不结球白菜开花相关基因BcGI的克隆、亚细胞定位及基因沉默功能验证
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摘要
[目的]本文旨在揭示不结球白菜GIGANTEA(GI)基因的功能,验证其与CONSTANS(CO)、FLOWERING LOCUS T(FT)基因之间的调控关系以及其在植物抽薹开花中的作用。[方法]以不结球白菜品种‘苏州青’为材料,提取RNA并反转录成cDNA,同源克隆BcGI基因,Gateway构建表达载体pEarleyGate101-BcGI-YFP,利用农杆菌介导法将表达载体注射入本氏烟草中,激光共聚焦显微镜观察其亚细胞定位。Gateway构建沉默载体BcGI-RNAi,利用农杆菌介导法将沉默载体导入拟南芥中,观察野生型和转基因植株抽薹期的表型变化。采用RT-qPCR技术,检测GI、CO、FT基因在转基因和野生型拟南芥植株抽薹期的基因表达量。[结果]通过同源克隆,得到BcGI基因,其含有1个3 516 bp的开放阅读框(ORF),编码1 171个氨基酸。亚细胞定位结果显示,BcGI定位于细胞核中。RT-qPCR结果表明,BcGI基因在沉默转基因植株中的相对表达量比野生型植株低;同时当GI基因表达受到抑制时,CO、FT基因在沉默转基因植株中的相对表达量也比野生型明显降低,沉默转基因植株与野生型相比表现为开花延迟且抽薹期莲座叶数明显增多。[结论]BcGI定位于细胞核,参与正向调控下游CO、FT基因的表达,进而影响不结球白菜光周期开花途径。
[Objectives]The paper aimed to explore the function of GI gene in Brassica campestris ssp. chinensis,and verify its regulatory relationship with CO and FT genes,and its role in plant flowering. [Methods]The RNA was extracted from the non-heading Chinese cabbage'Suzhouqing'and transcribed reversely into cDNA. The gene BcGI was obtained by homologous cloning method. Using Gateway method,we constructed the expression vector pEarleyGate101-BcGI-YFP. The expression vector was injected into Nicotiana benthamiana by Agrobacterium-mediated method,and its subcellular localization was observed by laser confocal microscopy. Also,we constructed the silencing vector BcGI-RNAi to explore the phenotypic changes of wild-type and silenced plants during the flowering period. RT-qPCR was used to detect the gene expression of GI,CO and FT in the flowering stage of transgenic and wild-type plants. [Results]By homologous cloning,we obtained BcGI including a 3 516 bp open reading frame(ORF)and encoding 1 171 amino acids. Subcellular localization analysis showed that BcGI was localized in the nucleus. The RT-qPCR results of wild-type and BcGI-RNAi silencing transgenic plants showed that the relative expression of BcGI gene in transgenic plants was lower than that in wild-type plants. When the expression of GI gene was inhibited,the relative expressions of CO and FT genes in transgenic plants were significantly lower than wild-type plants. The silenced plants showed late flowering compared with wild-type plants,and the number of rosette leaves significantly increased at blotting stage. [Conclusions]The BcGI was located in the nucleus and participated in the positive regulation of downstream CO and FT gene expression,which in turn affected the photoperiod flowering pathway of non-heading Chinese cabbage.
[Objectives]The paper aimed to explore the function of GI gene in Brassica campestris ssp. chinensis,and verify its regulatory relationship with CO and FT genes,and its role in plant flowering. [Methods]The RNA was extracted from the non-heading Chinese cabbage'Suzhouqing'and transcribed reversely into cDNA. The gene BcGI was obtained by homologous cloning method. Using Gateway method,we constructed the expression vector pEarleyGate101-BcGI-YFP. The expression vector was injected into Nicotiana benthamiana by Agrobacterium-mediated method,and its subcellular localization was observed by laser confocal microscopy. Also,we constructed the silencing vector BcGI-RNAi to explore the phenotypic changes of wild-type and silenced plants during the flowering period. RT-qPCR was used to detect the gene expression of GI,CO and FT in the flowering stage of transgenic and wild-type plants. [Results]By homologous cloning,we obtained BcGI including a 3 516 bp open reading frame(ORF)and encoding 1 171 amino acids. Subcellular localization analysis showed that BcGI was localized in the nucleus. The RT-qPCR results of wild-type and BcGI-RNAi silencing transgenic plants showed that the relative expression of BcGI gene in transgenic plants was lower than that in wild-type plants. When the expression of GI gene was inhibited,the relative expressions of CO and FT genes in transgenic plants were significantly lower than wild-type plants. The silenced plants showed late flowering compared with wild-type plants,and the number of rosette leaves significantly increased at blotting stage. [Conclusions]The BcGI was located in the nucleus and participated in the positive regulation of downstream CO and FT gene expression,which in turn affected the photoperiod flowering pathway of non-heading Chinese cabbage.
引文
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[16] Wu P,Wang W,Li Y,et al.Divergent evolutionary patterns of the MAPK cascade genes in Brassica rapa and plant phylogenetics[J].Horticulture Research,2017,4:17079.
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[18] Schwartz C J,Lee J,Amasino R.Variation in shade-induced flowering in Arabidopsis thaliana results from FLOWERING LOCUS T allelic variation[J].PLoS One,2017,12(11):e0187768.
[19] 陈福禄,傅永福,林辰涛.CO/FT调节元件与植物开花时间调节研究进展[J].中国农业科技导报,2009,11(2):17-22.Chen F L,Fu Y F,Lin C T.Research progress on CO/FT regulon and its role in adjusting plant flowering[J].China Agricultural Science and Technology Bulletin,2009,11(2):17-22(in Chinese with English abstract).
[20] Cha J Y,Kim J,Kim T S,et al.GIGANTEA is a co-chaperone which facilitates maturation of ZEITLUPE in the Arabidopsis circadian clock[J].Nature Communications,2017,8:3.
[21] Dalchau N,Baek S J,Briggs H M,et al.The circadian oscillator gene GIGANTEA mediates a long-term response of the Arabidopsis thaliana circadian clock to sucrose[J].Proc Natl Acad Sci USA,2011,108(12):5104-5109.
[22] Kim W Y,Ali Z,Park H J,et al.Release of SOS2 kinase from sequestration with GIGANTEA determines salt tolerance in Arabidopsis[J].Nature Communications,2013,4:1352.
[23] Ding J,B?hlenius H,Rühl M G,et al.GIGANTEA-like genes control seasonal growth cessation in Populus[J].New Phytologist,2018,218(4):1491-1503.
[24] Kardailsky I,Shukla V K,Ahn J H,et al.Activation tagging of the floral inducer FT[J].Science,1999,286(5446):1962-1965.
[25] Putterill J,Robson F,Lee K,et al.The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors[J].Cell,1995,80(6):847-857.
[26] Song X,Duan W,Huang Z,et al.Comprehensive analysis of the flowering genes in Chinese cabbage and examination of evolutionary pattern of CO-like genes in plant kingdom[J].Scientific Reports,2015,5:14631.
[27] Cao S,Ye M,Jiang S.Involvement of GIGANTEA gene in the regulation of the cold stress response in Arabidopsis[J].Plant Cell Reports,2005,24(11):683-690.
[28] Penfield S,Hall A.A role for multiple circadian clock genes in the response to signals that break seed dormancy in Arabidopsis[J].Plant Cell,2009,21(6):1722-1732.
[29] An H,Roussot C,Suárez-López P,et al.CONSTANS acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of Arabidopsis[J].Development,2004,131(15):3615-3626.
[2] Hayama R,Coupland G.Shedding light on the circadian clock and the photoperiodic control of flowering[J].Current Opinion in Plant Biology,2003,6(1):13-19.
[3] Rédei G P.Supervital mutants of Arabidopsis[J].Genetics,1962,47(4):443-460.
[4] Koornneef M,Hanhart C J,van der Veen J H.A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana[J].Molecular General Genetics,1991,229(1):57-66.
[5] Hecht V,Knowles C L,Schoor J K V,et al.Pea LATE BLOOMER1 is a GIGANTEA ortholog with roles in photoperiodic flowering,deetiolation,and transcriptional regulation of circadian clock gene homologs[J].Plant Physiology,2007,144(2):648-661.
[6] Yang S S,Weers B D,Morishige D T,et al.CONSTANS is a photoperiod regulated activator of flowering in sorghum[J].BMC Plant Biology,2014,14(1):148.
[7] Mizoguchi T,Wright L,Fujiwara S,et al.Distinct roles of GIGANTEA in promoting flowering and regulating circadian rhythms in Arabidopsis[J].Plant Cell,2005,17(8):2255-2270.
[8] Fire A,Xu S Q,Montgomery M K,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(6669):806-811.
[9] Kusaba M.RNA interference in crop plants[J].Current Opinion in Biotechnology,2004,15(2):139-143.
[10] Padmanaban S,Lin X,Perera I,et al.Differential expression of vacuolar H+-ATPase subunit c genes in tissues active in membrane trafficking and their roles in plant growth as revealed by RNAi[J].Plant Physiology,2004,134(4):1514-1526.
[11] Hartley J L,Temple G F,Brasch M A.DNA cloning using in vitro site-specific recombination[J].Genome Research,2000,10(11):1788-1795.
[12] Lai Z,Lin Y.Analysis of the global transcriptome of longan(Dimocarpus longan Lour.)embryogenic callus using Illumina paired-end sequencing[J].BMC Genomics,2013,14(1):561.
[13] Kumar S,Stecher G,Li M,et al.MEGA X:Molecular Evolutionary Genetics Analysis across computing platforms[J].Molecular Biology and Evolution,2018,35(6):1547-1549.
[14] 王聪颖,陈书霞,陈巧,等.利用Gateway技术构建黄瓜HPL基因的RNA干扰载体[J].西北农业学报,2013,22(2):152-158.Wang C Y,Chen S X,Chen Q,et al.Construction of RNAi vectors for HPL gene of cucumber using gateway technology[J].Acta Agriculturae Boreali-Occidentalis Sinica,2013,22(2):152-158(in Chinese with English abstract).
[15] 申希平,丁建生,李娟生,等.在SPSS中利用均数和标准差做两独立样本t检验[J].现代预防医学,2007,34(21):4066-4067,4069.Shen X P,Ding J S,Li J S,et al.2-independent-samples t test undertaking by the means of mean and standard deviation in SPSS[J].Modern Preventive Medicine,2007,34(21):4066-4067,4069(in Chinese with English abstract).
[16] Wu P,Wang W,Li Y,et al.Divergent evolutionary patterns of the MAPK cascade genes in Brassica rapa and plant phylogenetics[J].Horticulture Research,2017,4:17079.
[17] 帅敏敏,黄有军.光周期途径成花关键基因GIGANTEA和CONSTANS的研究进展[J].分子植物育种,2018,16(17):5601-5607.Shuai M M,Huang Y J.The evolution mechanism of the key genes GIGANTEA and CONSTANS in photoperidic pathway[J].Molecular Plant Breeding,2018,16(17):5601-5607(in Chinese with English abstract).
[18] Schwartz C J,Lee J,Amasino R.Variation in shade-induced flowering in Arabidopsis thaliana results from FLOWERING LOCUS T allelic variation[J].PLoS One,2017,12(11):e0187768.
[19] 陈福禄,傅永福,林辰涛.CO/FT调节元件与植物开花时间调节研究进展[J].中国农业科技导报,2009,11(2):17-22.Chen F L,Fu Y F,Lin C T.Research progress on CO/FT regulon and its role in adjusting plant flowering[J].China Agricultural Science and Technology Bulletin,2009,11(2):17-22(in Chinese with English abstract).
[20] Cha J Y,Kim J,Kim T S,et al.GIGANTEA is a co-chaperone which facilitates maturation of ZEITLUPE in the Arabidopsis circadian clock[J].Nature Communications,2017,8:3.
[21] Dalchau N,Baek S J,Briggs H M,et al.The circadian oscillator gene GIGANTEA mediates a long-term response of the Arabidopsis thaliana circadian clock to sucrose[J].Proc Natl Acad Sci USA,2011,108(12):5104-5109.
[22] Kim W Y,Ali Z,Park H J,et al.Release of SOS2 kinase from sequestration with GIGANTEA determines salt tolerance in Arabidopsis[J].Nature Communications,2013,4:1352.
[23] Ding J,B?hlenius H,Rühl M G,et al.GIGANTEA-like genes control seasonal growth cessation in Populus[J].New Phytologist,2018,218(4):1491-1503.
[24] Kardailsky I,Shukla V K,Ahn J H,et al.Activation tagging of the floral inducer FT[J].Science,1999,286(5446):1962-1965.
[25] Putterill J,Robson F,Lee K,et al.The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors[J].Cell,1995,80(6):847-857.
[26] Song X,Duan W,Huang Z,et al.Comprehensive analysis of the flowering genes in Chinese cabbage and examination of evolutionary pattern of CO-like genes in plant kingdom[J].Scientific Reports,2015,5:14631.
[27] Cao S,Ye M,Jiang S.Involvement of GIGANTEA gene in the regulation of the cold stress response in Arabidopsis[J].Plant Cell Reports,2005,24(11):683-690.
[28] Penfield S,Hall A.A role for multiple circadian clock genes in the response to signals that break seed dormancy in Arabidopsis[J].Plant Cell,2009,21(6):1722-1732.
[29] An H,Roussot C,Suárez-López P,et al.CONSTANS acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of Arabidopsis[J].Development,2004,131(15):3615-3626.