花及发育各时期种子的总RNA,利用转录组测序数据及Hmmer-3.0软件、Tbtools1_0692软件、Pfam数据库鉴定元宝枫KCS基因家族成员。利用元宝枫、蒜头果、文冠果、拟南芥、甘蓝型油菜的KCS基因家族成员数据,使用MEGA_X软件构建物种系统发育树。使用Expasy、plantCARE、GAIL、SWISS-MODEL等在线工具对元宝枫KCS家族基因成员进行蛋白质理化特性分析、染色体定位分析、基因结构分析、顺式作用元件分析及亚细胞定位预测。根据转录组数据进行基因表达特异性分析。【结果】从元宝枫基因组中共鉴定了35个AceKCS基因家族成员,分别命名为AceKCS1~AceKCS35,其编码蛋白质长度为156~571个氨基酸,保守结构域及Motif分析结果显示几乎所有AcerKCS均含有FAE1_CUT1_RppA和ACP_syn_Ⅲ_C蛋白保守结构域,启动区含多种与植物生长发育、激素响应、胁迫响应相关的顺式作用元件。亚细胞定位预测结果显示,AceKCS大多位于质膜、叶绿体和细胞质中。将21个蒜头果MoKCS基因、17个文冠果XsKCS基因、21个拟南芥AtKCS基因和58个甘蓝型油菜BnKCS基因共同构建了5个物种152个KCS基因的物种系统发育树,并将其分成了8个亚组,分别为α、β、γ、δ、ε、ζ、η、θ,元宝枫AceKCS仅分布于7个亚组,且大多分布于ε、η和θ亚组。基因表达谱分析结果表明,AceKCS15和AceKCS17在种仁中表达量较高,而在根、茎、叶和花中不表达,在4个发育时期的种仁中表达量变化趋势基本一致,在授粉后100 d时表达量最高,之后逐渐降低。【结论】AceKCS基因在不同组织及不同发育阶段种仁中以不同的水平表达,表现出较高的时空特异性。

【Objective】This study will provide reference for further study on the correlation between KCS gene and the function of nervonic acid biosynthesis and molecular breeding of high nervonic acid in Acer truncatum.【Method】Total RNA was extracted from roots,stems,leaves,flowers and seeds at different developmental stages by improved CTAB method.Members of KCS gene family were identified by transcriptomic sequencing data,HMER-3.0 software,Tbtools1_0692 software and Pfam database.Phylogenetic trees were constructed by MEGA_X software and KCS gene family members of 5 species,including A.truncatum Malania oleifera,Xantboceras sorbifolium,Arabidopsis thaliana and Brassica napus.The protein physicochemical characteristics,chromosome localization,gene structure,cis-acting element analysis and subcellular localization prediction of KCS family gene members were analyzed by online tools such as Expasy,plantCARE,GAIL and SWISS-MODEL.Gene expression specificity was analyzed by transcriptome data.【Result】We identified 35 AceKCSs,named AceKCS1-AceKCS35,whose coding protein length ranged 156-571 amino acids.Analysis of conserved domains and Motif showed that almost AceKCS contained conserved sequences of acyl-coA synthase.The promoter region contained a variety of cis-acting elements related to plant growth and development,hormone response,and stress response.The prediction of subcellular localization showed that most AceKCSs were located in the plasma membrane,chloroplast and cytoplasm.We constructed a phylogenetic tree of 152 KCS gene family members from five species,including 21 MoKCS genes,17 XsKCS genes,21 AtKCS genes and 58 BnKCS genes,and divided it into 8 subgroups,forα、β、γ、δ、ε、ζ、ηandθ.AceKCS were distributed in only 7 subgroups,and mostly inε,ηandθsubgroups.Gene expression analysis showed that AceKCS15 and AceKCS17 were highly expressed in kernels but not in roots,stems,leaves and flowers,and the variation trend of AceKCS15 and AceKCS17 expression levels were basically alike in the four stages of kernel development,the highest expression level on the 100th day after pollination,and then gradually decreased.【Conclusion】AceKCS genes is expressed at different levels in different tissues and kernel development stages,showing high temporal and spatial specificity.