甘蔗ScMOC1基因启动子的克隆与瞬时表达分析
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摘要
MOC1属于植物特有的GRAS家族蛋白基因,是调控植物腋芽形成发育的关键基因。启动子对基因转录效率起直接调控作用,其功能分析可以精确定位基因的表达部位、发育阶段和调控机制,克隆甘蔗腋芽形成发育关键基因ScMOC1的启动子序列,研究其功能对该基因表达调控机制具有重要意义。本研究以我国主栽甘蔗品种新台糖22号(ROC22)的基因组DNA为模板,通过基因组步移和巢式PCR技术克隆到ScMOC1起始密码子ATG上游1874 bp的启动子序列。PlantCARE在线分析预测表明,该序列包含多个真核生物启动子必需的核心元件TATA-box、CAAT-box以及与光响应、激素响应和分生组织表达等相关的顺式作用元件,推测ScMOC1启动子可通过激素诱导调控ScMOC1表达,且该启动子可能通过分生组织表达顺式调控元件CAT-box参与ScMOC1对甘蔗分蘖的调控。将获得的启动子序列替换pBI121质粒中的CaMV35S启动子驱动下游GUS基因表达进行活性分析,结果表明:本研究克隆的启动子片段能驱动GUS基因在甘蔗嫩叶中瞬时表达。5′缺失分析表明该启动子的基础启动子序列在起始密码子ATG上游350～500 bp之间。该结果为后续ScMOC1的调控机制研究奠定了良好的基础。
MOC1 encodes for a plant-specific GRAS family protein,and this gene plays key role in the formation and development of axillary bud.Since the gene promoter is directly involved into transcriptional regulation,functional analysis can accurately locate the expression site,development stage and regulation mechanism of gene.Cloning the sequence and function analysis of ScMOC1 promoter will be of great signi?cance in illustrating the regulation mechanism of ScMOC1.In this study,the 1874 bp promoter sequence upstream of ScMOC1 was isolated from the genomic DNA of sugarcane main cultivar ROC22 by using nested PCR and genomic walking methods.This isolated fragment was verified to be the promoter of ScMOC1 via sequence structure analysis.The results indicated that ScMOC1 promoter contained a few of core elements of the eukaryotic promoter such as TATA-box,CAAT-box,several light,hormone responsive elements and a cis-acting regulatory element related to meristem expression(CAT-box).We speculated that the ScMOC1 promoter could regulate the expression of ScMOC1 in a manner of hormone treatment,required for the sugarcane tillering regulation by the CAT-box cis-acting element.By constructing the target promoter into pBI121 plasmid that contains a GUS reporter,the results showed that this promoter could result in transient expression of GUS gene in sugarcane young leaves,and the deletion analysis indicated that the basic promoter sequence of the promoter was between350 bp and 500 bp upstream of the starting codon ATG of ScMOC1.Thus,these results obtained above will provide the foundation information for ScMOC1 on transcriptional regulation.
MOC1 encodes for a plant-specific GRAS family protein,and this gene plays key role in the formation and development of axillary bud.Since the gene promoter is directly involved into transcriptional regulation,functional analysis can accurately locate the expression site,development stage and regulation mechanism of gene.Cloning the sequence and function analysis of ScMOC1 promoter will be of great signi?cance in illustrating the regulation mechanism of ScMOC1.In this study,the 1874 bp promoter sequence upstream of ScMOC1 was isolated from the genomic DNA of sugarcane main cultivar ROC22 by using nested PCR and genomic walking methods.This isolated fragment was verified to be the promoter of ScMOC1 via sequence structure analysis.The results indicated that ScMOC1 promoter contained a few of core elements of the eukaryotic promoter such as TATA-box,CAAT-box,several light,hormone responsive elements and a cis-acting regulatory element related to meristem expression(CAT-box).We speculated that the ScMOC1 promoter could regulate the expression of ScMOC1 in a manner of hormone treatment,required for the sugarcane tillering regulation by the CAT-box cis-acting element.By constructing the target promoter into pBI121 plasmid that contains a GUS reporter,the results showed that this promoter could result in transient expression of GUS gene in sugarcane young leaves,and the deletion analysis indicated that the basic promoter sequence of the promoter was between350 bp and 500 bp upstream of the starting codon ATG of ScMOC1.Thus,these results obtained above will provide the foundation information for ScMOC1 on transcriptional regulation.
引文
[1]李晓君,武媛丽,张焱珍,杨本鹏,张树珍·甘蔗ShSAP1基因的启动子克隆与序列分析·中国农学通报,2014, 30(12):272-278Li X J, Wu Y L, Zhang Y Z, Yang B P, Zhang S Z.Cloning and sequence analysis of ShSAPl promoter from sugarcane-Chinese Agricultural Science Bulletin, 2014, 30(12):272-278
[2]Li X Y, Qian Q, Fu Z M, Wang Y H, Xiong G S, Zeng D L,Wang X Q, Liu X F, Teng S, Fujimoto Hiroshi, Yuan M, Da Luok, Bin H, Li J Y.Control of tillering in rice-Nature, 2003,422(6932):618
[3]涂田莉·小麦分蘖相关基因TaMOC1的分离及功能分析·泰安:山东农业大学,2017Tu T L-Isolation and functional analysis of TaMOC1 in Triticum aestivum L.Tai'an:Shandong Agricultural University, 2017
[4]李旭娟,李纯佳,徐超华,刘洪博,吴转娣,林秀琴,陆鑫,毛钧,字秋艳,刘新龙.甘蔗MOC1基因(ScMOC1)的克隆与表达分析.植物遗传资源学报,2017, 18(4):734-746LiXJ,LiC J,XuCH,LiuHB,WuZ D,Lin X Q,Lu X,Mao J,Zi Q Y, Liu X L-Cloning and expression analysis of the MOC1gene(ScMOC1)in sugarcane(Saccharum officinarum)·Journal of Plant Genetic Resources, 2017, 18(4):734-746
[5]李田,孙景宽,刘京涛·植物启动子研究进展·生物技术通报,2015,31(2):18-25Li T, Sun J K, Liu J T Research advances on plant promoter.Biotechnology Bulletin, 2015,31(2):18-25
[6]汤方,涂慧珍·真核启动子研究进展·林业工程学报,2015,29(2):7-12Tang F, Tu H Z Research advances on eukaryotic promoter.Journal of Forestry Engineering, 2015,29(2):7-12
[7]张锦,刘新龙,李纯佳,刘洪博,朱建荣,李旭娟.甘蔗启动子研究进展·中国糖料,2018(1):53-56Zhang J, Liu X L, Li C J, Liu H B, Zhu J R, Li X J Research progress on sugarcane promoter.Sugar Crops of China, 2018(1):53-56
[8]牛俊奇,王爱勤,黄静丽,朱惠,李杨瑞,杨丽涛.甘蔗可溶性酸性转化酶(SoSAI1)基因的克隆及表达分析·中国农业科学,2013,46(24):5248-5260Niu J Q, Wang A Q,Huang J L,Zhu H,Li Y R, Yang L T-Cloning and expression analysis of a soluble acid invertase gene(SoSAI1)of sugarcane.Scientia Agricultura Sinica, 2013,46(24):5248-5260
[9]牛俊奇,王爱勤,黄静丽,李杨瑞,杨丽涛.甘蔗中性/碱性转化酶基因SoNIN1的克隆和表达分析·作物学报,2014,40(2):253-263Niu J Q-Wang A Q, Huang J L, Li Y R, Yang L T-Cloning and expression analysis of sugarcane alkaline/neutral invertase gene SoNIN1.Acta Agronomica Sinica,2014, 40(2):253-263
[10]雷美华,叶冰莹,王冰梅,黄祖新,张华,许莉萍,陈由强,陈如凯.甘蔗蔗糖合成酶基因的克隆·应用与环境生物学报,2008, 14(2):177-179Lei M H, Ye B Y, Wang B M, Huang Z X, Zhang H,Xu L P, Chen Y Q, Chen R K-Cloning of sucrose synthase gene from sugarcane(Saccharum officenarum L.).China Journal of applied Environment Biology,2008, 14(2):177-179
[11]周平,何炜,金光,高玉娜,叶冰莹,陈由强,陈如凯.甘蔗SPSⅢ基因组DNA及5'侧翼序列的克隆与分析·农业生物技术学报,2014, 22(1):1-9Zhou P, He W, Jin G, Gao Y N, Ye B Y, Chen Y Q, Chen R K-Clonging and sequence analysis of sucrose phosphate synthase genomic DNA and 5'-flanking sequence from sugarcane(Saccharum spp.).Journal of Agricultural Biotechnology, 2014, 22(1):1-9
[12]吴自明,江安庆,张积森,叶冰莹,陈由强.甘蔗ShSUT4基因及其5'侧翼序列的克隆与序列分析·亚热带农业研究,2012,8(2):126-130Wu Z M, Jiang A Q, Zhang J S, Ye B Y, Chen Y Q-Cloning and sequence analysis of sucrose transporter 4 DNA and 5'flanking sequence from sugarcane.Subtropical Agriculture Research,2012,8(2):126-130
[13]许莉萍.一种提高农杆菌介导的甘蔗GUS瞬时表达率方法:中国,CN201110435405.2011-12-23Xu L P-An improved agrobacterium mediated method for transient expression rate of sugarcane GUS:China,CN201110435405-2011-12-23
[14] Li F, Han Y Y, Feng Y N, Xing S C, Zhao M R, Chen Y H,Wang W.Expression of wheat expansin driven by the RD29promoter in tobacco confers water-stress tolerance without impacting growth and development.Journal of Biotechnology,2013, 163:281-291
[15] Pino M T,Skinner J S,Park E J,Jeknic Z,Hayes P M,Thomashow M F, Chen T H-Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield-Plant Biotechnology Journal, 2007, 5:591-604
[16]秦丽霞,李静,张换样,李盛,竹梦婕,焦改丽,吴慎杰·棉花半乳糖基转移酶基因GhGalT1启动子的克隆及表达分析·作物学报,2018,44(2):218-226Qin L X, Li J, Zhang H Y, Li S, Zhu M J, Jiao G L, Wu S J-Cloning and expression analysis of galactosyl transferase gene GhGalTl promoter in cotton-Acta Agronomica Sinica, 2018,44(2):218-226
[17]张吉贞·水稻MOC1基因启动子的克隆与诱导物的筛选.儋州:华南热带农业大学,2005Zhang J Z.Clone of MOC1 promoter and its inductive material selection in rice-Danzhou:South China University of Tropical Agriculture, 2005
[18]杜丽君·小麦TaMOC1基因的启动子分析及CRISPR-Cas9突变体的获得·泰安:山东农业大学,2018Du L J-Promoter analysis of TaMOC1 gene and obtaining of the CRISPR-Cas9 mutants in Triticum aestivum L..Tai'an:Shandong Agricultural University, 2018
[2]Li X Y, Qian Q, Fu Z M, Wang Y H, Xiong G S, Zeng D L,Wang X Q, Liu X F, Teng S, Fujimoto Hiroshi, Yuan M, Da Luok, Bin H, Li J Y.Control of tillering in rice-Nature, 2003,422(6932):618
[3]涂田莉·小麦分蘖相关基因TaMOC1的分离及功能分析·泰安:山东农业大学,2017Tu T L-Isolation and functional analysis of TaMOC1 in Triticum aestivum L.Tai'an:Shandong Agricultural University, 2017
[4]李旭娟,李纯佳,徐超华,刘洪博,吴转娣,林秀琴,陆鑫,毛钧,字秋艳,刘新龙.甘蔗MOC1基因(ScMOC1)的克隆与表达分析.植物遗传资源学报,2017, 18(4):734-746LiXJ,LiC J,XuCH,LiuHB,WuZ D,Lin X Q,Lu X,Mao J,Zi Q Y, Liu X L-Cloning and expression analysis of the MOC1gene(ScMOC1)in sugarcane(Saccharum officinarum)·Journal of Plant Genetic Resources, 2017, 18(4):734-746
[5]李田,孙景宽,刘京涛·植物启动子研究进展·生物技术通报,2015,31(2):18-25Li T, Sun J K, Liu J T Research advances on plant promoter.Biotechnology Bulletin, 2015,31(2):18-25
[6]汤方,涂慧珍·真核启动子研究进展·林业工程学报,2015,29(2):7-12Tang F, Tu H Z Research advances on eukaryotic promoter.Journal of Forestry Engineering, 2015,29(2):7-12
[7]张锦,刘新龙,李纯佳,刘洪博,朱建荣,李旭娟.甘蔗启动子研究进展·中国糖料,2018(1):53-56Zhang J, Liu X L, Li C J, Liu H B, Zhu J R, Li X J Research progress on sugarcane promoter.Sugar Crops of China, 2018(1):53-56
[8]牛俊奇,王爱勤,黄静丽,朱惠,李杨瑞,杨丽涛.甘蔗可溶性酸性转化酶(SoSAI1)基因的克隆及表达分析·中国农业科学,2013,46(24):5248-5260Niu J Q, Wang A Q,Huang J L,Zhu H,Li Y R, Yang L T-Cloning and expression analysis of a soluble acid invertase gene(SoSAI1)of sugarcane.Scientia Agricultura Sinica, 2013,46(24):5248-5260
[9]牛俊奇,王爱勤,黄静丽,李杨瑞,杨丽涛.甘蔗中性/碱性转化酶基因SoNIN1的克隆和表达分析·作物学报,2014,40(2):253-263Niu J Q-Wang A Q, Huang J L, Li Y R, Yang L T-Cloning and expression analysis of sugarcane alkaline/neutral invertase gene SoNIN1.Acta Agronomica Sinica,2014, 40(2):253-263
[10]雷美华,叶冰莹,王冰梅,黄祖新,张华,许莉萍,陈由强,陈如凯.甘蔗蔗糖合成酶基因的克隆·应用与环境生物学报,2008, 14(2):177-179Lei M H, Ye B Y, Wang B M, Huang Z X, Zhang H,Xu L P, Chen Y Q, Chen R K-Cloning of sucrose synthase gene from sugarcane(Saccharum officenarum L.).China Journal of applied Environment Biology,2008, 14(2):177-179
[11]周平,何炜,金光,高玉娜,叶冰莹,陈由强,陈如凯.甘蔗SPSⅢ基因组DNA及5'侧翼序列的克隆与分析·农业生物技术学报,2014, 22(1):1-9Zhou P, He W, Jin G, Gao Y N, Ye B Y, Chen Y Q, Chen R K-Clonging and sequence analysis of sucrose phosphate synthase genomic DNA and 5'-flanking sequence from sugarcane(Saccharum spp.).Journal of Agricultural Biotechnology, 2014, 22(1):1-9
[12]吴自明,江安庆,张积森,叶冰莹,陈由强.甘蔗ShSUT4基因及其5'侧翼序列的克隆与序列分析·亚热带农业研究,2012,8(2):126-130Wu Z M, Jiang A Q, Zhang J S, Ye B Y, Chen Y Q-Cloning and sequence analysis of sucrose transporter 4 DNA and 5'flanking sequence from sugarcane.Subtropical Agriculture Research,2012,8(2):126-130
[13]许莉萍.一种提高农杆菌介导的甘蔗GUS瞬时表达率方法:中国,CN201110435405.2011-12-23Xu L P-An improved agrobacterium mediated method for transient expression rate of sugarcane GUS:China,CN201110435405-2011-12-23
[14] Li F, Han Y Y, Feng Y N, Xing S C, Zhao M R, Chen Y H,Wang W.Expression of wheat expansin driven by the RD29promoter in tobacco confers water-stress tolerance without impacting growth and development.Journal of Biotechnology,2013, 163:281-291
[15] Pino M T,Skinner J S,Park E J,Jeknic Z,Hayes P M,Thomashow M F, Chen T H-Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield-Plant Biotechnology Journal, 2007, 5:591-604
[16]秦丽霞,李静,张换样,李盛,竹梦婕,焦改丽,吴慎杰·棉花半乳糖基转移酶基因GhGalT1启动子的克隆及表达分析·作物学报,2018,44(2):218-226Qin L X, Li J, Zhang H Y, Li S, Zhu M J, Jiao G L, Wu S J-Cloning and expression analysis of galactosyl transferase gene GhGalTl promoter in cotton-Acta Agronomica Sinica, 2018,44(2):218-226
[17]张吉贞·水稻MOC1基因启动子的克隆与诱导物的筛选.儋州:华南热带农业大学,2005Zhang J Z.Clone of MOC1 promoter and its inductive material selection in rice-Danzhou:South China University of Tropical Agriculture, 2005
[18]杜丽君·小麦TaMOC1基因的启动子分析及CRISPR-Cas9突变体的获得·泰安:山东农业大学,2018Du L J-Promoter analysis of TaMOC1 gene and obtaining of the CRISPR-Cas9 mutants in Triticum aestivum L..Tai'an:Shandong Agricultural University, 2018