桂花扩展蛋白基因OfEXPA2、OfEXPA4和OfEXLA1启动子克隆及活性分析
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摘要
为研究扩展蛋白基因在桂花花开放过程中的作用机制,利用染色体步移法,从桂花(Osmanthus fragrans)中克隆得到花开放相关基因OfEXPA2、OfEXPA4和OfEXLA1起始密码子上游1 108、808、945 bp的启动子序列。利用在线数据库Plantcare预测启动子中的顺式作用元件,发现这3个启动子序列中均存在基本元件TATA盒和CAAT盒,还含有脱落酸、生长素、水杨酸等激素诱导元件及响应干旱、高温等多个与植物非生物胁迫相关的元件,如MBS、HSE。分别将这3个启动子与β-葡萄糖苷酸酶(β-glucuronidase, GUS)报告基因融合进行瞬时表达,结果显示:OfEXPA2、OfEXPA4和OfEXLA1的启动子均能够驱动GUS基因在转基因烟草叶片中的表达。
Osmanthus fragrans is not only one of the top ten traditional famous flowers of our country, but also one of the famous greening and perfume plants in China. In order to study the flower opening mechanism of O. fragrans,expansin genes were identified and selected from the obtained transcriptome of O. fragrans. Three expansin genes,Of EXPA2, Of EXPA4 and Of EXLA1, were performed, which contributed to flower opening mechanism of O. fragrans.Promoters of the three genes were cloned by the method of Genome Walker. The lengths of 1 108, 808 and 945 bp(OfEXPA2, OfEXPA4 and OfEXLA1) were obtained from O. fragrans, respectively. Online database‘Plantcare'analysis revealed that the sequences of the three promoters contained basic cis-elements, such as TATA-box and CAATbox. In addition, the promoter of Of EXPA2 contained hormone inducing elements ABRE, and the promoter of Of EXLA1 had Aux RR and TCA, indicating that the two genes would be regulated by corresponding hormones. Besides, there were many other elements involved in the plant abiotic stress. MBS, a cis-acting element in the promoter of Of EXPA2,was the MYB binding site, which responded to drought stress; there was a cis-acting element that responded to high temperature, HSE, in the promoter of OfEXLA1. In addition, the recombinant vectors were constructed and named Of EXPA2:: GUS, OfEXPA4:: GUS and OfEXLA1:: GUS, respectively. The result indicated that the promoters of Of EXPA2, Of EXPA4 and Of EXLA1 could drive the GUS gene exclusively to express in leaves of transgenic tobacco.
Osmanthus fragrans is not only one of the top ten traditional famous flowers of our country, but also one of the famous greening and perfume plants in China. In order to study the flower opening mechanism of O. fragrans,expansin genes were identified and selected from the obtained transcriptome of O. fragrans. Three expansin genes,Of EXPA2, Of EXPA4 and Of EXLA1, were performed, which contributed to flower opening mechanism of O. fragrans.Promoters of the three genes were cloned by the method of Genome Walker. The lengths of 1 108, 808 and 945 bp(OfEXPA2, OfEXPA4 and OfEXLA1) were obtained from O. fragrans, respectively. Online database‘Plantcare'analysis revealed that the sequences of the three promoters contained basic cis-elements, such as TATA-box and CAATbox. In addition, the promoter of Of EXPA2 contained hormone inducing elements ABRE, and the promoter of Of EXLA1 had Aux RR and TCA, indicating that the two genes would be regulated by corresponding hormones. Besides, there were many other elements involved in the plant abiotic stress. MBS, a cis-acting element in the promoter of Of EXPA2,was the MYB binding site, which responded to drought stress; there was a cis-acting element that responded to high temperature, HSE, in the promoter of OfEXLA1. In addition, the recombinant vectors were constructed and named Of EXPA2:: GUS, OfEXPA4:: GUS and OfEXLA1:: GUS, respectively. The result indicated that the promoters of Of EXPA2, Of EXPA4 and Of EXLA1 could drive the GUS gene exclusively to express in leaves of transgenic tobacco.
引文
[1]王英,张超,付建新,等.桂花花芽分化和花开放研究进展.浙江农林大学学报,2016,33(2):340-347.WANG Y,ZHANG C,FU J X,et al.Progresses on flower bud differentiation and flower opening in Osmanthus fragrans.Journal of Zhejiang A&F University,2016,33(2):340-347.(in Chinese with English abstract)
[2]VAN DOORN W G,VAN M U.Flower opening and closure:A review.Journal of Experimental Botany,2003,54(389):1801-1812.
[3]MCQUEEN-MASON S,COSGROVE D J.Disruption of hydrogen bonding between plant cell wall polymers by proteins that induce wall extension.Proceedings of the National Academy of Sciences of the United States of America,1994,91(14):6574-6578.
[4]KENDE H,BRADFORD K,BRUMMELL D,et al.Nomen‐clature for members of the expansin superfamily of genes and proteins.Plant Molecular Biology,2004,55(3):311-314.
[5]YAN A,WU M J,YAN L M,et al.AtEXP2 is involved in seed germination and abiotic stress response in Arabidopsis.PLoS One,2014,9(1):e85208.
[6]ZOU H Y,WENWEN Y H,ZANG G C,et al.OsEXPB2,aβ-expansin gene,is involved in rice root system architecture.Molecular Breeding,2015,35:41.
[7]XING S C,LI F,GUO Q F,et al.The involvement of an expansin gene TaEXPB23from wheat in regulating plant cell growth.Biologia Plantarum,2009,53(3):429-434.
[8]GOH H H.The role of expansin in leaf development:Amolecular genetics and AFM approach.Sheffield,UK:The University of Sheffield,2011.
[9]VALDIVIAE R,STEPHENSONAG,DURACHKO D M,et al.Class Bβ-expansins are needed for pollen separation and stigma penetration.Sexual Plant Reproduction,2009,22(3):141-152.
[10]PALAPOL Y,KUNYAMEE S,THONGKHUM M,et al.Expression of expansin genes in the pulp and the dehiscence zone of ripening durian(Durio zibethinus)fruit.Journal of Plant Physiology,2015,182:33-39.
[11]AZEEZ A,SANE A P,TRIPATHI S K,et al.The gladiolus GgEXPA1 is a GA-responsive alpha-expansin gene expressed ubiquitously during expansion of all floral tissues and leaves but repressed during organ senescence.Postharvest Biology&Technology,2010,58(1):48-56.
[12]HARADA T,TORII Y,MORITA S,et al.Cloning,characterization,and expression of xyloglucan endotransglucosylase/hydrolase and expansin genes associated with petal growth and development during carnation flower opening.Journal of Experimental Botany,2010,62(2):815-823.
[13]MA J,LI Z,WANG B,et al.Cloning of an expansin gene from Chimonanthus praecox flowers and its expression in flowers treated with ethephon or 1-methylcyclopropene.HortScience,2012,47(10):1472-1477.
[14]罗云,张超,付建新,等.桂花扩展蛋白基因家族的鉴定和表达分析.农业生物技术学报,2017,25(8):1289-1299.LUO Y,ZHANG C,FU J X,et al.Identification and expression analysis of expansin gene family in Osmanthus fragrans.Journal of Agricultural Biotechnology,2017,25(8):1289-1299.(in Chinese with English abstract)
[15]周小琼,丁一琼,左丽,等.大豆硫转运蛋白基因GmSULTR1;2b启动子的克隆及活性分析.中国农业科学,2015,48(8):1650-1659.ZHOU X Q,DING Y Q,ZUO L,et al.Cloning and activity analysis of the promoter of sulfate transporter gene GmSULTR1;2b.Scientia Agricultura Sinica,2015,48(8):1650-1659.(in Chinese with English abstract)
[16]YAMAGUCHI-SHINOZAKI K,SHINOZAKI K.Organization of cis-acting regulatory elements in osmotic-and cold-stress-responsive promoters.Trends in Plant Science,2005,10(2):88-94.
[17]TRIVEDI P K,NATH P.MaExp1,an ethylene-induced expansin from ripening banana fruit.Plant Science,2004,167(6):1351-1358.
[18]LI Y,TU L L,YE Z X,et al.A cotton fiber-preferential promoter,PGbEXPA2,is regulated by GA and ABA in Arabidopsis.Plant Cell Reports,2015,34(9):1539-1549.
[19]ZHAO M R,HAN Y Y,FENG Y N,et al.Expansins are involved in cell growth mediated by abscisic acid and indole-3-acetic acid under drought stress in wheat.Plant Cell Reports,2012,31(4):671-685.
[20]OCHIAI M,MATSUMOTO S,YAMADA K.Methyl jasmonate treatment promotes flower opening of cut Eustoma by inducing cell wall loosening proteins in petals.Postharvest Biology&Technology,2013,82:1-5.
[21]李娟.水稻温度诱导启动子POsUAH和POsACO1的结构与功能研究.北京:中国科学院大学,2014:20-24.LI J.Function analysis of two rice temperature-inducible promoters POsUAH and POsACO1.Beijing:University of Chinese Academy of Sciences,2014:20-24.(in Chinese with English abstract)
[22]AMBAWAT S,SHARMA P,YADAV N R,et al.MYBtranscription factor genes as regulators for plant responses:an overview.Physiology and Molecular Biology of Plants,2013,19(3):307-321.
[23]STRACKE R,ISHIHARA H,HUEP G,et al.Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling.The Plant Journal,2007,50(4):660-677.
[24]HUANG W J,SUN W,LüH Y,et al.A R2R3-MYBtranscription factor from Epimedium sagittatum regulates the flavonoid biosynthetic pathway.PLo S One,2013,8(8):e70778.
[25]COMINELLI E,GALBIATI M,VAVASSEUR A,et al.Aguard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance.Current Biology,2005,15(13):1196-1200.
[26]SEO P J,XIANG F N,QIAO M,et al.The MYB96transcription factor mediates abscisic acid signaling during drought stress response in Arabidopsis.Plant Physiology,2009,151(1):275-289.
[2]VAN DOORN W G,VAN M U.Flower opening and closure:A review.Journal of Experimental Botany,2003,54(389):1801-1812.
[3]MCQUEEN-MASON S,COSGROVE D J.Disruption of hydrogen bonding between plant cell wall polymers by proteins that induce wall extension.Proceedings of the National Academy of Sciences of the United States of America,1994,91(14):6574-6578.
[4]KENDE H,BRADFORD K,BRUMMELL D,et al.Nomen‐clature for members of the expansin superfamily of genes and proteins.Plant Molecular Biology,2004,55(3):311-314.
[5]YAN A,WU M J,YAN L M,et al.AtEXP2 is involved in seed germination and abiotic stress response in Arabidopsis.PLoS One,2014,9(1):e85208.
[6]ZOU H Y,WENWEN Y H,ZANG G C,et al.OsEXPB2,aβ-expansin gene,is involved in rice root system architecture.Molecular Breeding,2015,35:41.
[7]XING S C,LI F,GUO Q F,et al.The involvement of an expansin gene TaEXPB23from wheat in regulating plant cell growth.Biologia Plantarum,2009,53(3):429-434.
[8]GOH H H.The role of expansin in leaf development:Amolecular genetics and AFM approach.Sheffield,UK:The University of Sheffield,2011.
[9]VALDIVIAE R,STEPHENSONAG,DURACHKO D M,et al.Class Bβ-expansins are needed for pollen separation and stigma penetration.Sexual Plant Reproduction,2009,22(3):141-152.
[10]PALAPOL Y,KUNYAMEE S,THONGKHUM M,et al.Expression of expansin genes in the pulp and the dehiscence zone of ripening durian(Durio zibethinus)fruit.Journal of Plant Physiology,2015,182:33-39.
[11]AZEEZ A,SANE A P,TRIPATHI S K,et al.The gladiolus GgEXPA1 is a GA-responsive alpha-expansin gene expressed ubiquitously during expansion of all floral tissues and leaves but repressed during organ senescence.Postharvest Biology&Technology,2010,58(1):48-56.
[12]HARADA T,TORII Y,MORITA S,et al.Cloning,characterization,and expression of xyloglucan endotransglucosylase/hydrolase and expansin genes associated with petal growth and development during carnation flower opening.Journal of Experimental Botany,2010,62(2):815-823.
[13]MA J,LI Z,WANG B,et al.Cloning of an expansin gene from Chimonanthus praecox flowers and its expression in flowers treated with ethephon or 1-methylcyclopropene.HortScience,2012,47(10):1472-1477.
[14]罗云,张超,付建新,等.桂花扩展蛋白基因家族的鉴定和表达分析.农业生物技术学报,2017,25(8):1289-1299.LUO Y,ZHANG C,FU J X,et al.Identification and expression analysis of expansin gene family in Osmanthus fragrans.Journal of Agricultural Biotechnology,2017,25(8):1289-1299.(in Chinese with English abstract)
[15]周小琼,丁一琼,左丽,等.大豆硫转运蛋白基因GmSULTR1;2b启动子的克隆及活性分析.中国农业科学,2015,48(8):1650-1659.ZHOU X Q,DING Y Q,ZUO L,et al.Cloning and activity analysis of the promoter of sulfate transporter gene GmSULTR1;2b.Scientia Agricultura Sinica,2015,48(8):1650-1659.(in Chinese with English abstract)
[16]YAMAGUCHI-SHINOZAKI K,SHINOZAKI K.Organization of cis-acting regulatory elements in osmotic-and cold-stress-responsive promoters.Trends in Plant Science,2005,10(2):88-94.
[17]TRIVEDI P K,NATH P.MaExp1,an ethylene-induced expansin from ripening banana fruit.Plant Science,2004,167(6):1351-1358.
[18]LI Y,TU L L,YE Z X,et al.A cotton fiber-preferential promoter,PGbEXPA2,is regulated by GA and ABA in Arabidopsis.Plant Cell Reports,2015,34(9):1539-1549.
[19]ZHAO M R,HAN Y Y,FENG Y N,et al.Expansins are involved in cell growth mediated by abscisic acid and indole-3-acetic acid under drought stress in wheat.Plant Cell Reports,2012,31(4):671-685.
[20]OCHIAI M,MATSUMOTO S,YAMADA K.Methyl jasmonate treatment promotes flower opening of cut Eustoma by inducing cell wall loosening proteins in petals.Postharvest Biology&Technology,2013,82:1-5.
[21]李娟.水稻温度诱导启动子POsUAH和POsACO1的结构与功能研究.北京:中国科学院大学,2014:20-24.LI J.Function analysis of two rice temperature-inducible promoters POsUAH and POsACO1.Beijing:University of Chinese Academy of Sciences,2014:20-24.(in Chinese with English abstract)
[22]AMBAWAT S,SHARMA P,YADAV N R,et al.MYBtranscription factor genes as regulators for plant responses:an overview.Physiology and Molecular Biology of Plants,2013,19(3):307-321.
[23]STRACKE R,ISHIHARA H,HUEP G,et al.Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling.The Plant Journal,2007,50(4):660-677.
[24]HUANG W J,SUN W,LüH Y,et al.A R2R3-MYBtranscription factor from Epimedium sagittatum regulates the flavonoid biosynthetic pathway.PLo S One,2013,8(8):e70778.
[25]COMINELLI E,GALBIATI M,VAVASSEUR A,et al.Aguard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance.Current Biology,2005,15(13):1196-1200.
[26]SEO P J,XIANG F N,QIAO M,et al.The MYB96transcription factor mediates abscisic acid signaling during drought stress response in Arabidopsis.Plant Physiology,2009,151(1):275-289.