摘要
【目的】鉴定辣木中MoGAD基因,并对其进行克隆、生物信息学分析和表达水平分析,为探究辣木MoGAD基因的功能与γ-氨基丁酸(GABA)生物合成的关系奠定基础。【方法】以拟南芥(NP_197235.1)和葡萄(XP_002285268.1)GAD蛋白序列为参考,通过本地BLAST方法,利用TBtools软件在辣木转录组数据中挖掘MoGAD家族基因,采用PCR技术对其进行克隆,并通过生物信息学在线分析网站和分子对接技术分析MoGAD蛋白的理化性质和催化活性位点,采用实时荧光定量RT-qPCR技术分析MoGAD基因在不同组织中的表达水平。【结果】在转录组数据中挖掘到4个MoGAD基因并成功对其进行克隆,测序的CDS区长度分别为:1494、1212、1470和942 bp;MoGAD1、MoGAD3和MoGAD4蛋白不存在跨膜结构域,属于膜外蛋白。其中MoGAD2蛋白序列在第5~27和60~82氨基酸位置存在跨膜结构,此段序列编码蛋白为分泌蛋白;系统发育分析显示MoGAD1基因家族所在分支基因Motif数量最多;分子对接表明4个MoGAD蛋白存在磷酸吡哆醛(PLP)和L-谷氨酸结合位点,且每个蛋白有多个氨基酸残基与PLP,L-谷氨酸形成氢键;荧光定量分析显示MoGAD1和MoGAD2基因在嫩叶、成熟叶和花中的表达量显著高于MoGAD3和MoGAD4基因,但这些基因在茎中表达量较低,推测MoGAD酶主要参与辣木叶部位GABA的生物合成。【结论】本研究成功克隆了辣木的MoGAD基因,并分析其编码蛋白的理化性质及与底物结合的功能活性位点,为进一步研究MoGAD基因的功能提供了理论依据。
【Objective】The MoGAD gene in Moringa oleifera was identified,and subsequent cloning,bioinformatics analysis,and expression level analysis were conducted.These investigations have established a solid foundation for exploring the functional relationship between the MoGAD genes andγ-aminobutyric acid(GABA)biosynthesis in M.oleifera.【Method】Using the GAD protein sequences of Arabidopsis thaliana(NP_197235.1)and Vitis vinifera(XP_002285268.1)to be as references,MoGAD family genes were identified from M.oleifera transcriptome data through local BLAST method and TBtools software.The genes were subsequently cloned using PCR technology.Bioinformatics online analysis websites and molecular docking technology were employed to analyze the physical and chemical properties,as well as the catalytic active sites of the MoGAD protein.Additionally,the real-time fluorescence quantitative RT-qPCR technology was utilized to assess the expression levels of MoGAD genes in different tissues.【Result】The four MoGAD genes were excavated from transcriptome data and cloned successfully.The length of the CDS region sequenced was 1494,1212,1470 and 942 bp respectively.MoGAD1,MoGAD3 and MoGAD4 proteins had no transmembrane domains and belonged to extracellular proteins.The MoGAD2 gene had the transmembrane structure at amino acid positions 5-27 and 60-82,which encoded a kind of secreted proteins.The phylogenetic analysis showed that the largest number of motifs was in the branch of MoGAD1 gene family.The molecular docking revealed that four MoGAD proteins had binding sites for pyridoxal phosphate(PLP)and L-glutamate,and each protein had multiple amino acid residues that formed hydrogen bonds with PLP and L-glutamate.The results of fluorescence quantitative analysis showed that the expression levels of MoGAD1 and MoGAD2 genes in tender leaves,mature leaves and flowers were significantly higher than those of MoGAD3 and MoGAD4 genes,but the expression levels of these genes were relatively low in the stem,suggesting that MoGAD enzymes were mainly involved in GABA biosynthesis in the leaves of M.oleifera.【Conclusion】MoGAD gene is successfully cloned from M.oleifera,and the physical and chemical properties of its encoded protein and the functional active sites of substrate binding are analyzed,which provide a theoretical basis for further study on the function of MoGAD genes.
作者
敖冬慧
胡云飞
向贵生
刘佳
聂石圆
田洋
唐卿雁
AO Dong-hui;HU Yun-fei;XIANG Gui-sheng;LIU Jia;NIE Shi-yuan;TIAN Yang;TANG Qing-yan(College of Food Science and Technology,Yunnan Agricultural University,Kunming 650201,China;National-Local Joint Engineering Research Center on Germplasm Innovation&Utilization of Chinese Medicinal Materials in Southwestern China,Kunming 650201,China;Engineering Research Center of Development and Utilization of Food and Drug Homologous Resources,Ministry of Education,Kunming 650201,China;National Research and Development Professional Center for Moringa Processing Technology,Kunming 650201,China;Pu’er University,Simao,Yunnan 665000,China)
出处
《西南农业学报》
CSCD
北大核心
2024年第3期490-502,共13页
Southwest China Journal of Agricultural Sciences
基金
云南省科技厅基础研究专项-青年项目(202301AU070119)
云南省科技重大专项(202102AE090027-2、202002AA100005-2)。
