春性甘蓝型油菜sBnTCP2基因的克隆及表达分析
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摘要
克隆春性甘蓝型油菜相对于蕾期在苗期特异性表达的基因,并对其在各个发育时期以及各组织中的表达模式进行研究。用cDNA-AFLP技术筛选苗期特异性表达的基因片段,根据差异片段设计引物,用RACE技术扩增差异基因两端序列,再根据两端序列设计引物,进行全长cDNA克隆;用RT-qPCR法进行基因表达分析。结果获得了一个昼夜节律基因的CDS全长序列1 077 bp,该基因编码是一个由359个氨基酸残基组成的蛋白,暂命名为sBnTCP2。与油菜基因组序列比对结果显示,sBnTCP2基因位于C1染色体上。生物信息学分析显示,sBnTCP2的编码序列与电子预测的甘蓝型油菜TCP2 (XM_013815648.2)基因的编码序列相似度达99%,其CDS区的第52处、第53处、第54处3个碱基(AAC)缺失。sBnTCP2在油菜不同发育时的各组织中都有表达,但在苗期叶片中表达量最高。sBnTCP2在春性特早熟甘蓝型油菜发育中可能起到重要的调节作用。
The genes specifically expressed in the spring stage of Brassica napus L. were compared with those in the bud stage, and their expression patterns at various developmental stages and tissues were studied. The c DNA-AFLP technique was used to screen the gene fragments specifically expressed in the seedling stage, the primers were designed according to the different fragments, the two ends of the differential gene were amplified by RACE technology, and the primers were designed according to the sequence of the two ends to carry out full-length cDNA cloning. Gene expression analysis was performed by RT-qPCR. Finally, the full-length sequence of CDS of a circadian rhythm gene was obtained at 1 077 bp, which encodes a protein consisting of 359 amino acid residues, tentatively named sBnTCP2. The alignment with the rapeseed genome sequence revealed that the sBnTCP2 gene is located on the C1 chromosome. Bioinformatics analysis showed that the coding sequence of sBnTCP2 and the electronically predicted coding sequence of the Brassica napus L. TCP2(XM_013815648.2)gene were 99% similar, and the base of 52, 53 and 54 of the CDS region was 3 bases(AAC) missing. sBnTCP2 was expressed in all tissues of rapeseed at different developmental stages, but it was the highest in leaves at seedling stage. sBnTCP2 may play an important regulatory role in the development of spring-specific early-maturing Brassica napus L..
The genes specifically expressed in the spring stage of Brassica napus L. were compared with those in the bud stage, and their expression patterns at various developmental stages and tissues were studied. The c DNA-AFLP technique was used to screen the gene fragments specifically expressed in the seedling stage, the primers were designed according to the different fragments, the two ends of the differential gene were amplified by RACE technology, and the primers were designed according to the sequence of the two ends to carry out full-length cDNA cloning. Gene expression analysis was performed by RT-qPCR. Finally, the full-length sequence of CDS of a circadian rhythm gene was obtained at 1 077 bp, which encodes a protein consisting of 359 amino acid residues, tentatively named sBnTCP2. The alignment with the rapeseed genome sequence revealed that the sBnTCP2 gene is located on the C1 chromosome. Bioinformatics analysis showed that the coding sequence of sBnTCP2 and the electronically predicted coding sequence of the Brassica napus L. TCP2(XM_013815648.2)gene were 99% similar, and the base of 52, 53 and 54 of the CDS region was 3 bases(AAC) missing. sBnTCP2 was expressed in all tissues of rapeseed at different developmental stages, but it was the highest in leaves at seedling stage. sBnTCP2 may play an important regulatory role in the development of spring-specific early-maturing Brassica napus L..
引文
Anwesha N.,Stacey K.,and Thomas J.,2009,Mi R319a targeting of TCP4 is critical for petal growth and development in Arabidopsis,Proc.Natl.Acad.Sci.USA,106(52):22534-22539
Bei C.L.,Zhang X.Y.,Wen X.J.,and Liu X.,2006,Isolation of ta VHA-C a gene in wheat related to salt-tolerance via cDNA-AFLP,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),39(9):1736-1742(秘彩莉,张学勇,温小杰,刘旭,2006,利用cDNA-AFLP技术获得小麦耐盐性相关基因TaVHA-C,中国农业科学,39(9):1736-1742)
Christian W.B.,Rutger S.,van der H.,Steef M.,Dick V.,Marc Z.,and Richard G.F.V.,1996,Visualization of dif-ferential gene expression using a novel method of RNA fingerprinting based on AFLP:analysis of gene expression during potato tuber de-velopment,Plant Journal,9(6):745-753
Du D.Z.,Nie P.,Xu L.,Luo Y.X.,Yao Y.M.,Zhao H.W.,and Zhang X.M.,2010,Rapeseed heterosis of different ecotypes in Qinghai province,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Sciences),32(2):180-186(杜德志,聂平,徐亮,罗玉秀,姚艳梅,周红伟,张晓梅,2010,不同生态类型甘蓝型油菜在青海生态条件下的杂种优势表现,中国油料作物学报,32(2):180-186)
Estelle G.,Sophia N.,Chris C.,Owen D.,Jasmine L.,Chun P.L.,Van Aken O.,Millar A.H.,Monika M.,and James W.,2010,TCP transcription factors link the reguLation of genes encoding mitochondrial proteins with the circadian clock in Arabidopsis thaliana,Plant Cell,22(12):3921-3934
Han B.,and Peng J.Y.,2006,cDNA-AFLP and its application in research about gene expressions of plants,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),26(8):1753-1758(韩斌,彭建营,2006,cDNA-AFLP技术及其在植物基因表达研究中的应用,西北植物学报,26(8):1753-1758)
Haksong P.,Li Y.L.,Hyenchol K.,and Jiang L.X.,2017,cDNA-amplified fragment length polymorphism analysis reveals differential gene expression induced by exogenous MeJAand GA3 in oilseed rape(Brassica napus L.)flowers,Journal of Integrative Agriculture,16(1):47-56
Luo Y.X.,and Luo C.Y.,2015,Genetic analysis of flowering time of spring rape,Beifang Yuanyi(Northern Horticculture),6(19):1-6(罗玉秀,罗春燕,2015,春性甘蓝型油菜开花时间的遗传分析,北方园艺,6(19):1-6)
Luo Y.X.,Zhang S.P.,Xu C.C.,Ma X.G.,and Du D.Z.,2016,Cloning and expression of sBnFLD gene fromspring rapeseed(Brassica napus L.),Xibei Nonglin Keji Daxue Xuebao(Journal of northwest A&F university),44(11):1-7(罗玉秀,张生萍,许唱唱,马小岗,杜德志,2016,特早熟春性甘蓝油菜sBnFLD基因的克隆及表达研究,西北农林科技大学学报,44(11):1-7)
Ma G.,and Wang X.F.,2013,cDNA-AFLP analysis of genes related with desiccation to leranceduring development and germination in Brassica napus L.seeds,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),33(4):656-663(马刚,汪晓峰,2013,油菜种子发育和萌发过程中脱水耐性相关基因的cDNA-AFLP分析,西北植物学报,33(4):656-663)
Mimida N.,Kidou S.,and Iwanami H.,2011,Apple floering locus T proteins interact with transcription factors implicated in cell growth and organ development,Tree Physiology,31(5):555-566
Nazmul H.B.,GiuLia F.,Anton P.,Lalit P.,and Klaas J.W.,2015,MET1 is a Thylakoid-associated TPR protein involved in photosystemⅡsupercomplex formation and repair in Arabidopsis,Plant Cell,27(1):262-285
Ren H.Y.,Sun X.,Zheng C.S.,Wang W.L.,Sun X.Z.,and Shu H.R.,2011,Differential analysis of flowering related genes by cDNA-AFLP in chrysanthemum,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),44(16):3386-3394(任洪艳,孙霞,郑成淑,王文莉,孙宪芝,束怀瑞,2011,利用cD-NA-AFLP技术筛选菊花开花相关基因,中国农业科学,44(16):3386-3394)
Tang H.,Shuai A.H.,and Xiang F.Y.,2006,Review of methods and progress in plant gene differential expression researches,Hainan Daxue Xuebao(Naturat science Journal of Hainan Univeraity),24(3):309-316(汤华,帅爱华,向福英,2006,植物基因差异表达的研究方法及进展,海南大学学报,24(3):309-316)
Wang J.L.,Li X.X.,Qiu Y.,Zhang X.H.,Shen D.,Wang H.P.,Sun Y.Y.,and Duan M.M.,2015,Research advances on molecular mrkers and regulation mechanism forthe bolting and blooming characteristics of brassicaceae vegetables,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),16(6):1283-1289(汪精磊,李锡香,邱杨,张晓辉,沈镝,王海平,孙玉燕,段蒙蒙,2015,十字花科蔬菜抽薹开花性状的调控机理和分子育种研究进展,植物遗传资源学报,16(6):1283-1289)
Wei W.L.,Wang H.Z.,and Liu G.H.,2007,Differential expression analysis of genes at the stage before anther abortion of NCa CMS system in Brassica napus L.,Zuowu Xuebao(Acta Agronomica Sinica),33(10):1654-1661(危文亮,王汉中,刘贵华,2007,甘蓝型油菜NCa细胞质雄性不育系统花药败育前期的基因差异达,作物学报,33(10):165)
Xu C.C.,Zhang S.P.,Li P.,and Luo Y.X.,2018,Clone and expression analysis of BnMQ34 gene in spring rapeseed,Fengzi Zhiwu Yuzhong(Molecular Plant Breeding),16(3):718-723(许唱唱,张生萍,李潘,罗玉秀,2018,春性甘蓝型油菜BnMQ34基因的克隆及表达分析,分子植物育种,16(3):718-723
Xu Y.C.,Gu A.X.,Xuan S.X.,Wang Y.H.,Zhao J.J.,Zhang W.W.,and Sheng S.X.,2016,cDNA-AFLP analysis on differentially expressed genes associated with bolting in Chinese cabbage(Brassica oleracea var.capitata)translocation lines of AT4 Series,Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),24(1):26-34(许愿超,顾爱侠,轩淑欣,王彦华,赵建军,张巍巍,申书兴,2016,大白菜-结球甘蓝易位系AT4系列抽薹相关基因表达差异的cD-NA-AFLP分析,农业生物技术学报,24(1):26-34)
Zhang S.P.,Luo Y.X.,Du D.Z.,Luo C.Y.,Ma X.G.,Zhao Z.G.,Xu C.C.,Zhang H.S.,Wu P.,and Chao W.J.,2016,A novel alternative splicing isoform of FCA homologue in spring rapeseed,Xibei Nonglin Keji Daxue Xuebao(Journal of Northwest A&F University),44(4):64-73(张生萍,罗玉秀,杜德志,罗春燕,马小岗,赵志刚,许唱唱,张红生,吴鹏,晁文俊,2016,春性甘蓝型油菜FCA同源基因新可变剪接体的克隆及表达研究,西北农林科技大学学报,44(4):64-73)
Zhao J.R.,Li N.,Liu H.X.,Bi Y.,and Zhang Z.Y.,2009,Confirmation of cDNA-AFLP differential expressed fragments of related defense genes agaist BYDV in wheat,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),10(1):16-20(赵继荣,李宁,刘红霞,毕阳,张增艳,2009,小麦抗黄矮病相关基因cDNA-AFLP差异表达片段的验证,植物遗传资源学报,10(1):16-20)
Bei C.L.,Zhang X.Y.,Wen X.J.,and Liu X.,2006,Isolation of ta VHA-C a gene in wheat related to salt-tolerance via cDNA-AFLP,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),39(9):1736-1742(秘彩莉,张学勇,温小杰,刘旭,2006,利用cDNA-AFLP技术获得小麦耐盐性相关基因TaVHA-C,中国农业科学,39(9):1736-1742)
Christian W.B.,Rutger S.,van der H.,Steef M.,Dick V.,Marc Z.,and Richard G.F.V.,1996,Visualization of dif-ferential gene expression using a novel method of RNA fingerprinting based on AFLP:analysis of gene expression during potato tuber de-velopment,Plant Journal,9(6):745-753
Du D.Z.,Nie P.,Xu L.,Luo Y.X.,Yao Y.M.,Zhao H.W.,and Zhang X.M.,2010,Rapeseed heterosis of different ecotypes in Qinghai province,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Sciences),32(2):180-186(杜德志,聂平,徐亮,罗玉秀,姚艳梅,周红伟,张晓梅,2010,不同生态类型甘蓝型油菜在青海生态条件下的杂种优势表现,中国油料作物学报,32(2):180-186)
Estelle G.,Sophia N.,Chris C.,Owen D.,Jasmine L.,Chun P.L.,Van Aken O.,Millar A.H.,Monika M.,and James W.,2010,TCP transcription factors link the reguLation of genes encoding mitochondrial proteins with the circadian clock in Arabidopsis thaliana,Plant Cell,22(12):3921-3934
Han B.,and Peng J.Y.,2006,cDNA-AFLP and its application in research about gene expressions of plants,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),26(8):1753-1758(韩斌,彭建营,2006,cDNA-AFLP技术及其在植物基因表达研究中的应用,西北植物学报,26(8):1753-1758)
Haksong P.,Li Y.L.,Hyenchol K.,and Jiang L.X.,2017,cDNA-amplified fragment length polymorphism analysis reveals differential gene expression induced by exogenous MeJAand GA3 in oilseed rape(Brassica napus L.)flowers,Journal of Integrative Agriculture,16(1):47-56
Luo Y.X.,and Luo C.Y.,2015,Genetic analysis of flowering time of spring rape,Beifang Yuanyi(Northern Horticculture),6(19):1-6(罗玉秀,罗春燕,2015,春性甘蓝型油菜开花时间的遗传分析,北方园艺,6(19):1-6)
Luo Y.X.,Zhang S.P.,Xu C.C.,Ma X.G.,and Du D.Z.,2016,Cloning and expression of sBnFLD gene fromspring rapeseed(Brassica napus L.),Xibei Nonglin Keji Daxue Xuebao(Journal of northwest A&F university),44(11):1-7(罗玉秀,张生萍,许唱唱,马小岗,杜德志,2016,特早熟春性甘蓝油菜sBnFLD基因的克隆及表达研究,西北农林科技大学学报,44(11):1-7)
Ma G.,and Wang X.F.,2013,cDNA-AFLP analysis of genes related with desiccation to leranceduring development and germination in Brassica napus L.seeds,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),33(4):656-663(马刚,汪晓峰,2013,油菜种子发育和萌发过程中脱水耐性相关基因的cDNA-AFLP分析,西北植物学报,33(4):656-663)
Mimida N.,Kidou S.,and Iwanami H.,2011,Apple floering locus T proteins interact with transcription factors implicated in cell growth and organ development,Tree Physiology,31(5):555-566
Nazmul H.B.,GiuLia F.,Anton P.,Lalit P.,and Klaas J.W.,2015,MET1 is a Thylakoid-associated TPR protein involved in photosystemⅡsupercomplex formation and repair in Arabidopsis,Plant Cell,27(1):262-285
Ren H.Y.,Sun X.,Zheng C.S.,Wang W.L.,Sun X.Z.,and Shu H.R.,2011,Differential analysis of flowering related genes by cDNA-AFLP in chrysanthemum,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),44(16):3386-3394(任洪艳,孙霞,郑成淑,王文莉,孙宪芝,束怀瑞,2011,利用cD-NA-AFLP技术筛选菊花开花相关基因,中国农业科学,44(16):3386-3394)
Tang H.,Shuai A.H.,and Xiang F.Y.,2006,Review of methods and progress in plant gene differential expression researches,Hainan Daxue Xuebao(Naturat science Journal of Hainan Univeraity),24(3):309-316(汤华,帅爱华,向福英,2006,植物基因差异表达的研究方法及进展,海南大学学报,24(3):309-316)
Wang J.L.,Li X.X.,Qiu Y.,Zhang X.H.,Shen D.,Wang H.P.,Sun Y.Y.,and Duan M.M.,2015,Research advances on molecular mrkers and regulation mechanism forthe bolting and blooming characteristics of brassicaceae vegetables,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),16(6):1283-1289(汪精磊,李锡香,邱杨,张晓辉,沈镝,王海平,孙玉燕,段蒙蒙,2015,十字花科蔬菜抽薹开花性状的调控机理和分子育种研究进展,植物遗传资源学报,16(6):1283-1289)
Wei W.L.,Wang H.Z.,and Liu G.H.,2007,Differential expression analysis of genes at the stage before anther abortion of NCa CMS system in Brassica napus L.,Zuowu Xuebao(Acta Agronomica Sinica),33(10):1654-1661(危文亮,王汉中,刘贵华,2007,甘蓝型油菜NCa细胞质雄性不育系统花药败育前期的基因差异达,作物学报,33(10):165)
Xu C.C.,Zhang S.P.,Li P.,and Luo Y.X.,2018,Clone and expression analysis of BnMQ34 gene in spring rapeseed,Fengzi Zhiwu Yuzhong(Molecular Plant Breeding),16(3):718-723(许唱唱,张生萍,李潘,罗玉秀,2018,春性甘蓝型油菜BnMQ34基因的克隆及表达分析,分子植物育种,16(3):718-723
Xu Y.C.,Gu A.X.,Xuan S.X.,Wang Y.H.,Zhao J.J.,Zhang W.W.,and Sheng S.X.,2016,cDNA-AFLP analysis on differentially expressed genes associated with bolting in Chinese cabbage(Brassica oleracea var.capitata)translocation lines of AT4 Series,Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),24(1):26-34(许愿超,顾爱侠,轩淑欣,王彦华,赵建军,张巍巍,申书兴,2016,大白菜-结球甘蓝易位系AT4系列抽薹相关基因表达差异的cD-NA-AFLP分析,农业生物技术学报,24(1):26-34)
Zhang S.P.,Luo Y.X.,Du D.Z.,Luo C.Y.,Ma X.G.,Zhao Z.G.,Xu C.C.,Zhang H.S.,Wu P.,and Chao W.J.,2016,A novel alternative splicing isoform of FCA homologue in spring rapeseed,Xibei Nonglin Keji Daxue Xuebao(Journal of Northwest A&F University),44(4):64-73(张生萍,罗玉秀,杜德志,罗春燕,马小岗,赵志刚,许唱唱,张红生,吴鹏,晁文俊,2016,春性甘蓝型油菜FCA同源基因新可变剪接体的克隆及表达研究,西北农林科技大学学报,44(4):64-73)
Zhao J.R.,Li N.,Liu H.X.,Bi Y.,and Zhang Z.Y.,2009,Confirmation of cDNA-AFLP differential expressed fragments of related defense genes agaist BYDV in wheat,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),10(1):16-20(赵继荣,李宁,刘红霞,毕阳,张增艳,2009,小麦抗黄矮病相关基因cDNA-AFLP差异表达片段的验证,植物遗传资源学报,10(1):16-20)