辣椒细胞质雄性不育系和保持系蔗糖转化酶活性与相关基因表达分析
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摘要
蔗糖是植物光合作用的主要同化产物。植物生殖器官对蔗糖的有效利用,在植物的受精和坐果过程中发挥重要作用。蔗糖转化酶(invertase, INV)将蔗糖水解为葡萄糖和果糖,是蔗糖代谢的关键酶,根据细胞内位置不同,INV被分成细胞质蔗糖转化酶(cytoplasmic invertase, CIN)、液泡蔗糖转化酶(vacuole invertase, VIN)和细胞壁蔗糖转化酶(cell wall invertase, CWIN)。为探究蔗糖代谢在花粉败育导致辣椒(Capsicum annuum)雄性不育过程中的相关生理分子机理,本研究分析了INV的酶活性、基因表达模式与辣椒雄性不育性状之间的关系。本研究以1对辣椒细胞质雄性不育系131A和保持系131B为实验材料,利用INV酶活性测定、辣椒基因组INV鉴定与结构分析等,以及辣椒CWIN合成相关基因qRT-PCR等方法,对不同组织以及不同发育时期花中蔗糖转化酶的活性、转化酶合成基因表达模式等指标进行了比较分析。结果表明,在大花蕾和成花时期保持系131B的CWIN酶活性显著高于不育系131A,且差异显著性分别达到极其显著(P<0.001)和极显著(P<0.01),而这一现象与辣椒CWIN合成基因CaCWIN3和CaCWIN4在保持系131B雄蕊中的高表达水平紧密相关。据此推测,辣椒CaCWIN3和CaCWIN4可能是蔗糖转化酶参与调控辣椒雄性不育的重要功能基因。本研究结果为深入研究辣椒雄性不育分子调控机理提供了方向。
Sucrose is one of the main assimilates in plant photosynthesis process. The effective use of sucrose in plant reproduction plays important roles in the process of fertilization and fruit-setting. Invertase(INV), as a key enzyme of sucrose metabolism, regulate the hydrolysis of sucrose into glucose and fructose. According tothe cellular localization, INVs are further divided as cytoplasmic invertase(CIN), vacuolar invertase(VIN),and cell wall invertase(CWIN). In order to understand the physiological and molecular mechanism of sucrose metabolism involved in the process of male sterility of pepper(Capsicum annuum) induced by pollen abortion,the INV enzyme activities, INV genes expression pattern and their correlation with pepper male sterility were analyzed. A pair of pepper male sterile line 131 A and maintainer line 131 B(fertile) were used as investigating materials in this study. The parameters including INV enzyme activities, VIN and CWIN gene identification in pepper genome, and CWIN encoding gene expression pattern by qRT-PCR, were comparatively analyzed in different tissues and developmental stages of pepper flower. The results showed that the CWIN activities in the big bud and flower of the maintainer line 131 B were significantly higher than those in the male sterile line131 A(P<0.001 and P<0.01, respectively), which were closely correlated with the high expression level of pepper CaCWIN encoding genes CaCWIN3 and CaCWIN4. According to the results, CaCWIN3 and CaCWIN4 were likely the key functional genes involved in the process of male sterility of pepper. These results shed light on the further investigation of molecular regulation mechanism of capsicum male sterility.
Sucrose is one of the main assimilates in plant photosynthesis process. The effective use of sucrose in plant reproduction plays important roles in the process of fertilization and fruit-setting. Invertase(INV), as a key enzyme of sucrose metabolism, regulate the hydrolysis of sucrose into glucose and fructose. According tothe cellular localization, INVs are further divided as cytoplasmic invertase(CIN), vacuolar invertase(VIN),and cell wall invertase(CWIN). In order to understand the physiological and molecular mechanism of sucrose metabolism involved in the process of male sterility of pepper(Capsicum annuum) induced by pollen abortion,the INV enzyme activities, INV genes expression pattern and their correlation with pepper male sterility were analyzed. A pair of pepper male sterile line 131 A and maintainer line 131 B(fertile) were used as investigating materials in this study. The parameters including INV enzyme activities, VIN and CWIN gene identification in pepper genome, and CWIN encoding gene expression pattern by qRT-PCR, were comparatively analyzed in different tissues and developmental stages of pepper flower. The results showed that the CWIN activities in the big bud and flower of the maintainer line 131 B were significantly higher than those in the male sterile line131 A(P<0.001 and P<0.01, respectively), which were closely correlated with the high expression level of pepper CaCWIN encoding genes CaCWIN3 and CaCWIN4. According to the results, CaCWIN3 and CaCWIN4 were likely the key functional genes involved in the process of male sterility of pepper. These results shed light on the further investigation of molecular regulation mechanism of capsicum male sterility.
引文
戴雄泽,唐冬英.2004.辣椒雄性不育与花蕾内源激素含量变化的关系[J].辣椒杂志,2(1):10-13.(Dai X Z,Tang D Y.2004.Male sterility in relation to endogenous hormone change in buds in chili peppers[J].Journal of China Capsicum,2(1):10-13.)
郭爽,常绍东,黄贞,等.2012.辣椒雄性不育研究进展[J].辣椒杂志,(2):1-5.(Guo S,Chang S D,Huang Z,et al.2012.The research advance of pepper male sterile[J].Journal of China Capsicum,(2):1-5.)
刘科伟,王述彬,刘金兵,等.2009.辣椒细胞质雄性不育基因的AFLP分析[J].分子植物育种,7(4):736-742.(Liu K W,Wang S B,Liu J B,et al.2009.The AFLP analysis of cytoplasmic male sterility gene in pepper[J].Molecular Plant Breeding,7(4):736-742.)
阮美颖,李志邈,程远,等.2016.单性结实与非单性结实樱桃番茄的蔗糖转化酶与相关基因的表达分析[J].农业与生物技术学报,24(2):206-214.(Ruan M Y,Li Z M,Cheng Y,et al.2016.Invertase Activity and related genes expression analysis in parthenocarpic and non-parthenocarpic cherry tomato(Solanum lycopersicum var.cerasiforme)inbred lines[J].Journal of Agriculture and Biotechnology,24(2):206-214.)
孙日飞,方智远,张淑江,等.2000.萝卜胞质大白菜雄性不育系的生化分析[J].园艺学报,27(3):187-192.(Sun R F,Fang Z Y,Zhang S J,et al.2000.Biochemical analysis of coldtolerant ogura CMS Chinese cabbage lines[J].Acta Horticulturae Sinica,27(3):187-192.)
邹学校,侯喜林,刘荣云,等.2004.辣椒细胞质雄性不育基因对不育系及杂交一代农艺性状和生化特性的影响[J].园艺学报,31(6):732-736.(Zou X X,Hou X L,Liu R Y,et al.2004.Effect of cytop lasmic male sterile genes on agronomic and biochemical characters in CMS lines and their F1hybrid in pepper[J].Acta Horticulturae Sinica,31(6):732-736.)
Barratt D H,Derbyshire P,Findlay K,et al.2009.Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase[J].Proceedings of the National Academy of Sciences of the USA,106(31):13124-13129.
Bradford M.1976.A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,72:248-254.
Cheng Y,Ahammed G J,Yu J,et al.2016.Putative WRKYs associated with regulation of fruit ripening revealed by detailed expression analysis of the WRKY gene family in pepper[J].Scientific Reports,6:39000.
Fridman E,Zamir D.2003.Functional divergence of a syntenic invertase gene family in tomato,potato,and Arabidopsis[J].Plant Physiology,131(2):603-609.
Jia L,Zhang B,Mao C,et al.2008.OsCYT-INV1 for alkaline neutral invertase is involved in root cell development and reproductivity in rice(Oryza sativa L.)[J].Planta,228(1)51-59.
Jin Y,Ni D A,Ruan Y L.2009.Posttranslational elevation of cell wall invertase activity by silencing its inhibitor in tomato delays leaf senescence and increases seed weigh and fruit hexose level[J].Plant Cell,21(7):2072-2089.
Letunic I,Doerks T,Bork P,et al.2012.SMART 7:Recent updates to the protein domain annotation resource[J].Nucleic Acids Research,40:D302-305.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T))method[J].Methods,25(4):402-408.
Li Z,Palmer W M,Martin A P,et al.2012.High invertase activity in tomato reproductive organs correlates with enhanced sucrose import into,and heat tolerance of young fruit[J]Journal of Experimental Botany,63(3):1155-1166.
Punta M,Coggill P C,Eberhardt R Y,et al.2008.The Pfam protein families database[J].Nucleic Acids Research,32(1)D138.
Qin C,Yu C,Shen Y,et al.,2014.Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization[J].Proceedings of the National Academy of Sciences of the USA,111(14)5135-5140.
Roy K L,Vergauwen R,Struyf T,et al.2013.Understanding the role of defective invertases in plants:Tobacco Nin88fails to degrade sucrose[J].Plant Physiology,161(4)1670-1681.
Ruan Y L.2014.Sucrose metabolism:Gateway to diverse carbon use and sugar signaling[J].Annual Review of Plan Biology,65(1):33-67.
Ruan Y L,Jin Y,Yang Y J,et al.2010.Sugar input,metabolism,and signaling mediated by invertase:Roles in development,yield potential,and response to drought and heat[J].Molecular Plant,3(6):942-955.
Sturm A.1999.Invertases.Primary structures,functions,and roles in plant development and sucrose partitioning[J].Plant Physiology,121(1):1-8.
Tamura K,Peterson D,Peterson N,et al.2011.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology&Evolution,28(10):2731-2739.
Thompson J D,Gibson T J,Plewniak F,et al.1997.The CLUSTAL_X windows interface:Flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Research,25(25):4876-4882.
Tomlinson K L,McHugh S,Labbe H,et al.2004.Evidence that the hexose-to-sucrose ratio does not control the switch to storage product accumulation in oilseeds:Analysis of tobacco seed development and effects of overexpressing apoplastic invertase[J].Journal of Experimental Botany,55(406):2291-2303.
Wan H J,Yuan W,Ruan M Y,et al.2011.Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepper(Capsicum annuum L.)[J].Biochemical&Biophysical Research Communication,416(1):24-30.
Wang L,Li X R,Lian H,et al.2010.Evidence that high activity of vacuolar invertase is required for cotton fiber and Arabidopsis root elongation through osmotic dependent and independent pathways,respectively[J].Plant Physiology,154(2):744-756.
Zanor M I,Osorio S,Nunes-Nesi A,et al.2009.RNA interference of LIN5 in Solanum lycopersicum confirms its role in controlling Brix content,uncovers the influence of sugars on the levels of fruit hormones and demonstrates the importance of sucrose cleavage for normal fruit development and fertility[J].Plant Physiology,150(3):1204-1218.
郭爽,常绍东,黄贞,等.2012.辣椒雄性不育研究进展[J].辣椒杂志,(2):1-5.(Guo S,Chang S D,Huang Z,et al.2012.The research advance of pepper male sterile[J].Journal of China Capsicum,(2):1-5.)
刘科伟,王述彬,刘金兵,等.2009.辣椒细胞质雄性不育基因的AFLP分析[J].分子植物育种,7(4):736-742.(Liu K W,Wang S B,Liu J B,et al.2009.The AFLP analysis of cytoplasmic male sterility gene in pepper[J].Molecular Plant Breeding,7(4):736-742.)
阮美颖,李志邈,程远,等.2016.单性结实与非单性结实樱桃番茄的蔗糖转化酶与相关基因的表达分析[J].农业与生物技术学报,24(2):206-214.(Ruan M Y,Li Z M,Cheng Y,et al.2016.Invertase Activity and related genes expression analysis in parthenocarpic and non-parthenocarpic cherry tomato(Solanum lycopersicum var.cerasiforme)inbred lines[J].Journal of Agriculture and Biotechnology,24(2):206-214.)
孙日飞,方智远,张淑江,等.2000.萝卜胞质大白菜雄性不育系的生化分析[J].园艺学报,27(3):187-192.(Sun R F,Fang Z Y,Zhang S J,et al.2000.Biochemical analysis of coldtolerant ogura CMS Chinese cabbage lines[J].Acta Horticulturae Sinica,27(3):187-192.)
邹学校,侯喜林,刘荣云,等.2004.辣椒细胞质雄性不育基因对不育系及杂交一代农艺性状和生化特性的影响[J].园艺学报,31(6):732-736.(Zou X X,Hou X L,Liu R Y,et al.2004.Effect of cytop lasmic male sterile genes on agronomic and biochemical characters in CMS lines and their F1hybrid in pepper[J].Acta Horticulturae Sinica,31(6):732-736.)
Barratt D H,Derbyshire P,Findlay K,et al.2009.Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase[J].Proceedings of the National Academy of Sciences of the USA,106(31):13124-13129.
Bradford M.1976.A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,72:248-254.
Cheng Y,Ahammed G J,Yu J,et al.2016.Putative WRKYs associated with regulation of fruit ripening revealed by detailed expression analysis of the WRKY gene family in pepper[J].Scientific Reports,6:39000.
Fridman E,Zamir D.2003.Functional divergence of a syntenic invertase gene family in tomato,potato,and Arabidopsis[J].Plant Physiology,131(2):603-609.
Jia L,Zhang B,Mao C,et al.2008.OsCYT-INV1 for alkaline neutral invertase is involved in root cell development and reproductivity in rice(Oryza sativa L.)[J].Planta,228(1)51-59.
Jin Y,Ni D A,Ruan Y L.2009.Posttranslational elevation of cell wall invertase activity by silencing its inhibitor in tomato delays leaf senescence and increases seed weigh and fruit hexose level[J].Plant Cell,21(7):2072-2089.
Letunic I,Doerks T,Bork P,et al.2012.SMART 7:Recent updates to the protein domain annotation resource[J].Nucleic Acids Research,40:D302-305.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T))method[J].Methods,25(4):402-408.
Li Z,Palmer W M,Martin A P,et al.2012.High invertase activity in tomato reproductive organs correlates with enhanced sucrose import into,and heat tolerance of young fruit[J]Journal of Experimental Botany,63(3):1155-1166.
Punta M,Coggill P C,Eberhardt R Y,et al.2008.The Pfam protein families database[J].Nucleic Acids Research,32(1)D138.
Qin C,Yu C,Shen Y,et al.,2014.Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization[J].Proceedings of the National Academy of Sciences of the USA,111(14)5135-5140.
Roy K L,Vergauwen R,Struyf T,et al.2013.Understanding the role of defective invertases in plants:Tobacco Nin88fails to degrade sucrose[J].Plant Physiology,161(4)1670-1681.
Ruan Y L.2014.Sucrose metabolism:Gateway to diverse carbon use and sugar signaling[J].Annual Review of Plan Biology,65(1):33-67.
Ruan Y L,Jin Y,Yang Y J,et al.2010.Sugar input,metabolism,and signaling mediated by invertase:Roles in development,yield potential,and response to drought and heat[J].Molecular Plant,3(6):942-955.
Sturm A.1999.Invertases.Primary structures,functions,and roles in plant development and sucrose partitioning[J].Plant Physiology,121(1):1-8.
Tamura K,Peterson D,Peterson N,et al.2011.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology&Evolution,28(10):2731-2739.
Thompson J D,Gibson T J,Plewniak F,et al.1997.The CLUSTAL_X windows interface:Flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Research,25(25):4876-4882.
Tomlinson K L,McHugh S,Labbe H,et al.2004.Evidence that the hexose-to-sucrose ratio does not control the switch to storage product accumulation in oilseeds:Analysis of tobacco seed development and effects of overexpressing apoplastic invertase[J].Journal of Experimental Botany,55(406):2291-2303.
Wan H J,Yuan W,Ruan M Y,et al.2011.Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepper(Capsicum annuum L.)[J].Biochemical&Biophysical Research Communication,416(1):24-30.
Wang L,Li X R,Lian H,et al.2010.Evidence that high activity of vacuolar invertase is required for cotton fiber and Arabidopsis root elongation through osmotic dependent and independent pathways,respectively[J].Plant Physiology,154(2):744-756.
Zanor M I,Osorio S,Nunes-Nesi A,et al.2009.RNA interference of LIN5 in Solanum lycopersicum confirms its role in controlling Brix content,uncovers the influence of sugars on the levels of fruit hormones and demonstrates the importance of sucrose cleavage for normal fruit development and fertility[J].Plant Physiology,150(3):1204-1218.