过表达或沉默棉花GhPCBER基因株系的获得及其茎叶木质素和木脂素含量研究
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摘要
编码苯基香豆满苄基醚还原酶(phenylcoumaran benzylic ether reductase,PCBER)的基因PCBER属于PIP亚家族,是苯丙烷代谢途径中参与木脂素合成的关键基因。该研究构建了棉花GhPCBER基因的植物过表达载体并转化拟南芥,同时构建了VIGS(virus induced gene silencing,病毒诱导的基因沉默)载体转化棉花,采用实时荧光定量PCR技术对GhPCBER基因在不同组织中的表达进行分析;对野生型和转基因植株茎叶组织中的木质素和木脂素含量进行测定分析。结果表明:(1)成功构建了GhPCBER植物过表达载体pGWB17-GhPCBRE以及基因沉默重组载体pTRV2-GhPCBER;经遗传转化获得6株转棉花GhPCBER基因抗性拟南芥植株,同时获得15株GhPCBER基因沉默棉花植株(5株为一组)。(2)PCR检测表明,6株转基因拟南芥均为过表达株系,其中株系1、2、3相对表达量更高,且在茎、叶组织中的表达量分别较野生型提高了7～14倍和6～16倍,表明GhPCBER基因成功在拟南芥中过表达;GhPCBER基因沉默棉花植株的茎、叶组织中的表达量分别比野生型棉株约下降12%和26%,表明烟草脆裂病毒(TRV)体系(pTRV2-GhPCBER)成功抑制了GhPCBER基因的表达。(3)转GhPCBER基因拟南芥茎、叶中木质素和木脂素含量较野生型均显著降低;GhPCBER基因沉默棉花植株茎、叶中木质素和木脂素含量较野生型均极显著降低;组织化学染色观察发现GhPCBER基因沉默棉花植株茎秆颜色明显比野生型染色浅,也证明沉默基因棉花植株茎秆中的木质素含量减少。(4)苯丙烷代谢通路中8个相关基因的实时荧光定量PCR分析发现,过表达或抑制GhPCBRE基因均会导致苯丙烷代谢途径发生重新定向。
The PCBER gene belongs to the PIP subfamily and is an important gene involved in the synthesis of lignans in the phenylpropane metabolic pathway.In this study,The GhPCBERplant over-expression vector pGWB17-GhPCBRE was constructed and transformed into Arabidopsis thaliana,and gene silencing recombinant vector pTRV2-GhPCBER was also constructed and transformed into cotton.Real time quantitative PCR was used to analyze the expression of GhPCBERgene under different tissues.The lignin and lignan contents in stem and leaf tissues from transgenic A.thalianaand cotton plants were extracted and determined.The results showed that:(1)GhPCBERplant overexpression vector pGWB17-GhPCBREand gene silencing recombinant vectors pTRV2-GhPCBER were successfully constructed.Six transgenic A.thalianalines with overexpression GhPCBER were obtained by genetic transformation,and GhPCBERgene silenced cotton plants were obtained(5 strains as a group).(2)PCR analysis showed that six transgenic Arabidopsis thalianalines were overexpressing lines,among which the relative expression of lines 1,2 and 3 was higher,and the expression in stem and leaf tissues was 7-14 and 6-16 times higher than that of wild type,indicating that the GhPCBERgene was successfully overexpressed in A.thaliana;The expression levels of GhPCBERgene silencing cotton plants in stems and leaves were 12% and26%lower than those in wild-type cotton plants,respectively,indicating that the tobacco fragile virus(TRV)system(pTRV2-GhPCBER)successfully inhibited the expression of GhPCBER gene.(3)The contents of lignin and lignan in stems and leaves of GhPCBERtransgenic A.thaliana were significantly lower than that of wild type;the contents of lignin and lignan in stems and leaves of GhPCBERgene silencing cotton plants were significantly lower than that of wild type;Histochemical staining observation showed that the color of the stem of GhPCBERgene silencing cotton plants was significantly lighter than that of wild type,which also proved that the lignin content in the stems of silenced cotton plants decreased;(4)Real-time quantitative PCR analysis of eight related genes in the phenylpropanoid metabolic pathway revealed that overexpression or inhibition of the GhPCBREgene resulted the reorientation of the phenylpropanoid metabolic pathway.
The PCBER gene belongs to the PIP subfamily and is an important gene involved in the synthesis of lignans in the phenylpropane metabolic pathway.In this study,The GhPCBERplant over-expression vector pGWB17-GhPCBRE was constructed and transformed into Arabidopsis thaliana,and gene silencing recombinant vector pTRV2-GhPCBER was also constructed and transformed into cotton.Real time quantitative PCR was used to analyze the expression of GhPCBERgene under different tissues.The lignin and lignan contents in stem and leaf tissues from transgenic A.thalianaand cotton plants were extracted and determined.The results showed that:(1)GhPCBERplant overexpression vector pGWB17-GhPCBREand gene silencing recombinant vectors pTRV2-GhPCBER were successfully constructed.Six transgenic A.thalianalines with overexpression GhPCBER were obtained by genetic transformation,and GhPCBERgene silenced cotton plants were obtained(5 strains as a group).(2)PCR analysis showed that six transgenic Arabidopsis thalianalines were overexpressing lines,among which the relative expression of lines 1,2 and 3 was higher,and the expression in stem and leaf tissues was 7-14 and 6-16 times higher than that of wild type,indicating that the GhPCBERgene was successfully overexpressed in A.thaliana;The expression levels of GhPCBERgene silencing cotton plants in stems and leaves were 12% and26%lower than those in wild-type cotton plants,respectively,indicating that the tobacco fragile virus(TRV)system(pTRV2-GhPCBER)successfully inhibited the expression of GhPCBER gene.(3)The contents of lignin and lignan in stems and leaves of GhPCBERtransgenic A.thaliana were significantly lower than that of wild type;the contents of lignin and lignan in stems and leaves of GhPCBERgene silencing cotton plants were significantly lower than that of wild type;Histochemical staining observation showed that the color of the stem of GhPCBERgene silencing cotton plants was significantly lighter than that of wild type,which also proved that the lignin content in the stems of silenced cotton plants decreased;(4)Real-time quantitative PCR analysis of eight related genes in the phenylpropanoid metabolic pathway revealed that overexpression or inhibition of the GhPCBREgene resulted the reorientation of the phenylpropanoid metabolic pathway.
引文
[1]BOERJAN W,RALPH J,BAUCHER M.Lignin biosynthesis[J].Annual Review Plant Biology,2003,54:519-546.
[2]GRAND C,BOUDET A,BOUDET AM.Isoenzymes of hydroxycinnamate:CoA ligase from poplar stems properties and tissue distribution[J].Planta,1983,158(3):225-229.
[3]JOHN H G,JOHN RALPH,CATHERINE L,et al.Genetic and molecular basis of grass cell-wall degradability.I.Lignincell wall matrix interactions[J].Comptes Rendus Biologies,2004,327:455-465.
[4]BHUIYAN NH,SELVARA JG,WEI Y,et al.Gene expression profiling and silencing reveal that monolignol biosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion[J].Journal of Exprimental Botany,2009,60:509-521.
[5]LING F,RAPHAEL L,SHIMON G,et al.Progressive inhibition by water deficit of cell wall extensibility and growth along the elongation zone of maize roots is related to increased lignin metabolism and progressive stelar accumulation of wall phenolics[J].Plant Physiology,2006,140:603-612.
[6]TSUBASA S,ROBERT W,TADAYUKI I,et al.Expression patterns of two tobacco isoflavone reductase-like genes and their possible roles in secondary metabolism in tobacco[J].Plant Molecular Biology,2002,50:427-440.
[7]TANNER GJ,FRANCKI KT,ABRAHAMS S,et al.Proanthocyanidin biosynthesis in plants.purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA[J].The J Biol Chem,2003,278(3):31 647-31 656.
[8]DAVIN L B,LEWIS N G.Lignin and lignan biochemical pathways in plants:an unprecedented discovery in phenolic coupling[J].Anais Da Academia Brasileira De Ciencias,1995,67:363-378.
[9]PRASSINOS C,KO J H,YANG J,et al.Transcriptome profiling of vertical stem segments provides insights into the genetic regulation of secondary growth in hybrid aspen trees[J].Plant Cell Physiol,2005,46(8):1 213-1 225.
[10]涂礼莉,谭家福,郭凯,等.类黄酮代谢途径与棉花纤维发育.[J].中国科学,2014,44(8):758-765.TU L L,TAN J F,GUO K,et al.Flavonoid metabolic pathway and cotton fiber development.[J].Scientia Sinica Vitae,2014,44(8):758-765.
[11]GRABBER JH,RALPH J,LAPIERRE C,et al.Genetic and molecular basis of grass cell-wall degradability.I.Lignin-cell wall matrix interactions[J].Comptes Rendus Biologies,2004,327(5):455-465.
[12]BOERJAN W,POLLE A,VANDER MIJNSBRUGGE K.Role in lignification and growth for plant phenylcoumaran benzylic ether reductase:US,US20060015967[P].2006.
[13]喻树迅,范术丽,王寒涛,等.中国棉花高产育种研究进展[J].中国农业科学,2016,49(18):3 465-3 476.YU S X,FAN S L,WANG H T,WEI H L,et al.Progresses in research on cotton high yield breeding in China[J].Scientia Agricultura Sinica,2016,49(18):3 465-3 476.
[14]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 Research,2007,17(5):422-434.
[15]FAN L,SHI W J,HU W R,et al.Molecular and biochemical evidence for phenylpropanoid synthesis and presence of wall-linked phenolics in cotton fibers[J].Journal of Integrative Plant Biology,2009,51(7):626-637.
[16]HAN L B,LI Y B,WANG H Y,et al.The dual functions of WLIM1ain cell elongation and secondary wall formation in developing cotton fibers[J].The Plant Cell,2013,25(11):4 421-4 438.
[17]DOWD C,WILSON IW,MCFADDEN H.Gene expression profile changes in cotton root and hypocotyl tissues in response to infection with Fusarium oxysporum f.sp.vasinfectum[J].Molecular Plant-Microbe Interactions,2004,17(6):654-667.
[18]SUN J,AN H,SHI W,et al.Molecular cloning and characterization of GhWRKY11,agene implicated in pathogen responses from cotton[J].South African Journal of Botany,2012,81(4):113-123.
[19]DAVID RG,ALBENA T DK,LAURENCE B,et al.Phylogenetic links in plant defense systems:lignans,isoflavonoids,and their reductases[J].ACS Symposium Series,1997,658:58-89.
[20]STERKY F,REGAN S,KARLSSON J,et al.Gene discovery in the wood-formig tissues of poplar:analysis of 5,692expressed sequence tags[J].Proceedings of the National A-cademy of Science of the United States of America,1998,95(22):13 330-13 335.
[21]VANDER MIJNSBRUGGE K,MEYERMANS H,VANMONTAGU M,et al.Wood formation in poplar:identification,characterization,and seasonal variation of xylem proteins[J].Planta,2000,210(4):589-598.
[22]KWON M,DAVIN LB,LEWIS NG.In situ hybridization and immunolocalization of lignin reductases in woody tissues:implications heartwood formation and other forms of vascular tissue preservation[J].Phytochemistry,2001,57:899-914.
[23]NUOENDAGULA,KAMIMURA N,MORI T,et al.Expression and functional analyses of a putative phenylcoumaran benzylic ether reductase in Arabidopsis thaliana[J].Plant Cell Rep,2016,35(3):513-526.
[24]ZHAO Q,DIXON RA.Altering the cell wall and its impact on plant disease:from forage to bioenergy[J].Annu Rev Phytopathol,2014,52:69-91.
[25]SHOJI T,WINZ R,NAKAJIMA K,et al.Expression patterns of two tobacco isoflavone reductase-like genes and their possible roles in secondary metabolism in tobacco[J].Plant Molecular Biology,2002,50(3):427-440.
[26]R.B TURLEY.Expression of a phenylcoumaran benzylic ether reductase-like protein in the ovules of Gossypium hirsutum[J].Biologia Plantarum,2008,52(4):759-762.
[27]TALIERCIO E,ALLEN RD,ESSENBERG M,et al.Analysis of ESTs from multiple Gossypium hirsutumtissues and identification of SSRs[J].Genome,2006,49(4):306-319.
[28]UDALL JA,SWANSON JM,HALLER K,et al.A global assembly of cotton ESTs[J].Genome Research,2006,16(3):441-450.
[29]齐雯雯,宫晓琳,王洋,等.蘸花法在植物遗传转化上的应用研究进展[J].现代农业科技,2014,(24):9-10,12.QI W W,GONG X L,WANG Y,et al.Application and research progress of floral dip in plant genetic transformation[J].Modern Agricultural Sciences and Technology,2014,(24):9-10,12.
[30]王建庆,曹佃元,张玉.乙酰溴法测定棉籽壳中木质素的含量[J].纺织学报,2013,34(9):12-16.WANG J Q,CAO D Y,ZHANG Y.cetyl bromide test method for determining amount of lignin in cottonseed coat[J].Journal of Textile Research,2013,34(9):12-16.
[31]闫洪颖.棉花纤维发育过程超微结构观察和次生壁木质素含量的分析[D].乌鲁木齐:新疆农业大学,2009.
[32]陈韵,石展望,黄晓敏.超声波辅助提取大豆总木脂素及其含量分析[J].大豆科学,2010,29(1):168-170,173.CHEN Y,SHI Z W,HUANG X M.Ultrasonic-assisted extraction of total lignans and its content analysis in soybeans[J].Soybean Science,2010,29(1):168-170,173.
[33]XIE M,WELLINGTON M,ANTHONY C.A 5-enolpyruvylshikimate 3-phosphate synthase functions as a transcriptional repressor in Populus[J].Plant Cell,2018,30(7):1 645-1 660.
[34]VASSAO D G,KIM S J,MILHOLLAN J K,et al.A pinoresinol-lariciresinol reductase homologue from the creosote bush(Larrea tridentata)catalyzes the efficient in vitro conversion of p-coumaryl/coniferyl alcohol esters into the allylphenols chavicol/eugenol,but not the propenylphenols panol/isoeuge[J].Archives of Biochemistry&Biophysics,2007,465(1):209-218.
[35]BAYINDIR U,ALFERMANN AW,FUSS E.Hinokinin biosynthesis in Linum corymbulosum Reichenb[J].The Plant Journal,2008,55(5):810-820.
[36]李芳军.利用技术研究棉花抗逆基因功能[D].北京:中国农业大学,2014.
[37]BECKER A,LANGE M.VIGS-Genomics goes functional[J].Trends in Plant Science.2010,15(1):1-4.
[38]HU Q,MIN L,YANG X Y,et al.Laccase GhLac1modulates broad-spectrum biotic stress tolerance via Manipulating Phenylpropanoid Pathway and Jasmonic Acid Synthesis[J].Plant Physiology,2017,176(2):1 808-1 823.
[39]唐英才.下调棉花3-酮基二氢鞘氨醇还原酶基因(GhKDSR2-1)的表达对棉纤维发育的影响[D].重庆:西南大学,2016.
[40]秦超.棉花肉桂酰辅酶A还原酶基因(GhCCR)的遗传转化和功能分析[D].乌鲁木齐:新疆农业大学,2009.
[41]刘珍.棉花木脂素代谢相关基因GhPCBER的克隆及功能分析[D].武汉:华中农业大学,2016.
[2]GRAND C,BOUDET A,BOUDET AM.Isoenzymes of hydroxycinnamate:CoA ligase from poplar stems properties and tissue distribution[J].Planta,1983,158(3):225-229.
[3]JOHN H G,JOHN RALPH,CATHERINE L,et al.Genetic and molecular basis of grass cell-wall degradability.I.Lignincell wall matrix interactions[J].Comptes Rendus Biologies,2004,327:455-465.
[4]BHUIYAN NH,SELVARA JG,WEI Y,et al.Gene expression profiling and silencing reveal that monolignol biosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion[J].Journal of Exprimental Botany,2009,60:509-521.
[5]LING F,RAPHAEL L,SHIMON G,et al.Progressive inhibition by water deficit of cell wall extensibility and growth along the elongation zone of maize roots is related to increased lignin metabolism and progressive stelar accumulation of wall phenolics[J].Plant Physiology,2006,140:603-612.
[6]TSUBASA S,ROBERT W,TADAYUKI I,et al.Expression patterns of two tobacco isoflavone reductase-like genes and their possible roles in secondary metabolism in tobacco[J].Plant Molecular Biology,2002,50:427-440.
[7]TANNER GJ,FRANCKI KT,ABRAHAMS S,et al.Proanthocyanidin biosynthesis in plants.purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA[J].The J Biol Chem,2003,278(3):31 647-31 656.
[8]DAVIN L B,LEWIS N G.Lignin and lignan biochemical pathways in plants:an unprecedented discovery in phenolic coupling[J].Anais Da Academia Brasileira De Ciencias,1995,67:363-378.
[9]PRASSINOS C,KO J H,YANG J,et al.Transcriptome profiling of vertical stem segments provides insights into the genetic regulation of secondary growth in hybrid aspen trees[J].Plant Cell Physiol,2005,46(8):1 213-1 225.
[10]涂礼莉,谭家福,郭凯,等.类黄酮代谢途径与棉花纤维发育.[J].中国科学,2014,44(8):758-765.TU L L,TAN J F,GUO K,et al.Flavonoid metabolic pathway and cotton fiber development.[J].Scientia Sinica Vitae,2014,44(8):758-765.
[11]GRABBER JH,RALPH J,LAPIERRE C,et al.Genetic and molecular basis of grass cell-wall degradability.I.Lignin-cell wall matrix interactions[J].Comptes Rendus Biologies,2004,327(5):455-465.
[12]BOERJAN W,POLLE A,VANDER MIJNSBRUGGE K.Role in lignification and growth for plant phenylcoumaran benzylic ether reductase:US,US20060015967[P].2006.
[13]喻树迅,范术丽,王寒涛,等.中国棉花高产育种研究进展[J].中国农业科学,2016,49(18):3 465-3 476.YU S X,FAN S L,WANG H T,WEI H L,et al.Progresses in research on cotton high yield breeding in China[J].Scientia Agricultura Sinica,2016,49(18):3 465-3 476.
[14]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 Research,2007,17(5):422-434.
[15]FAN L,SHI W J,HU W R,et al.Molecular and biochemical evidence for phenylpropanoid synthesis and presence of wall-linked phenolics in cotton fibers[J].Journal of Integrative Plant Biology,2009,51(7):626-637.
[16]HAN L B,LI Y B,WANG H Y,et al.The dual functions of WLIM1ain cell elongation and secondary wall formation in developing cotton fibers[J].The Plant Cell,2013,25(11):4 421-4 438.
[17]DOWD C,WILSON IW,MCFADDEN H.Gene expression profile changes in cotton root and hypocotyl tissues in response to infection with Fusarium oxysporum f.sp.vasinfectum[J].Molecular Plant-Microbe Interactions,2004,17(6):654-667.
[18]SUN J,AN H,SHI W,et al.Molecular cloning and characterization of GhWRKY11,agene implicated in pathogen responses from cotton[J].South African Journal of Botany,2012,81(4):113-123.
[19]DAVID RG,ALBENA T DK,LAURENCE B,et al.Phylogenetic links in plant defense systems:lignans,isoflavonoids,and their reductases[J].ACS Symposium Series,1997,658:58-89.
[20]STERKY F,REGAN S,KARLSSON J,et al.Gene discovery in the wood-formig tissues of poplar:analysis of 5,692expressed sequence tags[J].Proceedings of the National A-cademy of Science of the United States of America,1998,95(22):13 330-13 335.
[21]VANDER MIJNSBRUGGE K,MEYERMANS H,VANMONTAGU M,et al.Wood formation in poplar:identification,characterization,and seasonal variation of xylem proteins[J].Planta,2000,210(4):589-598.
[22]KWON M,DAVIN LB,LEWIS NG.In situ hybridization and immunolocalization of lignin reductases in woody tissues:implications heartwood formation and other forms of vascular tissue preservation[J].Phytochemistry,2001,57:899-914.
[23]NUOENDAGULA,KAMIMURA N,MORI T,et al.Expression and functional analyses of a putative phenylcoumaran benzylic ether reductase in Arabidopsis thaliana[J].Plant Cell Rep,2016,35(3):513-526.
[24]ZHAO Q,DIXON RA.Altering the cell wall and its impact on plant disease:from forage to bioenergy[J].Annu Rev Phytopathol,2014,52:69-91.
[25]SHOJI T,WINZ R,NAKAJIMA K,et al.Expression patterns of two tobacco isoflavone reductase-like genes and their possible roles in secondary metabolism in tobacco[J].Plant Molecular Biology,2002,50(3):427-440.
[26]R.B TURLEY.Expression of a phenylcoumaran benzylic ether reductase-like protein in the ovules of Gossypium hirsutum[J].Biologia Plantarum,2008,52(4):759-762.
[27]TALIERCIO E,ALLEN RD,ESSENBERG M,et al.Analysis of ESTs from multiple Gossypium hirsutumtissues and identification of SSRs[J].Genome,2006,49(4):306-319.
[28]UDALL JA,SWANSON JM,HALLER K,et al.A global assembly of cotton ESTs[J].Genome Research,2006,16(3):441-450.
[29]齐雯雯,宫晓琳,王洋,等.蘸花法在植物遗传转化上的应用研究进展[J].现代农业科技,2014,(24):9-10,12.QI W W,GONG X L,WANG Y,et al.Application and research progress of floral dip in plant genetic transformation[J].Modern Agricultural Sciences and Technology,2014,(24):9-10,12.
[30]王建庆,曹佃元,张玉.乙酰溴法测定棉籽壳中木质素的含量[J].纺织学报,2013,34(9):12-16.WANG J Q,CAO D Y,ZHANG Y.cetyl bromide test method for determining amount of lignin in cottonseed coat[J].Journal of Textile Research,2013,34(9):12-16.
[31]闫洪颖.棉花纤维发育过程超微结构观察和次生壁木质素含量的分析[D].乌鲁木齐:新疆农业大学,2009.
[32]陈韵,石展望,黄晓敏.超声波辅助提取大豆总木脂素及其含量分析[J].大豆科学,2010,29(1):168-170,173.CHEN Y,SHI Z W,HUANG X M.Ultrasonic-assisted extraction of total lignans and its content analysis in soybeans[J].Soybean Science,2010,29(1):168-170,173.
[33]XIE M,WELLINGTON M,ANTHONY C.A 5-enolpyruvylshikimate 3-phosphate synthase functions as a transcriptional repressor in Populus[J].Plant Cell,2018,30(7):1 645-1 660.
[34]VASSAO D G,KIM S J,MILHOLLAN J K,et al.A pinoresinol-lariciresinol reductase homologue from the creosote bush(Larrea tridentata)catalyzes the efficient in vitro conversion of p-coumaryl/coniferyl alcohol esters into the allylphenols chavicol/eugenol,but not the propenylphenols panol/isoeuge[J].Archives of Biochemistry&Biophysics,2007,465(1):209-218.
[35]BAYINDIR U,ALFERMANN AW,FUSS E.Hinokinin biosynthesis in Linum corymbulosum Reichenb[J].The Plant Journal,2008,55(5):810-820.
[36]李芳军.利用技术研究棉花抗逆基因功能[D].北京:中国农业大学,2014.
[37]BECKER A,LANGE M.VIGS-Genomics goes functional[J].Trends in Plant Science.2010,15(1):1-4.
[38]HU Q,MIN L,YANG X Y,et al.Laccase GhLac1modulates broad-spectrum biotic stress tolerance via Manipulating Phenylpropanoid Pathway and Jasmonic Acid Synthesis[J].Plant Physiology,2017,176(2):1 808-1 823.
[39]唐英才.下调棉花3-酮基二氢鞘氨醇还原酶基因(GhKDSR2-1)的表达对棉纤维发育的影响[D].重庆:西南大学,2016.
[40]秦超.棉花肉桂酰辅酶A还原酶基因(GhCCR)的遗传转化和功能分析[D].乌鲁木齐:新疆农业大学,2009.
[41]刘珍.棉花木脂素代谢相关基因GhPCBER的克隆及功能分析[D].武汉:华中农业大学,2016.