彩色棉查尔酮合成酶基因GhCHS1的克隆和功能分析
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摘要
查尔酮合成酶基因(chalcone synthase, CHS)是黄酮类生物合成途径中的第1个限速酶基因。为探讨彩色棉(Gossypium hirsutum)花青素通路中关键酶基因GhCHS与彩色纤维色泽形成的关系,本研究克隆了GhCHS1基因,采用病毒介导基因干涉(virus induced gene silencing, VIGS)技术,获得彩色棉棕絮1号GhCHS1干涉株系,使用qRT-PCR分析干涉株系不同发育时期纤维的GhCHS1基因表达水平,测定干涉株系的纤维、叶片及棉仁花青素含量。从已知棉属基因组筛选到CHS家族的24个成员,根据同源性可归属7个类型和6种三级空间结构类型,其中GhCHS1 (GenBank No. LOC107897841)在棕絮1号发育的纤维和叶片中优势表达。GhCHS1蛋白主要分布在胞液中,二级结构以无规卷曲和α螺旋为主。棕絮1号GhCHS1干涉株系不同发育时期纤维中GhCHS1基因表达水平显著降低(P<0.05),纤维色泽显著淡化变白,明显区别于野生型棕色纤维,GhCHS1基因的表达水平越低,纤维色泽越浅。花青素含量分析表明,纤维种皮和棉仁花青素含量越少纤维色泽越浅。该研究结果表明GhCHS1基因参与彩色棉植株体内和纤维中花青素的合成和累积,GhCHS1基因在纤维色泽形成中起着关键作用。本研究为开展彩色棉纤维色泽形成机理的研究提供了一定的理论基础。
The chalcone synthase gene(CHS) were the first committed enzyme gene of flavonoid biosynthesis, CHS condensed 3 molecules of malonyl-CoA and 1 molecule of p-coumaroyl-CoA to form naringenin chalcone. The study was to investigate the relationship between GhCHS1 gene and the formation of fiber color through virus induced gene silencing(VIGS) in colored cotton ZongXu1(Gossypium hirsutum). The expression of GhCHS1 gene in interference lines(GhCHS1 i) was measured by qRT-PCR, and the anthocyanins contents in the fibers and seedcoat, leaves and cotton kernels were determined. Bioinformatics analysis indicated that GhCHS1 proteins were mainly distributed in vacuoles, and the secondary structure of GhCHS1 proteins was mainly composed of random coil and alpha helix. 24 CHS members were screened in the known genomes of Gossypium, belonging to 7 types according to homology, and 6 types of tertiary spatial structures.GhCHS1(GenBank No. LOC107897841) was predominantly expressed in the developing fibers and leaves of Zongxu1. The expression of GhCHS1 gene was increased in the early developing fibers(0～9 DPA, day post anthesis), reached the peak at 9 DPA, and then decreased from 12 DPA to 20 DPA. The GhCHS1 gene was interference by VIGS technology, and a large number of transgenic interference lines were obtained. Phytoene desaturase(GhPDS) was silenced by VIGS as the positive control, vector-empty CK was used as the negative control, and Zongxu 1 WT was used as the wild type control. The GhCHS1 i lines had slightly curly leaves the same as the phytoene desaturase gene interference(GhPDSi) and vector-empty plants compared with the WT plants. The GhCHS1 expression level was significantly decreased in the GhCHS1 i plants, the fiber color of GhCHS1 i plants was significantly lighter compared with the brown fiber of WT Zongxu1, and different from the white fiber of upland cotton C312. The expression level of GhCHS1 gene in the developing fibers of GhCHS1 i lines was significantly lower than that in the WT, the fiber color of the GhCHS1 i lines was lighter brown. The expression level of GhCHS1 gene was indicated the positive correlation with the fiber color.Compared with the WT, the GhCHS1 i lines had the lower the expression level of GhCHS1 gene, the fiber color appeared significantly lighter(P<0.05). The analysis of anthocyanin content showed that the less the anthocyanin content of fiber and seed coat, cotton kernel, the fiber color became significantly lighter. The results indicated that GhCHS1 gene was involved in the biosynthesis and accumulation of anthocyanins in colored cotton leaves, fiber and seed coat, cotton kernel, and also played an important role in the fiber pigmentation. This study would have important reference value for the formation mechanism of fiber color formation in colored cotton.
The chalcone synthase gene(CHS) were the first committed enzyme gene of flavonoid biosynthesis, CHS condensed 3 molecules of malonyl-CoA and 1 molecule of p-coumaroyl-CoA to form naringenin chalcone. The study was to investigate the relationship between GhCHS1 gene and the formation of fiber color through virus induced gene silencing(VIGS) in colored cotton ZongXu1(Gossypium hirsutum). The expression of GhCHS1 gene in interference lines(GhCHS1 i) was measured by qRT-PCR, and the anthocyanins contents in the fibers and seedcoat, leaves and cotton kernels were determined. Bioinformatics analysis indicated that GhCHS1 proteins were mainly distributed in vacuoles, and the secondary structure of GhCHS1 proteins was mainly composed of random coil and alpha helix. 24 CHS members were screened in the known genomes of Gossypium, belonging to 7 types according to homology, and 6 types of tertiary spatial structures.GhCHS1(GenBank No. LOC107897841) was predominantly expressed in the developing fibers and leaves of Zongxu1. The expression of GhCHS1 gene was increased in the early developing fibers(0～9 DPA, day post anthesis), reached the peak at 9 DPA, and then decreased from 12 DPA to 20 DPA. The GhCHS1 gene was interference by VIGS technology, and a large number of transgenic interference lines were obtained. Phytoene desaturase(GhPDS) was silenced by VIGS as the positive control, vector-empty CK was used as the negative control, and Zongxu 1 WT was used as the wild type control. The GhCHS1 i lines had slightly curly leaves the same as the phytoene desaturase gene interference(GhPDSi) and vector-empty plants compared with the WT plants. The GhCHS1 expression level was significantly decreased in the GhCHS1 i plants, the fiber color of GhCHS1 i plants was significantly lighter compared with the brown fiber of WT Zongxu1, and different from the white fiber of upland cotton C312. The expression level of GhCHS1 gene in the developing fibers of GhCHS1 i lines was significantly lower than that in the WT, the fiber color of the GhCHS1 i lines was lighter brown. The expression level of GhCHS1 gene was indicated the positive correlation with the fiber color.Compared with the WT, the GhCHS1 i lines had the lower the expression level of GhCHS1 gene, the fiber color appeared significantly lighter(P<0.05). The analysis of anthocyanin content showed that the less the anthocyanin content of fiber and seed coat, cotton kernel, the fiber color became significantly lighter. The results indicated that GhCHS1 gene was involved in the biosynthesis and accumulation of anthocyanins in colored cotton leaves, fiber and seed coat, cotton kernel, and also played an important role in the fiber pigmentation. This study would have important reference value for the formation mechanism of fiber color formation in colored cotton.
引文
杜雄明,张天真.1997.有色棉的研究利用现状及展望[J].中国农学通报,(3):30-32.(Du X M,Zhang T Z.1997.Current status and prospects of research and utilization of colored cotton[J].Chinese Agricultural Science Bulletin,(3):30-32.)
杜雄明,刘方,王坤波,等.2017.棉花种质资源收集鉴定与创新利用[J].棉花学报,29(z1):51-61.(Du X M,Liu F,Wang K B,et al.2017.Collection,evolution and utilization of cotton germplasm[J].Cotton Science,29(z1):51-61.)
梁明炜,刘海峰,陆雪莹,等.2011.棕色棉类黄酮3'-羟化酶基因(F3'H)的克隆及色素合成途径中相关基因表达特性研究[J].农业生物技术学报,19(5):808-814.(Liang M W,Liu H F,Lu X Y,et al.2011.Cloning of flavonoid3'-hydroxylase gene(GhF3'H)and expressional characteristics of several genes associated with pigment synthesis in brown cotton(Gossypium hirsutum L.)fibers[J].Journal of Agricultural Biotechnology,19(5):808-814.)
邱新棉.2004.天然彩色棉研究进展与发展前景[J].棉花学报,16(4):249-254.(Qiu X M,2004.Research progress and prospects on naturally-colored cotton[J].Cotton Science,16(4):249-254.)
王宏芝,李瑞芬,王国英,等.2005.病毒诱导的基因沉默及其在植物功能基因组学研究中的应用[J].自然科学进展15(1):8-14.(Wang H Z,Li R F,Wang G Y,et al.2005.Virus induced gene silence and its application in plant gene functional genomics[J].Progress in Natural Science,15(1):8-14.)
Burch-Smith T M,Anderson J C,Martin G B,et al.2004.Applications and advantages of virus-induced gene silencing for gene function studies in plants[J].Plant Journal,39(5):734-746.
Fauquet C M,Briddon R W,Brown J K,et al.2008.Geminivirus strain demarcation and nomenclature[J].Archives of Virology,153(4):783-821.
Feng H J,Tian X H,Liu Y C,et al.2013.Analysis of flavonoids and the flavonoid structural genes in brown fiber of upland cotton[J].PloS One,8(3):e58820.
Feng H,Li Y,Wang S,et al.2014.Molecular analysis of proanthocyanidins related to pigmentation in brown cotton fibre(Gossypium hirsutum L.)[J].Journal of Experimental Botany,65(20):5759-5769.
Ferrer J L,Jez J M,Bowman M E,et al.1999.Structure of chalcone synthase and the molecular basis of plant polyketide pathway[J].Nature Structural Biology,6(8):775-784.
Ge X,Jie W,Zhang C,et al.2016.Prediction of VIGS efficiency by the sfold program and its reliability analysis in Gossypium hirsutum[J].Science Bulletin,61(7):1-9.
Gu Z,Huang C,Li F,et al.2014.A versatile system for functional analysis of genes and microRNAs in cotton[J].Plant Biotechnology Journal,12(5):638-649.
Jeske H.2009.Geminiviruses[J].Current Topics Microbiology and Immunology,331(1):185-226.
Koes R E,Quattrocchio F,Mol J N.1994.The flavonoid biosynthetic pathway in plants:Function and evolution[J].BioEssays,16(2):123-132.
Lorenc-Kuku?A K,Jafra S,Oszmia?Ski J,et al.2005.Ectopic expression of anthocyanin 5-o-glucosyltransferase in potato tuber causes increased resistance to bacteria[J].Journal of Agriculture Food Chemistry,53(2):272-281.
Ohkawa K,Cha D I,Kim H,et al.2010.Electrospinning of Chitosan[J].Macromolecular Rapid Communications,25(18):1600-1605.
Pang J,Yue Z,Li Q,et al.2013.Development of agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense[J].PLoS ONE,8(9):e73211.
Ratcliff F,Harrison B D,Baulcombe D C.1997.A similarity between viral defense and gene silencing in plants[J].Science,276(5318):1558-1560.
Sarma A D,Sharma R.1999.Anthocyanin-DNA copigmentation complex:Mutual protection against oxidative damage[J].Phytochemistry(Oxford),52(7):1313-1318.
Schmittgen T D,Livak K J.2008.Analyzing real-time PCRdata by the comparative C(T)method[J].Nature Protocols,3(6):1101-1108.
Scofield S R,Li H,Brandt A S,et al.2005.Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway[J].Plant Physiology,138(4):2165-2173.
Tuttle J R,Idris A M,Brown J K,et al.2008.Geminivirus-mediated gene silencing from cotton leaf crumple virus is enhanced by low temperature in cotton[J].Plant Physiology,148(1):41-50.
Wrolstad R E,Acree T E,Decker E A,et al.2001.Characterization and measurement of anthocyanins by UV-Visible spectroscopy[J].Current Protocols in Food Analytical Chemistry,ppF1.2.1-F1.2.13.
Xiao Y H,Zhang Z S,Yin M H,et al.2007.Cotton flavonoid structural genes related to the pigmentation in brown fibers[J].Biochemical and Biophysical Research Communications,358(1):73-78.
Yan Q,Wang Y,Li Q,et al.2018.Upregulation of ghtt2-3A in cotton fibers during secondary wall thickening results in brown fibers with improved quality[J].Plant Biotechnology Journal,16(10):1735-1747.
Zha J,Koffas M A G.2017.Production of anthocyanins in metabolically engineered microorganisms:Current status and perspectives[J].Synthetic and Systems Biotechnology,2(4):259-266.
Zhou X.2013.Advances in understanding begomovirus satellites[J].Annual Review of Phytopathology,51(1):357-381.
杜雄明,刘方,王坤波,等.2017.棉花种质资源收集鉴定与创新利用[J].棉花学报,29(z1):51-61.(Du X M,Liu F,Wang K B,et al.2017.Collection,evolution and utilization of cotton germplasm[J].Cotton Science,29(z1):51-61.)
梁明炜,刘海峰,陆雪莹,等.2011.棕色棉类黄酮3'-羟化酶基因(F3'H)的克隆及色素合成途径中相关基因表达特性研究[J].农业生物技术学报,19(5):808-814.(Liang M W,Liu H F,Lu X Y,et al.2011.Cloning of flavonoid3'-hydroxylase gene(GhF3'H)and expressional characteristics of several genes associated with pigment synthesis in brown cotton(Gossypium hirsutum L.)fibers[J].Journal of Agricultural Biotechnology,19(5):808-814.)
邱新棉.2004.天然彩色棉研究进展与发展前景[J].棉花学报,16(4):249-254.(Qiu X M,2004.Research progress and prospects on naturally-colored cotton[J].Cotton Science,16(4):249-254.)
王宏芝,李瑞芬,王国英,等.2005.病毒诱导的基因沉默及其在植物功能基因组学研究中的应用[J].自然科学进展15(1):8-14.(Wang H Z,Li R F,Wang G Y,et al.2005.Virus induced gene silence and its application in plant gene functional genomics[J].Progress in Natural Science,15(1):8-14.)
Burch-Smith T M,Anderson J C,Martin G B,et al.2004.Applications and advantages of virus-induced gene silencing for gene function studies in plants[J].Plant Journal,39(5):734-746.
Fauquet C M,Briddon R W,Brown J K,et al.2008.Geminivirus strain demarcation and nomenclature[J].Archives of Virology,153(4):783-821.
Feng H J,Tian X H,Liu Y C,et al.2013.Analysis of flavonoids and the flavonoid structural genes in brown fiber of upland cotton[J].PloS One,8(3):e58820.
Feng H,Li Y,Wang S,et al.2014.Molecular analysis of proanthocyanidins related to pigmentation in brown cotton fibre(Gossypium hirsutum L.)[J].Journal of Experimental Botany,65(20):5759-5769.
Ferrer J L,Jez J M,Bowman M E,et al.1999.Structure of chalcone synthase and the molecular basis of plant polyketide pathway[J].Nature Structural Biology,6(8):775-784.
Ge X,Jie W,Zhang C,et al.2016.Prediction of VIGS efficiency by the sfold program and its reliability analysis in Gossypium hirsutum[J].Science Bulletin,61(7):1-9.
Gu Z,Huang C,Li F,et al.2014.A versatile system for functional analysis of genes and microRNAs in cotton[J].Plant Biotechnology Journal,12(5):638-649.
Jeske H.2009.Geminiviruses[J].Current Topics Microbiology and Immunology,331(1):185-226.
Koes R E,Quattrocchio F,Mol J N.1994.The flavonoid biosynthetic pathway in plants:Function and evolution[J].BioEssays,16(2):123-132.
Lorenc-Kuku?A K,Jafra S,Oszmia?Ski J,et al.2005.Ectopic expression of anthocyanin 5-o-glucosyltransferase in potato tuber causes increased resistance to bacteria[J].Journal of Agriculture Food Chemistry,53(2):272-281.
Ohkawa K,Cha D I,Kim H,et al.2010.Electrospinning of Chitosan[J].Macromolecular Rapid Communications,25(18):1600-1605.
Pang J,Yue Z,Li Q,et al.2013.Development of agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense[J].PLoS ONE,8(9):e73211.
Ratcliff F,Harrison B D,Baulcombe D C.1997.A similarity between viral defense and gene silencing in plants[J].Science,276(5318):1558-1560.
Sarma A D,Sharma R.1999.Anthocyanin-DNA copigmentation complex:Mutual protection against oxidative damage[J].Phytochemistry(Oxford),52(7):1313-1318.
Schmittgen T D,Livak K J.2008.Analyzing real-time PCRdata by the comparative C(T)method[J].Nature Protocols,3(6):1101-1108.
Scofield S R,Li H,Brandt A S,et al.2005.Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway[J].Plant Physiology,138(4):2165-2173.
Tuttle J R,Idris A M,Brown J K,et al.2008.Geminivirus-mediated gene silencing from cotton leaf crumple virus is enhanced by low temperature in cotton[J].Plant Physiology,148(1):41-50.
Wrolstad R E,Acree T E,Decker E A,et al.2001.Characterization and measurement of anthocyanins by UV-Visible spectroscopy[J].Current Protocols in Food Analytical Chemistry,ppF1.2.1-F1.2.13.
Xiao Y H,Zhang Z S,Yin M H,et al.2007.Cotton flavonoid structural genes related to the pigmentation in brown fibers[J].Biochemical and Biophysical Research Communications,358(1):73-78.
Yan Q,Wang Y,Li Q,et al.2018.Upregulation of ghtt2-3A in cotton fibers during secondary wall thickening results in brown fibers with improved quality[J].Plant Biotechnology Journal,16(10):1735-1747.
Zha J,Koffas M A G.2017.Production of anthocyanins in metabolically engineered microorganisms:Current status and perspectives[J].Synthetic and Systems Biotechnology,2(4):259-266.
Zhou X.2013.Advances in understanding begomovirus satellites[J].Annual Review of Phytopathology,51(1):357-381.