棉花GhCAD6基因表达及其功能分析研究
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摘要
为研究GhCAD6基因在转基因棉花纤维中的表达及功能,通过农杆菌介导法获得新陆早36号转GhCAD6基因材料,经过连续多代的选择得到T6代植株材料,对该T6代材料植株叶片基因组DNA进行PCR检测,确定其阳性植株,并对阳性植株叶片基因组DNA进行Southern杂交,结果发现GhCAD6以单拷贝的形式整合到受体棉花基因组中。利用热硼酸-蛋白酶K法提取出棉花纤维RNA,结合实时荧光定量PCR建立了棉花纤维中GhCAD6基因的表达量的检测方法,并测定了GhCAD6基因在不同发育阶段棉纤维中的表达量。转基因植株纤维中GhCAD6的表达量低于相同发育阶段对照样品。GhCAD6的表达量在转基因植株和其对照在15 DPA(开花后天数)时变化趋势不一致,其他时期大致相同。15 DPA中GhCAD6基因的表达量的变化可能是造成转基因棉花纤维品质变化的主要原因。研究结果可为深入分析GhCAD6基因在改良棉花纤维品质的作用机理提供理论依据。
To study the expression and function of GhCAD6 gene in transgenic cotton fibers,the transgenic material was obtained by Agrobacterium-mediated transformation into Xinluzao 36,and the T6 generation plant material was obtained after successive generations of selection. The genomic DNA from cotton leaf of the T6 generation material plants was detected by PCR,and the positive plants were identified. The genomic DNA of the positive plants were hybridized by Southern blotting. The results showed that GhCAD6 was integrated into the cotton genome in different genes loci in the form of single copy. Total RNA from cotton fiber was extracted by hot borate-proteinase K method. The method for detecting the expression of GhCAD6 gene in cotton fiber was established by quantitative real-time PCR. The expression of GhCAD6 gene in cotton fiber at different developmental stages was determined by the method. The expression of GhCAD6 in transgenic plant fiber was lower than that in control samples at the same developmental stage. The expression of GhCAD6 was inconsistent with that in the control samples at 15 DPA( day post-anthesis),and the other periods were approximately the same. The change of GhCAD6 gene expression at 15 DPA may be the main reason for the variation of fiber quality of transgenic cotton. The results of this research could provide theoretical basis for further analysis of the mechanism of GhCAD6 gene in improving cotton fiber quality.
To study the expression and function of GhCAD6 gene in transgenic cotton fibers,the transgenic material was obtained by Agrobacterium-mediated transformation into Xinluzao 36,and the T6 generation plant material was obtained after successive generations of selection. The genomic DNA from cotton leaf of the T6 generation material plants was detected by PCR,and the positive plants were identified. The genomic DNA of the positive plants were hybridized by Southern blotting. The results showed that GhCAD6 was integrated into the cotton genome in different genes loci in the form of single copy. Total RNA from cotton fiber was extracted by hot borate-proteinase K method. The method for detecting the expression of GhCAD6 gene in cotton fiber was established by quantitative real-time PCR. The expression of GhCAD6 gene in cotton fiber at different developmental stages was determined by the method. The expression of GhCAD6 in transgenic plant fiber was lower than that in control samples at the same developmental stage. The expression of GhCAD6 was inconsistent with that in the control samples at 15 DPA( day post-anthesis),and the other periods were approximately the same. The change of GhCAD6 gene expression at 15 DPA may be the main reason for the variation of fiber quality of transgenic cotton. The results of this research could provide theoretical basis for further analysis of the mechanism of GhCAD6 gene in improving cotton fiber quality.
引文
[1]唐淑荣,金石桥,付小琼,等.“六五”至“十二五”期间我国棉花品种纤维品质评价分析[J].中国棉花,2015,42(9):18-24.
[2]许乃银,李健.我国主产棉区棉花纤维品质性状的区域分布特征[J].中国生态农业学报,2016,24(11):1547-1554.
[3]Basra A S,Malik C P.Development of the cotton fiber[J].Int.Rev.Cytol.,1984,89:65-113.
[4]焦改丽,张换祥,王娇娟,等.转伸展蛋白基因改良彩色棉纤维品质研究[A].见:全国作物生物技术与诱变技术学术研讨会论文摘要集[C].福建,2005.
[5]Joohyun L,Teresa H B,Ginger L,et al..Xyloglucan endotransglycosylase/hydrolase genes in cotton and their role in fiber elongation[J].Planta,2010,232:1191-1205.
[6]张震林,刘正銮,周宝良,等.转兔角蛋白基因改良棉纤维品质研究[J].棉花学报,2004,16(2):72-76.
[7]李飞飞,吴慎杰,吕芬妮,等.转蚕丝心蛋白基因改良了棉花纤维品质[J].科学通报,2009,54(4):457-462.
[8]上官小霞,王凌健,李燕娥,等.对转蚕丝芯蛋白轻链基因棉花的分析[J].作物学报,2007,33(5):697-702.
[9]Yang Z R,Zhang C J,Yang X J,et al..PAG1,a cotton brassinosteroid catabolism gene,modulates fiber elongation[J].New Phytol.,2014,203(2):437-448.
[10]Sun Y,Veerabomma S,Fokar M,et al..Brassinosteroid regulates fiber development on cultured cotton ovules[J].Plant Cell Physiol.,2005,46(8):1384-1391.
[11]Luo M,Xiao Y H,Li X B.GhDET2,a steroid 5a-reductase,plays an important role in cotton fiber cell initiation and elongation[J].Plant J.,2007,51:419-430.
[12]Xiao Y H,Li D E,Yin M H,et al..Gibberellin 20-oxidase promotes initiation and elongation of cotton fibers by regulating gibberellin synthesis[J].J.Plant Physiol.,2010,167(10):829-837.
[13]Li Y,Liu D,Xu W L,et al..Suppression of GhAGP4 gene expression repressed the initiation and elongation of cotton fiber[J].Plant Cell Rep.,2002,29:193-202.
[14]Zeng Q W,Qin S,Song S Q,et al..Molecular cloning and characterization of a cytokinin dehydrogenase gene from upland cotton(Gossypium hirsutum L.)[J].Plant Mol.Biol.Rep.,2012,30(1):1-9.
[15]Li X B,Fan X P,Wang X L,et al..The cotton ACTIN1 gene is functionally expressed in fibers and participates in fiber elongation[J].Plant Cell,2005,17:859-875.
[16]范小平,李学宝,徐子勤,等.肌动蛋白基因在棉花纤维中的作用研究[J].华北农学报,2008,23(5):73-75.
[17]罗明.GhDET2和GhKTN1在棉花纤维细胞发育中的功能[D].重庆:西南大学,博士学位论文,2007.
[18]南芝润,李红方,张换祥,等.转反义GhADF基因对陆地棉纤维品质的影响[J].中国农业科学,2009,42(12):4139-4144.
[19]Divya K,Jami S K,Kirti P B.Constitutive expression of mustard annexin,AnnBj1 enhances abiotic stress tolerance and fiber quality in cotton under stress[J].Plant Mol.Biol.,2010,73(3):293-308.
[20]左开井,王劲.提高棉花纤维品质的GbAnn9基因及其应用[P].中国,ZL201110253458.4,2013.
[21]Li X,Wang X D,Dutt Y,et al..Improvement of cotton fiber quality by transforming the acsA and acs B genes in to Gossypium hirsutum L.by means of vacuum in filtration[J].Plant Cell Rep.,2004,22(9):691-697.
[22]张海平,王学德,邵明彦,等.外源纤维素合酶基因对棉纤维品质的改良作用[J].棉花学报,2008(2):110-115.
[23]Haigler C H,Singh B,Zhang D S,et al..Transgenic cotton overproducing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions[J].Plant Mol.Biol.,2007,63(6):815-832.
[24]Ruan Y L,Llewellyn D J,Furbank R T.Suppression of sucrose synthase gene expression represses cotton fiber cell initiation,elongation,and seed development[J].Plant Cell,2003,15:952-964.
[25]Jiang Y J,Guo W Z,Zhu H Y,et al..Overexpression of GhSusA1 increases plant biomass and improves cotton fiber yield and quality[J].Plant Biotechnol.J.,2011,10(3):301-312.
[26]李晓荣,秦咏梅,李晓东,等.GhUGP1基因在改良棉花纤维品质中的应用方法[P].中国,201310613879.2,2016.
[27]夏桂先,韩立波,焦改丽,等.GhLIM2蛋白及其基因在其在改良棉花纤维品质中的应用[P].中国,ZL201210505575.X,2014.
[28]Fan L,Shi W J,Hu W R,et al..Molecular and biochemical evidence for phenylpropanoid synthesis and presence of walllinked phenolics in cotton fibers[J].J.Integr.Plant Biol.,2009,5:626-637.
[29]Gou J Y,Wang L J,Chen S P,et al..Gene expression and metabolite profiles of cotton fiber during cell elongation and secondary cell wall synthesis[J].Cell Res.,2007,17:422-434.
[30]Han L B,Li Y B,Wang H Y,et al..The dual functions of WLIM1a in cell elongation and secondary wall formation in developing cotton fibers[J].Plant Cell,2013,25:4421-4438.
[31]Shi Y H,Zhu S W,Mao X Z,et al..Transcriptome profiling,molecular biological,and physiological studies reveal a major role for ethylene in cotton fiber cell elongation[J].Plant Cell,2006,18:651-664.
[32]倪志勇,王娟,李波,等.转GhCAD基因棉花的获得及其后代的初步鉴定[A].见:植物免疫机制研究及其调控研讨会论文集[C].广州:2010,120-123.
[33]萨姆布鲁克J,弗里奇E F,曼尼阿蒂斯T,金冬雁等译.分子克隆实验指南(第2版)[M].北京:科学出版社,1992,475-782.
[34]武耀廷,刘进元.一种高效提取棉花不同组织总RNA的热硼酸改良法[J].棉花学报,2004,16(2):67-71.
[35]敖振超,王丕武,宋阳.大豆促苗期根系发育相关基因Gmr937在烟草中的功能分析[J].吉林农业大学学报,2018,doi:1013327/j.jjlau.2018.3782.
[36]王新桐.转基因棉花中新霉素磷酸转移酶(NPTⅡ)双抗体夹心ELISA定量检测方法的建立[D].山东泰安:山东农业大学,硕士学位论文,2014.
[37]Santiago-FelipeL S,Tortajada-Genaro L A,Puchades R,et al..Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis[J].Anal.Chim.Acta,2014,811(6):81-87.
[38]Pardridge W M,Boado R J,Farrell C R.Brain-type glucose transporter(GLUT-1)is selectively localized to the blood-brain barrier.Studies with quantitative western blotting and in situ hybridization[J].J.Biol.Chem.,1990,265(29):18035-18040.
[39]Wang Y,Xiao M,Chen X,et al..WT1 recruits TET2 to regulate its target gene expression and suppress leukemia cell proliferation[J].Mol.Cell,2015,57(4):662-673.
[40]Hayward-Lester A,Oefner P S,Sabatini S,et al..Accurate and absolute quantitative measurement of gene expression by single-tube RT-PCR and HPLC[J].Genome Res.,1995,5:494-499.
[41]Sabari B R,Zhang D,Allis C D,et al..Metabolic regulation of gene expression through histone acylations[J].Nat.Rev.Mol.Cell Biol.,2017,18(2):90-101.
[42]Tanpure R S,Barbole R S,Dawkar V V,et al..Improved tolerance against Helicoverpa armigera in transgenic tomato overexpressing multi-domain proteinase inhibitor gene from Capsicum annuum[J].Physiol.Mol.Biol.Plants,2017,23(3):597-604.
[43]He J,Xu M L,Willmann M R,et al..Threshold-dependent repression of SPL gene expression by miR156/miR157 controls vegetative phase change in Arabidopsis thaliana[J].PLoSGenet.,2018,14(4):e1007337.
[44]Pardatscher G,Schwarz-Schilling M,Daube Dr S S,et al..Gene expression on DNA biochips patterned with strand-displacement lithography[J].Angew.Chem.Int.Edit,2018,57(17):4783-4786.
[45]Mchugh K,Tremaine R,Sapone A,et al..Effects of salt concentration on Tetrahymena thermophila growth and CRP1 gene expression[J/OL].Life Environ.Sci.Course-based Res.Projects,2018,https://scholars.carroll.edu/lifesci_undergrad/13.
[46]Zhang Y X,Han X J,Chen S S,et al..Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stressesb[J].Sci.Rep.,2017,doi:10.1038/srep4029.
[47]Whetten R,Sederoff R.Lignin biosynthesis[J].Plant Cell,1995,7(7):1001-1013.
[48]Li X M,Yuan D J,Zhang J F,et al..Genetic mapping and characteristics of genes specifically or preferentially expressed during fiber development in cotton[J].PLoS ONE,2013,8,e54444.
[49]袁辉.棉花纤维发育相关功能基因相对表达量和品质关系的研究[D].乌鲁木齐:新疆农业大学,硕士学位论文,2009.
[2]许乃银,李健.我国主产棉区棉花纤维品质性状的区域分布特征[J].中国生态农业学报,2016,24(11):1547-1554.
[3]Basra A S,Malik C P.Development of the cotton fiber[J].Int.Rev.Cytol.,1984,89:65-113.
[4]焦改丽,张换祥,王娇娟,等.转伸展蛋白基因改良彩色棉纤维品质研究[A].见:全国作物生物技术与诱变技术学术研讨会论文摘要集[C].福建,2005.
[5]Joohyun L,Teresa H B,Ginger L,et al..Xyloglucan endotransglycosylase/hydrolase genes in cotton and their role in fiber elongation[J].Planta,2010,232:1191-1205.
[6]张震林,刘正銮,周宝良,等.转兔角蛋白基因改良棉纤维品质研究[J].棉花学报,2004,16(2):72-76.
[7]李飞飞,吴慎杰,吕芬妮,等.转蚕丝心蛋白基因改良了棉花纤维品质[J].科学通报,2009,54(4):457-462.
[8]上官小霞,王凌健,李燕娥,等.对转蚕丝芯蛋白轻链基因棉花的分析[J].作物学报,2007,33(5):697-702.
[9]Yang Z R,Zhang C J,Yang X J,et al..PAG1,a cotton brassinosteroid catabolism gene,modulates fiber elongation[J].New Phytol.,2014,203(2):437-448.
[10]Sun Y,Veerabomma S,Fokar M,et al..Brassinosteroid regulates fiber development on cultured cotton ovules[J].Plant Cell Physiol.,2005,46(8):1384-1391.
[11]Luo M,Xiao Y H,Li X B.GhDET2,a steroid 5a-reductase,plays an important role in cotton fiber cell initiation and elongation[J].Plant J.,2007,51:419-430.
[12]Xiao Y H,Li D E,Yin M H,et al..Gibberellin 20-oxidase promotes initiation and elongation of cotton fibers by regulating gibberellin synthesis[J].J.Plant Physiol.,2010,167(10):829-837.
[13]Li Y,Liu D,Xu W L,et al..Suppression of GhAGP4 gene expression repressed the initiation and elongation of cotton fiber[J].Plant Cell Rep.,2002,29:193-202.
[14]Zeng Q W,Qin S,Song S Q,et al..Molecular cloning and characterization of a cytokinin dehydrogenase gene from upland cotton(Gossypium hirsutum L.)[J].Plant Mol.Biol.Rep.,2012,30(1):1-9.
[15]Li X B,Fan X P,Wang X L,et al..The cotton ACTIN1 gene is functionally expressed in fibers and participates in fiber elongation[J].Plant Cell,2005,17:859-875.
[16]范小平,李学宝,徐子勤,等.肌动蛋白基因在棉花纤维中的作用研究[J].华北农学报,2008,23(5):73-75.
[17]罗明.GhDET2和GhKTN1在棉花纤维细胞发育中的功能[D].重庆:西南大学,博士学位论文,2007.
[18]南芝润,李红方,张换祥,等.转反义GhADF基因对陆地棉纤维品质的影响[J].中国农业科学,2009,42(12):4139-4144.
[19]Divya K,Jami S K,Kirti P B.Constitutive expression of mustard annexin,AnnBj1 enhances abiotic stress tolerance and fiber quality in cotton under stress[J].Plant Mol.Biol.,2010,73(3):293-308.
[20]左开井,王劲.提高棉花纤维品质的GbAnn9基因及其应用[P].中国,ZL201110253458.4,2013.
[21]Li X,Wang X D,Dutt Y,et al..Improvement of cotton fiber quality by transforming the acsA and acs B genes in to Gossypium hirsutum L.by means of vacuum in filtration[J].Plant Cell Rep.,2004,22(9):691-697.
[22]张海平,王学德,邵明彦,等.外源纤维素合酶基因对棉纤维品质的改良作用[J].棉花学报,2008(2):110-115.
[23]Haigler C H,Singh B,Zhang D S,et al..Transgenic cotton overproducing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions[J].Plant Mol.Biol.,2007,63(6):815-832.
[24]Ruan Y L,Llewellyn D J,Furbank R T.Suppression of sucrose synthase gene expression represses cotton fiber cell initiation,elongation,and seed development[J].Plant Cell,2003,15:952-964.
[25]Jiang Y J,Guo W Z,Zhu H Y,et al..Overexpression of GhSusA1 increases plant biomass and improves cotton fiber yield and quality[J].Plant Biotechnol.J.,2011,10(3):301-312.
[26]李晓荣,秦咏梅,李晓东,等.GhUGP1基因在改良棉花纤维品质中的应用方法[P].中国,201310613879.2,2016.
[27]夏桂先,韩立波,焦改丽,等.GhLIM2蛋白及其基因在其在改良棉花纤维品质中的应用[P].中国,ZL201210505575.X,2014.
[28]Fan L,Shi W J,Hu W R,et al..Molecular and biochemical evidence for phenylpropanoid synthesis and presence of walllinked phenolics in cotton fibers[J].J.Integr.Plant Biol.,2009,5:626-637.
[29]Gou J Y,Wang L J,Chen S P,et al..Gene expression and metabolite profiles of cotton fiber during cell elongation and secondary cell wall synthesis[J].Cell Res.,2007,17:422-434.
[30]Han L B,Li Y B,Wang H Y,et al..The dual functions of WLIM1a in cell elongation and secondary wall formation in developing cotton fibers[J].Plant Cell,2013,25:4421-4438.
[31]Shi Y H,Zhu S W,Mao X Z,et al..Transcriptome profiling,molecular biological,and physiological studies reveal a major role for ethylene in cotton fiber cell elongation[J].Plant Cell,2006,18:651-664.
[32]倪志勇,王娟,李波,等.转GhCAD基因棉花的获得及其后代的初步鉴定[A].见:植物免疫机制研究及其调控研讨会论文集[C].广州:2010,120-123.
[33]萨姆布鲁克J,弗里奇E F,曼尼阿蒂斯T,金冬雁等译.分子克隆实验指南(第2版)[M].北京:科学出版社,1992,475-782.
[34]武耀廷,刘进元.一种高效提取棉花不同组织总RNA的热硼酸改良法[J].棉花学报,2004,16(2):67-71.
[35]敖振超,王丕武,宋阳.大豆促苗期根系发育相关基因Gmr937在烟草中的功能分析[J].吉林农业大学学报,2018,doi:1013327/j.jjlau.2018.3782.
[36]王新桐.转基因棉花中新霉素磷酸转移酶(NPTⅡ)双抗体夹心ELISA定量检测方法的建立[D].山东泰安:山东农业大学,硕士学位论文,2014.
[37]Santiago-FelipeL S,Tortajada-Genaro L A,Puchades R,et al..Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis[J].Anal.Chim.Acta,2014,811(6):81-87.
[38]Pardridge W M,Boado R J,Farrell C R.Brain-type glucose transporter(GLUT-1)is selectively localized to the blood-brain barrier.Studies with quantitative western blotting and in situ hybridization[J].J.Biol.Chem.,1990,265(29):18035-18040.
[39]Wang Y,Xiao M,Chen X,et al..WT1 recruits TET2 to regulate its target gene expression and suppress leukemia cell proliferation[J].Mol.Cell,2015,57(4):662-673.
[40]Hayward-Lester A,Oefner P S,Sabatini S,et al..Accurate and absolute quantitative measurement of gene expression by single-tube RT-PCR and HPLC[J].Genome Res.,1995,5:494-499.
[41]Sabari B R,Zhang D,Allis C D,et al..Metabolic regulation of gene expression through histone acylations[J].Nat.Rev.Mol.Cell Biol.,2017,18(2):90-101.
[42]Tanpure R S,Barbole R S,Dawkar V V,et al..Improved tolerance against Helicoverpa armigera in transgenic tomato overexpressing multi-domain proteinase inhibitor gene from Capsicum annuum[J].Physiol.Mol.Biol.Plants,2017,23(3):597-604.
[43]He J,Xu M L,Willmann M R,et al..Threshold-dependent repression of SPL gene expression by miR156/miR157 controls vegetative phase change in Arabidopsis thaliana[J].PLoSGenet.,2018,14(4):e1007337.
[44]Pardatscher G,Schwarz-Schilling M,Daube Dr S S,et al..Gene expression on DNA biochips patterned with strand-displacement lithography[J].Angew.Chem.Int.Edit,2018,57(17):4783-4786.
[45]Mchugh K,Tremaine R,Sapone A,et al..Effects of salt concentration on Tetrahymena thermophila growth and CRP1 gene expression[J/OL].Life Environ.Sci.Course-based Res.Projects,2018,https://scholars.carroll.edu/lifesci_undergrad/13.
[46]Zhang Y X,Han X J,Chen S S,et al..Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stressesb[J].Sci.Rep.,2017,doi:10.1038/srep4029.
[47]Whetten R,Sederoff R.Lignin biosynthesis[J].Plant Cell,1995,7(7):1001-1013.
[48]Li X M,Yuan D J,Zhang J F,et al..Genetic mapping and characteristics of genes specifically or preferentially expressed during fiber development in cotton[J].PLoS ONE,2013,8,e54444.
[49]袁辉.棉花纤维发育相关功能基因相对表达量和品质关系的研究[D].乌鲁木齐:新疆农业大学,硕士学位论文,2009.