棉花肉桂醇脱氢酶基因在棉纤维中的表达及对其结构组分的影响
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摘要
利用农杆菌介导法获得‘新陆早36号’转棉花肉桂醇脱氢酶基因(GhCAD6)材料,以转基因T_6代植株为试材,对GhCAD6基因在叶片基因组中的整合情况和不同发育阶段棉花纤维中的表达量进行分析,研究该基因对棉纤维中结构多糖和苯丙烷类化合物含量及纤维中苯丙烷类结构单体的影响。结果显示:(1)GhCAD6基因以单拷贝的形式整合到受体棉花基因组中。(2)转基因植株纤维中GhCAD6基因的表达量低于相同发育阶段对照样品,在对照样品中GhCAD6基因的表达量表现为先升高,在20 DPA(开花后天数)时表达量最高,之后下降,而在转基因植株纤维中先上升,并于发育15 DPA时表达量下降,20 DPA时又上升至最高,之后再次下降。(3)成熟纤维中,转GhCAD6基因植株纤维中苯丙烷类化合物含量低于对照,但结构多糖含量的差别不明显。(4)转GhCAD6基因纤维中苯丙烷类结构单体——紫丁香基木质素(S-木质素)和愈疮木基木质素(G-木质素)的比值下降。研究表明,棉花转入GhCAD6基因后,纤维发育(15 DPA)中GhCAD6基因的表达量变化可能导致棉花中苯丙烷类化合物含量及其结构单体比率变化,从而造成棉花纤维品质改变。该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。
The material is 'Xinluzao 36' transformed with GhCAD6 gene obtained by Agrobacterium-mediated transformation. In this study, the T_6 generation of the transgenic material was used as experimental material. The integration of GhCAD6 gene in the genome of cotton, the expression in different developmental stages of cotton fiber, the content of structural polysaccharides and phenylpropanoid compounds in the cotton fiber, and the influence of the fiber in the phenylpropanoid monomer were analyzed. The results showed:(1) GhCAD6 was integrated in the form of single copy to the recipient cotton genome.(2) The expression level of GhCAD6 in the transgenic plants was lower than that in the control samples at the same developmental stage. The expression of GhCAD6 was increased firstly, reached the highest at 20 DPA and then decreased in the control samples. While the expression decreased at 15 DPA in the transgenic plant fibers, rose to the highest level at 20 DPA, and then decreased.(3) In mature fibers, the content of phenylpropanoid compounds in the transgenic plants was lower than that in the control samples, and the content of structural polysaccharides had no significant difference.(4) The ratio of S/G decreased in cotton fiber of transgenic plants. After the GhCAD6 gene transferred into cotton, the change of GhCAD6 gene expression at 15 DPA may be the cause of the change of content of phenylpropanoid compounds and ratio of structural monomer, which result in the variation of cotton fiber quality. The results of this research can provide theoretical basis for further analysis of the mechanism of GhCAD6 gene in improving cotton fiber quality.
The material is 'Xinluzao 36' transformed with GhCAD6 gene obtained by Agrobacterium-mediated transformation. In this study, the T_6 generation of the transgenic material was used as experimental material. The integration of GhCAD6 gene in the genome of cotton, the expression in different developmental stages of cotton fiber, the content of structural polysaccharides and phenylpropanoid compounds in the cotton fiber, and the influence of the fiber in the phenylpropanoid monomer were analyzed. The results showed:(1) GhCAD6 was integrated in the form of single copy to the recipient cotton genome.(2) The expression level of GhCAD6 in the transgenic plants was lower than that in the control samples at the same developmental stage. The expression of GhCAD6 was increased firstly, reached the highest at 20 DPA and then decreased in the control samples. While the expression decreased at 15 DPA in the transgenic plant fibers, rose to the highest level at 20 DPA, and then decreased.(3) In mature fibers, the content of phenylpropanoid compounds in the transgenic plants was lower than that in the control samples, and the content of structural polysaccharides had no significant difference.(4) The ratio of S/G decreased in cotton fiber of transgenic plants. After the GhCAD6 gene transferred into cotton, the change of GhCAD6 gene expression at 15 DPA may be the cause of the change of content of phenylpropanoid compounds and ratio of structural monomer, which result in the variation of cotton fiber quality. The results of this research can provide theoretical basis for further analysis of the mechanism of GhCAD6 gene in improving cotton fiber quality.
引文
[1] BASRA A S,MALIK C P.Development of the Cotton Fiber[M]//BASRA A S,MALIK C P.eds.International Review of Cytology.Elsevier,1984:65-113.DOI:10.1016/s0074-7696(08)61300-5
[2] 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.
[3] 胡文冉.棉花纤维中苯丙烷类化合物的沉积及结构研究[D].北京:中国农业大学,2016.
[4] 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].The Plant Cell,2013,25(11):4 421-4 438.
[5] BOERJAN W,RALPH J,BAUCHER M.Lignin biosynthesis[J].Annual Review of Plant Biology,2003,54:519-546.
[6] AL-GHAZI Y,BOUROT S,ARIOLI T,et al.Transcript profiling during fiber development identifies pathways in secondary metabolism and cell wall structure that may contribute to cotton fiber quality[J].Plant & Cell Physiology,2009,50(7):1 364-1 381.
[7] 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.
[8] FENG H,LI X,CHEN H,et al.GhHUB2,a ubiquitin ligase,is involved in cotton fiber development via the ubiquitin-26S proteasome pathway[J].Journal of Experimental Botany,2018,69(21):5 059-5 075.
[9] WHETTEN R,SEDEROFF R.Lignin biosynthesis[J].The Plant Cell,1995,7(7):1 001-1 013.
[10] MANSELL R L,GROSS G G,ST?CKIGT J,et al.Purification and properties of cinnamyl alcohol dehydrogenase from higher plants involved in lignin biosynthesis[J].Phytochemistry,1974,13(11):2 427-2 435.
[11] 胡文冉,范玲,孙涛,等.棉花纤维中苯丙烷类结构单体的检测方法[J].棉花学报,2016,28(4):407-412.HU W R,FAN L,SUN T,et al.A method for detecting phenylpropanoid monomers in cotton fiber[J].Cotton Science,2016,28(4):407-412.
[12] 张经博,李波,杨洋,等.陆地棉CAD基因家族的进化和表达分析[J].新疆农业科学,2016,53(7):1 177-1 187.ZHANG J B,LI B,YANG Y,et al.Genomi-wide investigation and expression analysis of CAD gene family in Gossypium hirsutum[J].Xinjiang Agricultural Sciences,2016,53(7):1 177-1 187.
[13] 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(1):e54444.
[14] 张辉,汤文开,谭新,等.棉纤维发育及其相关基因表达调控研究进展[J].植物学通报,2007,24(2):127-133.ZHANG H,TANG W K,TAN X,et al.Progresses in the study of gene regulation of cotton fiber development[J].Chinese Bulletin of Botany,2007,24(2):127-133.
[15] 胡文冉,李晓东,周小云,等.棉花GhCAD6基因表达及其功能分析研究[J].生物技术进展,2019,9(1):46-53.HU W R,LI X D,ZHOU X Y,et al.Study on expression and functional analysis of GhCAD6 gene in transgenic cotton[J].Current Biotechnology,2019,9(1):46-53.
[16] 胡文冉,范玲,谢丽霞,等.不同发育阶段棉纤维中木质素的沉积变化[J].新疆农业科学,2016,53(3):467-472.HU W R,FAN L,XIE L X,et al.Dynamic changes of lignin deposition during cotton fiber development stage[J].Xinjiang Agricultural Sciences,2016,53(3):467-472.
[17] J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯.分子克隆实验指南(第2版)[M].金冬雁,黎孟枫主译.北京:科学出版社,1992:475-782.
[18] 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.WU Y T,LIU J Y.A modified hot borate method for efficient isolation of total RNA from different cotton tissues[J].Cotton Science,2004,16(2):67-71.
[19] 蒋淑丽,王学德.棉花纤维突变体胚珠发育过程中主要生化物质的积累特征[J].浙江大学学报(农业与生命科学版),2002,28(1):16-21.JIANG S L,WANG X D.The accumulation of biochemical components in ovule of cotton fiber mutants during the ovule development[J].Journal of Zhejiang University (Agriculture & Life Sciences),2002,28(1):16-21.
[20] HU W R,FAN L,TIAN X L,et al.Modified methods for the analysis of the lignin-like phenolic polymer contents of cotton fibers[J].JAPS,2015,25(3):232-239.
[21] 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,2009.
[22] NAPOLI C,LEMIEUX C,JORGENSEN R.Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans[J].The Plant Cell,1990,2(4):279-289.
[23] VAN DER KROL A R,MUR L A,BELD M,et al.Flavonoid genes in petunia:addition of a limited number of gene copies may lead to a suppression of gene expression[J].Plant Cell,1990,2(4):291-299.
[24] SMITH C J S,WATSON C F,BIRD C R,et al.Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants[J].Molecular & General Genetics,1990,224(3):477-481.
[25] BAUCHER M,BERNARD-VAILHé M A,CHABBERT B,et al.Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility[J].Plant Molecular Biology,1999,39(3):437-447.
[2] 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.
[3] 胡文冉.棉花纤维中苯丙烷类化合物的沉积及结构研究[D].北京:中国农业大学,2016.
[4] 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].The Plant Cell,2013,25(11):4 421-4 438.
[5] BOERJAN W,RALPH J,BAUCHER M.Lignin biosynthesis[J].Annual Review of Plant Biology,2003,54:519-546.
[6] AL-GHAZI Y,BOUROT S,ARIOLI T,et al.Transcript profiling during fiber development identifies pathways in secondary metabolism and cell wall structure that may contribute to cotton fiber quality[J].Plant & Cell Physiology,2009,50(7):1 364-1 381.
[7] 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.
[8] FENG H,LI X,CHEN H,et al.GhHUB2,a ubiquitin ligase,is involved in cotton fiber development via the ubiquitin-26S proteasome pathway[J].Journal of Experimental Botany,2018,69(21):5 059-5 075.
[9] WHETTEN R,SEDEROFF R.Lignin biosynthesis[J].The Plant Cell,1995,7(7):1 001-1 013.
[10] MANSELL R L,GROSS G G,ST?CKIGT J,et al.Purification and properties of cinnamyl alcohol dehydrogenase from higher plants involved in lignin biosynthesis[J].Phytochemistry,1974,13(11):2 427-2 435.
[11] 胡文冉,范玲,孙涛,等.棉花纤维中苯丙烷类结构单体的检测方法[J].棉花学报,2016,28(4):407-412.HU W R,FAN L,SUN T,et al.A method for detecting phenylpropanoid monomers in cotton fiber[J].Cotton Science,2016,28(4):407-412.
[12] 张经博,李波,杨洋,等.陆地棉CAD基因家族的进化和表达分析[J].新疆农业科学,2016,53(7):1 177-1 187.ZHANG J B,LI B,YANG Y,et al.Genomi-wide investigation and expression analysis of CAD gene family in Gossypium hirsutum[J].Xinjiang Agricultural Sciences,2016,53(7):1 177-1 187.
[13] 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(1):e54444.
[14] 张辉,汤文开,谭新,等.棉纤维发育及其相关基因表达调控研究进展[J].植物学通报,2007,24(2):127-133.ZHANG H,TANG W K,TAN X,et al.Progresses in the study of gene regulation of cotton fiber development[J].Chinese Bulletin of Botany,2007,24(2):127-133.
[15] 胡文冉,李晓东,周小云,等.棉花GhCAD6基因表达及其功能分析研究[J].生物技术进展,2019,9(1):46-53.HU W R,LI X D,ZHOU X Y,et al.Study on expression and functional analysis of GhCAD6 gene in transgenic cotton[J].Current Biotechnology,2019,9(1):46-53.
[16] 胡文冉,范玲,谢丽霞,等.不同发育阶段棉纤维中木质素的沉积变化[J].新疆农业科学,2016,53(3):467-472.HU W R,FAN L,XIE L X,et al.Dynamic changes of lignin deposition during cotton fiber development stage[J].Xinjiang Agricultural Sciences,2016,53(3):467-472.
[17] J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯.分子克隆实验指南(第2版)[M].金冬雁,黎孟枫主译.北京:科学出版社,1992:475-782.
[18] 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.WU Y T,LIU J Y.A modified hot borate method for efficient isolation of total RNA from different cotton tissues[J].Cotton Science,2004,16(2):67-71.
[19] 蒋淑丽,王学德.棉花纤维突变体胚珠发育过程中主要生化物质的积累特征[J].浙江大学学报(农业与生命科学版),2002,28(1):16-21.JIANG S L,WANG X D.The accumulation of biochemical components in ovule of cotton fiber mutants during the ovule development[J].Journal of Zhejiang University (Agriculture & Life Sciences),2002,28(1):16-21.
[20] HU W R,FAN L,TIAN X L,et al.Modified methods for the analysis of the lignin-like phenolic polymer contents of cotton fibers[J].JAPS,2015,25(3):232-239.
[21] 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,2009.
[22] NAPOLI C,LEMIEUX C,JORGENSEN R.Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans[J].The Plant Cell,1990,2(4):279-289.
[23] VAN DER KROL A R,MUR L A,BELD M,et al.Flavonoid genes in petunia:addition of a limited number of gene copies may lead to a suppression of gene expression[J].Plant Cell,1990,2(4):291-299.
[24] SMITH C J S,WATSON C F,BIRD C R,et al.Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants[J].Molecular & General Genetics,1990,224(3):477-481.
[25] BAUCHER M,BERNARD-VAILHé M A,CHABBERT B,et al.Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility[J].Plant Molecular Biology,1999,39(3):437-447.
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