茉莉花MVD基因及其启动子的克隆与表达
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摘要
根据茉莉花转录组数据,采用RT-PCR技术,克隆了茉莉花甲羟戊酸焦磷酸脱羧酶(MVD)基因,命名为JsMVD(GenBank登录号为MH311041.1).采用染色体步移技术分离JsMVD基因5′端上游调控序列,通过实时荧光定量PCR技术检测JsMVD基因在茉莉花植株不同组织及不同激素处理下的表达水平.测序结果表明,JsMVD基因全长cDNA序列的长度为1 500 bp,包含长度为1 269 bp的开放阅读框(ORF),共编码422个氨基酸,亚细胞定位预测该蛋白可能位于细胞质上.JsMVD蛋白含有GHMP激酶N-端和C-端保守结构域,具有多个保守氨基酸残基及ATP结合位点,与野生油橄榄的相似性最高,相似系数达到88%,且进化树显示两者的亲缘关系最近.JsMVD基因5′端启动子序列长度为893 bp,该调控序列包含多种植物激素响应元件和光响应元件.实时荧光定量PCR技术检测结果表明,JsMVD基因在成熟花中的表达量最高,从高到低依次为成熟花、花蕾、茎、叶、根,且受GA、IAA和ABA不同程度的诱导.
Based on jasmine transcriptome, diphosphomevalonate decarboxylase(MVD) gene was cloned using RT-PCR. The upstream regulation sequences of Jasminum sambac 5′ end was isolated by Genome Walking Technique, and its expression treated with different hormones in different tissues were quantified using RT-qPCR. The results showed that JsMVD gene(GenBank accession number MH311041.1) was 1 500 bp in length, which contained 1 269 bp open read frame and encoded 422 amino acids. Subcellular localization prediction showed that JsMVD protein may be located in the cytoplasm. The JsMVD contained GHMP_ kinases_N domain and GHMP_ kinases_C domain, many conserved amino acid residues and ATP binding sites. JsMVD was most homologous to Olea europaea based on phylogenetic tree, with an identity up to 88%. Plantcare analysis indicated that the upstream 5′ sequence of JsMVD was 893 bp in length. It contained multiple light-responsive cis-regulatory elements and hormones responsive cis-regulatory elements such as auxin(IAA), gibberellin(GA) and abscisic acid(ABA). RT-qPCR analysis showed that the expression of JsMVD was highest in mature flower, followed by bud, stem, leaf and root, and it was induced by GA, IAA, ABA to some extent.
Based on jasmine transcriptome, diphosphomevalonate decarboxylase(MVD) gene was cloned using RT-PCR. The upstream regulation sequences of Jasminum sambac 5′ end was isolated by Genome Walking Technique, and its expression treated with different hormones in different tissues were quantified using RT-qPCR. The results showed that JsMVD gene(GenBank accession number MH311041.1) was 1 500 bp in length, which contained 1 269 bp open read frame and encoded 422 amino acids. Subcellular localization prediction showed that JsMVD protein may be located in the cytoplasm. The JsMVD contained GHMP_ kinases_N domain and GHMP_ kinases_C domain, many conserved amino acid residues and ATP binding sites. JsMVD was most homologous to Olea europaea based on phylogenetic tree, with an identity up to 88%. Plantcare analysis indicated that the upstream 5′ sequence of JsMVD was 893 bp in length. It contained multiple light-responsive cis-regulatory elements and hormones responsive cis-regulatory elements such as auxin(IAA), gibberellin(GA) and abscisic acid(ABA). RT-qPCR analysis showed that the expression of JsMVD was highest in mature flower, followed by bud, stem, leaf and root, and it was induced by GA, IAA, ABA to some extent.
引文
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[15] 姚雪倩,陈丹,岳川,等.茶树烯醇酶基因CsENO的克隆及其在非生物胁迫中的表达分析[J].园艺学报,2017,44(3):537-546.
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[17] ZOU Z,YANG L,AN F,et al.Genomewide identification and analysis of MVD gene family in Euphorbiaceae plants [J].Agricultural Biotechnology,2013,2(6):1-6.
[18] WEERASINGHE S,DASSANAYAKE R S.Simulation of structural and functional properties of mevalonate diphosphate decarboxylase (MVD) [J].Journal of Molecular Modeling,2010,16(3):489-498.
[19] JIN K A,YOUNGKI P,WI Y L,et al.Increasement of eleutherosides and antioxidant activity in Eleutherococcus senticosus adventitious root by jasmonic acid [J].Journal of Korean Forestry Society,2007,96(5):539-542.
[20] YONEKURA-SAKAKIBARA K,SAITO K.Functional genomics for plant natural product biosynthesis [J].Natural Product Reports,2009,26(11):1 466-1 487.
[2] 刘力恒,王立升,沈芳,等.茉莉花精油的研究进展[J].大众科技,2007,97(9):124-125.
[3] 库尔班江,欧青海,阿布都萨拉木.中药茉莉花的研究进展[J].科技信息(科学教研),2008(5):43-44.
[4] 邹瑶,齐桂年.茉莉花渣多糖降血糖、改善糖尿病症状作用的研究[J].食品科技,2011,36(2):157-160.
[5] ANDREASSI J L,LEYH T S.Molecular functions of conserved aspects of the GHMP kinase family [J].Biochemistry,2004,43(46):14 594-14 601.
[6] SHI L,QIN L,XU Y,et al.Molecular cloning,characterization,and function analysis of a mevalonate pyrophosphate decarboxylase gene from Ganoderma lucidum [J].Molecular Biology Reports,2012,39(5):6 149-6 159.
[7] 李清,李标,郭顺星.金钗石斛焦磷酸甲羟戊酸脱羧酶基因的克隆及表达分析[J].基因组学与应用生物学,2018,37(2):850-858.
[8] 张晓东,李彩霞,王元忠.滇龙胆甲羟戊酸二磷酸脱羧酶基因的克隆与表达分析[J].贵州农业科学,2015,43(12):164-169.
[9] 李志栋,杨果,尤鹏升,等.刺五加MDD基因启动子的克隆与生物信息学分析[J].中草药,2016,47(21):3 871-3 875.
[10] VRANOVA E,COMAN D,GRUISSEM W.Network analysis of the MVA and MEP pathways for isopenoidsynthesis [J].Annual Revier of Plant Biology,2013,64:665-770.
[11] 邢朝斌,龙月红,何闪,等.刺五加甲羟戊酸焦磷酸脱羧酶基因的克隆与表达分析[J].西北植物学报,2012,32(10):1 950-1 956.
[12] GAO W,SUN H X,XIAO H,et al.Combining metabolomics and transcriptomics to characterize tanshinone biosythesis in Salvia miltiorrhiza [J].BMC Genomics,2014,15:73-86.
[13] 李鹤,俞滢,陈桂信,等.双瓣茉莉花[Jasminum sambac (L.) Ait]开放过程香气成分的动态变化[J].茶叶学报,2015,56(1):29-38.
[14] YU Y,LYU S H,CHEN D,et al.Olatiles emitted at different flowering stages of Jasminum sambac and expression of genes related to α-farnesenebiosynthesis [J].Molecules,2017,22:546-558.
[15] 姚雪倩,陈丹,岳川,等.茶树烯醇酶基因CsENO的克隆及其在非生物胁迫中的表达分析[J].园艺学报,2017,44(3):537-546.
[16] BARTA M L,MCWHORTER W J,MIZIORKO H M,et al.Structural basis for nucleotide binding and reaction catalysis in mevalonate diphosphate decarboxylase [J].Biochemistry,2012,51(28):5 611-5 621.
[17] ZOU Z,YANG L,AN F,et al.Genomewide identification and analysis of MVD gene family in Euphorbiaceae plants [J].Agricultural Biotechnology,2013,2(6):1-6.
[18] WEERASINGHE S,DASSANAYAKE R S.Simulation of structural and functional properties of mevalonate diphosphate decarboxylase (MVD) [J].Journal of Molecular Modeling,2010,16(3):489-498.
[19] JIN K A,YOUNGKI P,WI Y L,et al.Increasement of eleutherosides and antioxidant activity in Eleutherococcus senticosus adventitious root by jasmonic acid [J].Journal of Korean Forestry Society,2007,96(5):539-542.
[20] YONEKURA-SAKAKIBARA K,SAITO K.Functional genomics for plant natural product biosynthesis [J].Natural Product Reports,2009,26(11):1 466-1 487.