基于cDNA-AFLP分析桂花开花进程中差异表达基因
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摘要
桂花是我国重要的传统名花香花植物,花色与花香是桂花两个重要的观赏品质,受开花进程的影响。为了解其开花进程中色与香的合成过程及相关分子基础,该研究利用不同花期的桂花花瓣和幼叶建立了适用于多个样本的cDNA-AFLP体系,并分析开花进程中基因的表达差异。结果表明:共获得了283个在花瓣中特异且差异表达的转录衍生片段(transcript-derived fragments,TDFs)。其中,120个TDFs在数据库中无同源序列; 12个有同源序列,但功能未知; 150个已知生物学功能的序列,其主要功能包括次级代谢、初级代谢和发育过程等。对其中已知功能的6个TDFs进行qRT-PCR验证,4个转录水平较高的TDFs表达模式与AFLP分析结果基本一致。这对了解开花进程中桂花色与香的合成过程相关基因的表达奠定了基础,也为进一步研究桂花色香形成的分子机制提供了参考。
Osmanthus fragrans is an important traditional fragrant flower in China. Floral color and scent as the important ornamental characters are influenced by flowering process. Reports on the components of floral color and scent and related synthetic genes have been reported. However,studies on the synthesis and molecular basis of floral color and scent during flowering process are rarely reported. The study established a c DNA-AFLP system suitable for multiple samples and analyzed genes differential expression at different flowering stages. Finally,283 TDFs with specific expression in petals and differential expression during flowering were successfully cloned and sequenced. Public databases blast showed that 120 TDFs had no homologous sequences,12 TDFs had homologous sequences but their functions were unknown,and 150 TDFs had known biological functions,including the secondary metabolism,primary metabolism and development process,ect. qRT-PCR results of 6 TDFs with known functions comparing with c DNA-AFLP displayed that 4 TDFs with higher transcriptional level had consistent expression level. These results would be helpful to understand the gene expression of petals during flowering processes,and provide useful information for exploiting the mechanism of the floral color and scent formation of O. fragrans from molecular level.
Osmanthus fragrans is an important traditional fragrant flower in China. Floral color and scent as the important ornamental characters are influenced by flowering process. Reports on the components of floral color and scent and related synthetic genes have been reported. However,studies on the synthesis and molecular basis of floral color and scent during flowering process are rarely reported. The study established a c DNA-AFLP system suitable for multiple samples and analyzed genes differential expression at different flowering stages. Finally,283 TDFs with specific expression in petals and differential expression during flowering were successfully cloned and sequenced. Public databases blast showed that 120 TDFs had no homologous sequences,12 TDFs had homologous sequences but their functions were unknown,and 150 TDFs had known biological functions,including the secondary metabolism,primary metabolism and development process,ect. qRT-PCR results of 6 TDFs with known functions comparing with c DNA-AFLP displayed that 4 TDFs with higher transcriptional level had consistent expression level. These results would be helpful to understand the gene expression of petals during flowering processes,and provide useful information for exploiting the mechanism of the floral color and scent formation of O. fragrans from molecular level.
引文
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BALDERMANN S,KATO M,KUROSAWA M,et al.,2016.Functional characterization of a carotenoid cleavage dioxygenase 1 and its relation to the carotenoid accumulation and volatile emission during the floral development of Osmanthus fragrans Lour[J].J Exp Bot,61(11):2967-2977.
CAI X,SU F,JIN HX,et al.,2010.Components and extraction methods for petal pigments of Osmanthus fragrans‘Siji Gui’[J].J Zhejiang For Coll,27(4):559-564.[蔡璇,苏蘩,金荷仙,等,2010.四季桂花瓣色素的初步鉴定与提取方法[J].浙江林学院学报,27(4):559-564.]
CAI X,MAI RZ,ZOU JJ,et al.,2014.Analysis of aroma-active compounds in three sweet osmanthus(Osmanthus fragrans)cultivars by gas-chromatography-olfactometry and GC-mass spectrometry[J].J Zhejiang Univ Sci B,15(7):638-648.
DUDAREVA N,KLEMPIEN A,MUHLEMANN JK,et al.,2013.Biosynthesis,function and metabolic engineering of plant volatile organic compounds[J].New Phytol,198(1):16-32.
GONG MJ,TIAN M,WANG CX,2011.Establishment of c DNA-AFLP system in Oncidium Orchid and TDFs analysis[J].J Nucl Agric Sci,25(6):1142-1147.[龚茂江,田敏,王彩霞,2011.文心兰c DNA-AFLP体系的建立及TDFs分析[J].核农学报,25(6):1142-1147.]
HAN B,PENG JY,2006.c DNA-AFLP and its application in research about gene expressions of plants[J].Acta Bot Boreal-Occident Sin,26(8):1753-1758.[韩斌,彭建营,2006.c DNA-AFLP技术及其在植物基因表达研究中的应用[J].西北植物学报,26(8):1753-1758.]
HAN Y,WANG X,CHEN W,et al.,2014.Differential expression of carotenoid-related genes determines diversified carotenoid coloration in flower petal of Osmanthus fragrans[J].Tree Genet Genomes,10:329-338.
HAN Y,CHEN W,YANG F,et al.,2015.c DNA-AFLP analysis on 2 Osmanthus fragrans cultivars with different flower color and molecular characteristics of OfMYB1 gene[J].Trees,29(3):931-940.
HOU D,2014.Analysis of floral scent and pigment constituents and the reflectance to temperature fluctuation in Osmanthus fragrans(Thunb.)Lour.[D].Hangzhou:Zhejiang University of Agriculture and Forestry.[侯丹,2014.桂花主要品种花香与花色及其温度变化的响应[D].杭州:浙江农林大学.]
HUANG FC,MOLNR P,SCHWABW,2009.Cloning and functional characterization of carotenoid cleavage dioxygenase4 genes[J].J Exp Bot,60(11):3011-3022.
LENG CX,LI FG,CHEN GY,et al.,2007.c DNA-AFLP analysis of somatic embryogenesis at early stage in TM-1(Gossypium hirsutum L.)[J].Acta Bot Boreal-Occident Sin,27(2):233-237.[冷春旭,李付广,陈国跃,等,2007.陆地棉体细胞胚胎发生过程的CDNA-AFLP分析[J].西北植物学报,27(2):233-237.]
LI ZH,TANG ML,LIU J,et al.,2008.Establishment of c D-NA-AFLP system of Malus zumi Mats[J].J Nucl Agric Sci,22(5):607-610.[李志红,唐美玲,刘佳,等,2008.珠眉海棠c DNA-AFLP分析体系的建立[J].核农学报,22(5):607-610.]
REN HY,SUN X,ZHENG CS,et al.,2011.Differential analysis of flowering related genes by c DNA-AFLP in Chrysanthemum[J].Agric Sci Chin,44(16):3386-3394.[任洪艳,孙霞,郑成淑,等,2011.利用c DNA-AFLP技术筛选菊花开花相关基因[J].中国农业科学,44(16):3386-3394.]
SUN XM,YANG PP,LU XJ,et al.,2015.Establishment of c DNA-AFLP system of Paeonia lactiflora seed[J].Acta Hortic Sin,42(3):576-584.[孙晓梅,杨盼盼,陆秀君,等,2015.芍药种子c DNA-AFLP体系的建立[J].园艺学报,42(3):576-584.]
SUN YD,ZHANG XG,LUO WR,et al.,2008.Clone and analysis of expansin gene of cucumber fruit[J].Acta Bot Boreal-Occident Sin,28(2):0233-0236.[孙涌栋,张兴国,罗未蓉,等,2008.黄瓜果实扩张蛋白基因克隆与分析[J].西北植物学报,28(2):233-236.]
TANAKA Y,SASAKI N,OHMIYA A,2008.Biosynthesis of plant pigments:Anthocyanins,betalains and carotenoids[J].Plant J,54:733-749.
WANG JF,2009.Research of differential gene expression in the developing seed of Brassica napus under high temperatures stress[D].Chongqing:Southwest University.[王家丰,2009.高温胁迫下甘蓝型油菜发育种子相关基因的差异表达研究[D].重庆:西南大学.]
XIANG QB,LIU YL,2008.An illustrated monograph of the sweet osmanthus cultivars in China[M].Hangzhou:Zhejiang Science&Technology Press.[向其柏,刘玉莲,2008.中国桂花品种图志[M].杭州:浙江科学技术出版社.]
XIAO XF,WANG H,HUANG M,et al.,2013.Optimization and establishment of c DNA-AFLP analysis system for flowering Chinese cabbage[J].Acta Agric Univ Jiangxi,35(2):296-300.[肖旭峰,王恒,黄敏,等,2013.菜心c DNA-AFLP分析体系的优化与建立[J].江西农业大学学报,35(2):296-300.]
XIN H,WU B,ZHANG H,et al.,2013.Characterization of volatile compounds in flowers from four groups of sweet osmanthus(Osmanthus fragrans)cultivars[J].Can J Plant Sci,93:923-931.
YANG KM,2011.Chinese Osmanthus[M].Beijing:China Forestry Publishing House.[杨康民,2011.中国桂花[M].北京:中国林业出版社.]
ZENG X,LIU C,ZHENG R,et al.,2015.Emission and accumμlation of monoterpene and the key terpene synthase(TPS)associated with monoterpene biosynthesis in Osmanthus fragrans Lour[J].Front Plant Sci,6:1232.doi:,10.3389/fpls.2015.01232.
ZENG XL,ZHENG RR,LUO J,et al.,2016.Cloning and characterization of cinnamate 4-hydroxylase(C4H)genes from Osmanthus fragrans[J].Acta Hortic Sin,43(3):525-537.[曾祥玲,郑日如,罗靖,等,2016.桂花C4H基因的克隆与表达特性分析[J].园艺学报,43(3):525-537.]
ZHANG Y,2009.Separation of fragrance-related genes of Osmanthus fragrans by using c DNA-AFLP technology[D].Fuzhou:Fujian Agriculture and Forestry University.[张园,2009.c DNA-AFLP技术分离桂花香气相关基因[D].福州:福建农林大学.]
ZOU JJ,ZENG XL,CHEN HG,et al.,2017.Analysis on characteristic color compounds in different varieties of Osmanthus fragrans Lour.during flowering and senescence[J].J S Agric,48(9):1683-1690.[邹晶晶,曾祥玲,陈洪国,等,2017.不同桂花品种开花及衰老过程中的花色物质成分分析[J].南方农业学报,48(9):1683-1690.
BALDERMANN S,KATO M,KUROSAWA M,et al.,2016.Functional characterization of a carotenoid cleavage dioxygenase 1 and its relation to the carotenoid accumulation and volatile emission during the floral development of Osmanthus fragrans Lour[J].J Exp Bot,61(11):2967-2977.
CAI X,SU F,JIN HX,et al.,2010.Components and extraction methods for petal pigments of Osmanthus fragrans‘Siji Gui’[J].J Zhejiang For Coll,27(4):559-564.[蔡璇,苏蘩,金荷仙,等,2010.四季桂花瓣色素的初步鉴定与提取方法[J].浙江林学院学报,27(4):559-564.]
CAI X,MAI RZ,ZOU JJ,et al.,2014.Analysis of aroma-active compounds in three sweet osmanthus(Osmanthus fragrans)cultivars by gas-chromatography-olfactometry and GC-mass spectrometry[J].J Zhejiang Univ Sci B,15(7):638-648.
DUDAREVA N,KLEMPIEN A,MUHLEMANN JK,et al.,2013.Biosynthesis,function and metabolic engineering of plant volatile organic compounds[J].New Phytol,198(1):16-32.
GONG MJ,TIAN M,WANG CX,2011.Establishment of c DNA-AFLP system in Oncidium Orchid and TDFs analysis[J].J Nucl Agric Sci,25(6):1142-1147.[龚茂江,田敏,王彩霞,2011.文心兰c DNA-AFLP体系的建立及TDFs分析[J].核农学报,25(6):1142-1147.]
HAN B,PENG JY,2006.c DNA-AFLP and its application in research about gene expressions of plants[J].Acta Bot Boreal-Occident Sin,26(8):1753-1758.[韩斌,彭建营,2006.c DNA-AFLP技术及其在植物基因表达研究中的应用[J].西北植物学报,26(8):1753-1758.]
HAN Y,WANG X,CHEN W,et al.,2014.Differential expression of carotenoid-related genes determines diversified carotenoid coloration in flower petal of Osmanthus fragrans[J].Tree Genet Genomes,10:329-338.
HAN Y,CHEN W,YANG F,et al.,2015.c DNA-AFLP analysis on 2 Osmanthus fragrans cultivars with different flower color and molecular characteristics of OfMYB1 gene[J].Trees,29(3):931-940.
HOU D,2014.Analysis of floral scent and pigment constituents and the reflectance to temperature fluctuation in Osmanthus fragrans(Thunb.)Lour.[D].Hangzhou:Zhejiang University of Agriculture and Forestry.[侯丹,2014.桂花主要品种花香与花色及其温度变化的响应[D].杭州:浙江农林大学.]
HUANG FC,MOLNR P,SCHWABW,2009.Cloning and functional characterization of carotenoid cleavage dioxygenase4 genes[J].J Exp Bot,60(11):3011-3022.
LENG CX,LI FG,CHEN GY,et al.,2007.c DNA-AFLP analysis of somatic embryogenesis at early stage in TM-1(Gossypium hirsutum L.)[J].Acta Bot Boreal-Occident Sin,27(2):233-237.[冷春旭,李付广,陈国跃,等,2007.陆地棉体细胞胚胎发生过程的CDNA-AFLP分析[J].西北植物学报,27(2):233-237.]
LI ZH,TANG ML,LIU J,et al.,2008.Establishment of c D-NA-AFLP system of Malus zumi Mats[J].J Nucl Agric Sci,22(5):607-610.[李志红,唐美玲,刘佳,等,2008.珠眉海棠c DNA-AFLP分析体系的建立[J].核农学报,22(5):607-610.]
REN HY,SUN X,ZHENG CS,et al.,2011.Differential analysis of flowering related genes by c DNA-AFLP in Chrysanthemum[J].Agric Sci Chin,44(16):3386-3394.[任洪艳,孙霞,郑成淑,等,2011.利用c DNA-AFLP技术筛选菊花开花相关基因[J].中国农业科学,44(16):3386-3394.]
SUN XM,YANG PP,LU XJ,et al.,2015.Establishment of c DNA-AFLP system of Paeonia lactiflora seed[J].Acta Hortic Sin,42(3):576-584.[孙晓梅,杨盼盼,陆秀君,等,2015.芍药种子c DNA-AFLP体系的建立[J].园艺学报,42(3):576-584.]
SUN YD,ZHANG XG,LUO WR,et al.,2008.Clone and analysis of expansin gene of cucumber fruit[J].Acta Bot Boreal-Occident Sin,28(2):0233-0236.[孙涌栋,张兴国,罗未蓉,等,2008.黄瓜果实扩张蛋白基因克隆与分析[J].西北植物学报,28(2):233-236.]
TANAKA Y,SASAKI N,OHMIYA A,2008.Biosynthesis of plant pigments:Anthocyanins,betalains and carotenoids[J].Plant J,54:733-749.
WANG JF,2009.Research of differential gene expression in the developing seed of Brassica napus under high temperatures stress[D].Chongqing:Southwest University.[王家丰,2009.高温胁迫下甘蓝型油菜发育种子相关基因的差异表达研究[D].重庆:西南大学.]
XIANG QB,LIU YL,2008.An illustrated monograph of the sweet osmanthus cultivars in China[M].Hangzhou:Zhejiang Science&Technology Press.[向其柏,刘玉莲,2008.中国桂花品种图志[M].杭州:浙江科学技术出版社.]
XIAO XF,WANG H,HUANG M,et al.,2013.Optimization and establishment of c DNA-AFLP analysis system for flowering Chinese cabbage[J].Acta Agric Univ Jiangxi,35(2):296-300.[肖旭峰,王恒,黄敏,等,2013.菜心c DNA-AFLP分析体系的优化与建立[J].江西农业大学学报,35(2):296-300.]
XIN H,WU B,ZHANG H,et al.,2013.Characterization of volatile compounds in flowers from four groups of sweet osmanthus(Osmanthus fragrans)cultivars[J].Can J Plant Sci,93:923-931.
YANG KM,2011.Chinese Osmanthus[M].Beijing:China Forestry Publishing House.[杨康民,2011.中国桂花[M].北京:中国林业出版社.]
ZENG X,LIU C,ZHENG R,et al.,2015.Emission and accumμlation of monoterpene and the key terpene synthase(TPS)associated with monoterpene biosynthesis in Osmanthus fragrans Lour[J].Front Plant Sci,6:1232.doi:,10.3389/fpls.2015.01232.
ZENG XL,ZHENG RR,LUO J,et al.,2016.Cloning and characterization of cinnamate 4-hydroxylase(C4H)genes from Osmanthus fragrans[J].Acta Hortic Sin,43(3):525-537.[曾祥玲,郑日如,罗靖,等,2016.桂花C4H基因的克隆与表达特性分析[J].园艺学报,43(3):525-537.]
ZHANG Y,2009.Separation of fragrance-related genes of Osmanthus fragrans by using c DNA-AFLP technology[D].Fuzhou:Fujian Agriculture and Forestry University.[张园,2009.c DNA-AFLP技术分离桂花香气相关基因[D].福州:福建农林大学.]
ZOU JJ,ZENG XL,CHEN HG,et al.,2017.Analysis on characteristic color compounds in different varieties of Osmanthus fragrans Lour.during flowering and senescence[J].J S Agric,48(9):1683-1690.[邹晶晶,曾祥玲,陈洪国,等,2017.不同桂花品种开花及衰老过程中的花色物质成分分析[J].南方农业学报,48(9):1683-1690.