草莓AP2/ERF转录因子AD-cDNA文库的构建
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摘要
【目的】探究AP2/ERF家族成员在果实成熟过程中发挥的作用。【方法】利用CTAB法提取栽培草莓(Fragaria×ananassa)品种‘越心’的果实RNA,逆转录第1链c DNA;根据AP2 DNA-binding domain序列信息利用CDART工具在Genbank中获得草莓中所有AP2/ERF家族成员的序列信息,设计基因编码区全长引物并获得所有家族成员全长序列;将AP2/ERF家族成员基因全长序列从测序载体p GEM-T easy转移至p GADT7载体上,转化至大肠杆菌并保存菌株,构建完整的AP2/ERF AD-c DNA文库;参照YeastmakerTMyeast transformation system2(Clontech)说明书,利用Dk PDC2启动子进行文库筛选和单一验证。【结果】从数据库获得了草莓(Fragaria×ananassa)基因组120个非冗余AP2/ERF家族成员基因全长序列,通过设计的7条引物将AP2/ERF家族成员基因全长序列从测序载体p GEM-T easy转移至p GADT7载体上,转化至大肠杆菌并保存菌株,构建了完整的AP2/ERF的AD-c DNA文库。利用已报道能与Dk ERF19互作的柿子基因Dk PDC2启动子对该文库进行酵母单杂交筛选,获得了2个草莓AP2/ERF家族成员Fa ERF#83和Fa ERF#87,且证明了两者均能与Dk PDC2启动子互作。【结论】成功构建了一个只含有草莓AP2/ERF转录因子全长序列的AD-c DNA文库,用Dk PDC2启动子进行筛选获得了Dk ERF19直系同源基因Fa ERF#87和同亚家族基因Fa ERF#83,证明了该文库的有效性,也为进一步研究潜在的AP2/ERF调控提供了手段。
【Objective】As one of the most numerous transcription factors in plants, the members of AP2/ERF superfamily were reported extensively to be involved both in the regulation of the process of growth and development in plant, and in the regulation of ripening process and accompanying physiological changes of fruit. Rosaceae is of great importance to be investigated, because it consists of many economically important fruits such as apple, peach, pear and strawberry. But compared with the total amount of numbers of AP2/ERF superfamily in Rosaceae, the quantity of the members reported to be functional in the process of fruit development and ripening is too small, which means large work needs to do before a comprehensive understanding of the role of AP2/ERF in fruits' development and ripening process is made. To investigate the potential targets of transcription factors like AP2/ERF, Y1 H assay is one of the most effective methods to use. It is a rapid and efficient way to build an AD-c DNA library only consisting of transcription factors to be screened, which reduces the false-positive rate and great labor when using a traditional c DNA library instead.【Methods】With the use of CTAB, RNA was extracted from strawberry(Fragaria × ananassa)‘Yuexin'fruits, which was then used as template in the process of reverse transcription for the synthesis of the first strand c DNA. With the help of bioinformatics, the details of the sequences of the members of AP2/ERF superfamily in strawberry were ob-tained, according to which the special primers of each member were designed. Each CDS(coding sequence) was cloned to p GEM-T easy vector for sequencing. After the plasmid profiles of p GEM-T easy and p GADT7 vector were compared, seven special primers were designed to transfer the CDS from p GEM-T easy to p GADT7 vector, which was then transformed to E. coli and sequenced. The strains containing the CDS of AP2/ERF family members were preserved with 50% glycerol separately at-80 ℃ before use. To avoid the potential loss of certain plasmids when all of the strains were mixed to proliferate and extracted, each strain was cultured and extracted separately. The plasmids were mixed with the same molar quantity for completing the construction of the library. To verify the effectiveness of Fa ERFAD library, the promoter of Dk PDC2 which was reported to interact with an AP2/ERF transcription factor Dk ERF19, was screened by Y1 H assay with the use of the library. The detail process of Y1 H assay refered to YeastmakerTMYeast Transformation System 2 User Manual(Clontech) and MatchmakerTM Gold Yeast One-Hybrid Library Screening System User Manual(Clontech). After cultivation of 3 days,the colonies of Y1 H transformants were recorded and photographed.【Results】120 items of AP2/ERF in strawberry were obtained from DNA nucleotide database of NCBI after repeat records were omitted.Special primers were designed, and specific coding sequences of 120 AP2/ERF members in‘Yuexin'strawberry fruit were cloned with c DNA of strawberry fruits at 4 developmental stages(G, green stage;T, turning stage; IR, intermediate red stage; R, full red stage) as template. The AD-c DNA library of AP2/ERF transcription factors of strawberry was constructed successfully after all of p GADT7 vectors with specific AP2/ERF coding sequence were obtained and corresponding strains were preserved. After the screen of the library by Y1 H assay with the promoter of Dk PDC2 as a bait, dozens of colonies were obtained from SD/-Leu media with 200 ng· m L-1 Aureobasidin A(Ab A). The PCR results of the colonies showed that only two bands with about 650 bp and 1 000 bp separately emerged on the agarose gel,which indicated at least two members of AP2/ERF superfamily in strawberry could interact with the promoter of DKPDC2. After sequencing of all the bands, the bands with almost the same length proved to be the same. The larger bands with 1 000 bp was Fa ERF#87 and the smaller ones was Fa ERF#83. The interaction of the promoter of Dk PDC2 with Fa ERF#83 and Fa ERF#87 were verified by Y1 H assays with AD-Fa ERF#83 and AD-Fa ERF#87 as prey. Both the transformants containing AD-Fa ERF#83 and AD-Fa ERF#87 could survive on the plate with SD/-Leu+Ab A(200 ng· m L-1). To investigate the relationship between the reported Dk ERF19 and two AP2/ERFs obtained in this research, a phylogenetic tree was constructed, which showed that they were all members of ERF Ⅸ clade, and Dk ERF19 had a closest relationship with Fa ERF#87 compared with the other members of AP2/ERF in strawberry.【Conclusion】An AD-c DNA library of AP2/ERF transcription factors of strawberry was constructed. The screen of the library with the promoter of Dk PDC2 as the bait showed two members of AP2/ERF superfamily could interact with the promoter of Dk PDC2. The protein of Fa ERF#83, the ortholog gene of Dk ERF19, could interact with the promoter of Dk PDC2, verifying the effectiveness of the library to prove interaction among different species. Also, Fa ERF#87 interacted with the promoter of Dk PDC2, which could validated the potential application of the library to investigate transcription regulation of AP2/ERF.
【Objective】As one of the most numerous transcription factors in plants, the members of AP2/ERF superfamily were reported extensively to be involved both in the regulation of the process of growth and development in plant, and in the regulation of ripening process and accompanying physiological changes of fruit. Rosaceae is of great importance to be investigated, because it consists of many economically important fruits such as apple, peach, pear and strawberry. But compared with the total amount of numbers of AP2/ERF superfamily in Rosaceae, the quantity of the members reported to be functional in the process of fruit development and ripening is too small, which means large work needs to do before a comprehensive understanding of the role of AP2/ERF in fruits' development and ripening process is made. To investigate the potential targets of transcription factors like AP2/ERF, Y1 H assay is one of the most effective methods to use. It is a rapid and efficient way to build an AD-c DNA library only consisting of transcription factors to be screened, which reduces the false-positive rate and great labor when using a traditional c DNA library instead.【Methods】With the use of CTAB, RNA was extracted from strawberry(Fragaria × ananassa)‘Yuexin'fruits, which was then used as template in the process of reverse transcription for the synthesis of the first strand c DNA. With the help of bioinformatics, the details of the sequences of the members of AP2/ERF superfamily in strawberry were ob-tained, according to which the special primers of each member were designed. Each CDS(coding sequence) was cloned to p GEM-T easy vector for sequencing. After the plasmid profiles of p GEM-T easy and p GADT7 vector were compared, seven special primers were designed to transfer the CDS from p GEM-T easy to p GADT7 vector, which was then transformed to E. coli and sequenced. The strains containing the CDS of AP2/ERF family members were preserved with 50% glycerol separately at-80 ℃ before use. To avoid the potential loss of certain plasmids when all of the strains were mixed to proliferate and extracted, each strain was cultured and extracted separately. The plasmids were mixed with the same molar quantity for completing the construction of the library. To verify the effectiveness of Fa ERFAD library, the promoter of Dk PDC2 which was reported to interact with an AP2/ERF transcription factor Dk ERF19, was screened by Y1 H assay with the use of the library. The detail process of Y1 H assay refered to YeastmakerTMYeast Transformation System 2 User Manual(Clontech) and MatchmakerTM Gold Yeast One-Hybrid Library Screening System User Manual(Clontech). After cultivation of 3 days,the colonies of Y1 H transformants were recorded and photographed.【Results】120 items of AP2/ERF in strawberry were obtained from DNA nucleotide database of NCBI after repeat records were omitted.Special primers were designed, and specific coding sequences of 120 AP2/ERF members in‘Yuexin'strawberry fruit were cloned with c DNA of strawberry fruits at 4 developmental stages(G, green stage;T, turning stage; IR, intermediate red stage; R, full red stage) as template. The AD-c DNA library of AP2/ERF transcription factors of strawberry was constructed successfully after all of p GADT7 vectors with specific AP2/ERF coding sequence were obtained and corresponding strains were preserved. After the screen of the library by Y1 H assay with the promoter of Dk PDC2 as a bait, dozens of colonies were obtained from SD/-Leu media with 200 ng· m L-1 Aureobasidin A(Ab A). The PCR results of the colonies showed that only two bands with about 650 bp and 1 000 bp separately emerged on the agarose gel,which indicated at least two members of AP2/ERF superfamily in strawberry could interact with the promoter of DKPDC2. After sequencing of all the bands, the bands with almost the same length proved to be the same. The larger bands with 1 000 bp was Fa ERF#87 and the smaller ones was Fa ERF#83. The interaction of the promoter of Dk PDC2 with Fa ERF#83 and Fa ERF#87 were verified by Y1 H assays with AD-Fa ERF#83 and AD-Fa ERF#87 as prey. Both the transformants containing AD-Fa ERF#83 and AD-Fa ERF#87 could survive on the plate with SD/-Leu+Ab A(200 ng· m L-1). To investigate the relationship between the reported Dk ERF19 and two AP2/ERFs obtained in this research, a phylogenetic tree was constructed, which showed that they were all members of ERF Ⅸ clade, and Dk ERF19 had a closest relationship with Fa ERF#87 compared with the other members of AP2/ERF in strawberry.【Conclusion】An AD-c DNA library of AP2/ERF transcription factors of strawberry was constructed. The screen of the library with the promoter of Dk PDC2 as the bait showed two members of AP2/ERF superfamily could interact with the promoter of Dk PDC2. The protein of Fa ERF#83, the ortholog gene of Dk ERF19, could interact with the promoter of Dk PDC2, verifying the effectiveness of the library to prove interaction among different species. Also, Fa ERF#87 interacted with the promoter of Dk PDC2, which could validated the potential application of the library to investigate transcription regulation of AP2/ERF.
引文
[1]STOCKINGER E J,GILMOUR S J,THOMASHOW M F.Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE,a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(3):1035-1040.
[2]OHME-TAKAGI M,SHINSHI H.Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[J].Plant Cell,1995,7(2):173-182.
[3]KAGAYA Y,OHMIYA K,HATTORI T.RAV1,a novel DNAbinding protein,binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants[J].Nucleic Acids Research,1999,27(2):470-478.
[4]NAKANO T,SUZUKI K,FUJIMURA T,SHINSHI H.Genomewide analysis of the ERF gene family in Arabidopsis and rice[J].Plant Physiology,2006,140(2):411-432.
[5]PARK J M,PARK C J,LEE S B,HAM B K,SHIN R,PAEK K H.Overexpression of the tobacco Tsi1 gene encoding an EREBP/AP2-type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco[J].Plant Cell,2001,13(5):1035-1046.
[6]ZHANG G Y,CHEN M,LI L C,XU Z S,CHEN X P,GUO J M,MA Y Z.Overexpression of the soybean Gm ERF3 gene,an AP2/ERF type transcription factor for increased tolerances to salt,drought,and diseases in transgenic tobacco[J].Journal of Experimental Botany,2009,60(13):3781-3796.
[7]YIN X R,XIE X X,XIA X J,YU J Q,FERGUSON I B,GIOVANNONI J J.Involvement of an ethylene response factor in chlorophyll degradation during citrus fruit degreening[J].Plant Journal,2016,86(5):403-412.
[8]XIAO Y Y,CHEN J Y,KUANG J F,SHAN W,XIE H,JIANG Y M,LU W J.Banana ethylene response factors are involved in fruit ripening through their interactions with ethylene biosynthesis genes[J].Journal of Experimental Botany,2013,64(8):2499-2510.
[9]WANG M M,ZHU Q G,DENG C L,LUO Z R,SUN N J,GRIERSON D,YIN X R,CHEN K S.Hypoxia-responsive ERFs involved in postdeastringency softening of persimmon fruit[J].Plant Biotechnology Journal,2017,15(11):1409-1419.
[10]REECE-HOYES J S,MARIAN WALHOUT A J.Yeast one-hybrid assays:a historical and technical perspective[J].Methods,2012,57(4):441-447.
[11]SHULAEV V,KORBAN S S,SOSINSKI B,ABBOTT A G,ALDWINCKLE H S,FOLTA K M,IEZZONI A,MAIN D,ARUS P,DANDEKAR A M,LEWERS K,BROWN S K,DAVIS T M,GARDINER S E,POTTER D,VEILLEUX R E.Multiple models for Rosaceae genomics[J].Plant Physiology,2008,147(3):985-1003.
[12]MIN T,FANG F,GE H,SHI Y N,LUO Z R,YAO Y C.Two novel anoxia-induced ethylene response factors that interact with promoters of deastringency-related genes from persimmon[J].PLo S One,2014,9(5):e97043.
[13]ZHANG B,CHEN K S,BOWEN J,ALLAN A,ESPLEY R,KARUNAIRETNAM S,FERGUSON I.Differential expression within the lox gene family in ripening kiwifruit[J].Journal of Experimental Botany,2006,57(14):3825-3836.
[14]SHULAEV V,SARGENT D J,CROWHURST R N,MOCKLER T C,FOLKERTS O,DELCHER A L,JAISWAL P,……,FOLTA K M.The genome of woodland strawberry(Fragaria vesca)[J].Nature Genetics,2011,43(2):109-118.
[15]DARWISH O,SLOVIN J P,KANG C,HOLLENDER C A,GERETZ A,HOUSTON S,LIU Z.SGR:an online genomic resource for the woodland strawberry[J].BMC Plant Biology,2013,13(1):223-223.
[16]HIRAKAWA H,SHIRASAWA K,KOSUGI S,TASHIRO K,NAKAYAMA S,YAMADA M,KOHARA M,WATANABE A,KISHIDA Y,FUJISHIRO T,TSURUOKA H,MINAMI C,SASAMOTO S,KATO M,NANRI K,KOMAKI A,YANAGI T,QIN G,MAEDA F,ISHIKAWA M,KUHARA S,SATO S,TABATA S,ISOBE S N.Dissection of the octoploid strawberry genome by deep sequencing of the genomes of Fragaria species[J].DNA Research,2014,21(2):169-181.
[17]DARWISH O,SHAHAN R,LIU Z,SLOVIN J P,ALKHAROUF N W.Re-annotation of the woodland strawberry(Fragaria vesca)genome[J].BMC Genomics,2015,16(1):29-37.
[18]MITUSDA N,IKEDA M,TAKADA S,TAKIGUCHI Y,KONDOU Y,YOSHIZUMI T,FUJITA M,SHINOZAKI K,MATSUI M,OHME-TAKAGI M.Efficient yeast one-/two-hybrid screening using a library composed only of transcription factors in Arabidopsis thaliana[J].Plant&Cell Physiology,2010,51(12):2145-2151.
[2]OHME-TAKAGI M,SHINSHI H.Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[J].Plant Cell,1995,7(2):173-182.
[3]KAGAYA Y,OHMIYA K,HATTORI T.RAV1,a novel DNAbinding protein,binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants[J].Nucleic Acids Research,1999,27(2):470-478.
[4]NAKANO T,SUZUKI K,FUJIMURA T,SHINSHI H.Genomewide analysis of the ERF gene family in Arabidopsis and rice[J].Plant Physiology,2006,140(2):411-432.
[5]PARK J M,PARK C J,LEE S B,HAM B K,SHIN R,PAEK K H.Overexpression of the tobacco Tsi1 gene encoding an EREBP/AP2-type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco[J].Plant Cell,2001,13(5):1035-1046.
[6]ZHANG G Y,CHEN M,LI L C,XU Z S,CHEN X P,GUO J M,MA Y Z.Overexpression of the soybean Gm ERF3 gene,an AP2/ERF type transcription factor for increased tolerances to salt,drought,and diseases in transgenic tobacco[J].Journal of Experimental Botany,2009,60(13):3781-3796.
[7]YIN X R,XIE X X,XIA X J,YU J Q,FERGUSON I B,GIOVANNONI J J.Involvement of an ethylene response factor in chlorophyll degradation during citrus fruit degreening[J].Plant Journal,2016,86(5):403-412.
[8]XIAO Y Y,CHEN J Y,KUANG J F,SHAN W,XIE H,JIANG Y M,LU W J.Banana ethylene response factors are involved in fruit ripening through their interactions with ethylene biosynthesis genes[J].Journal of Experimental Botany,2013,64(8):2499-2510.
[9]WANG M M,ZHU Q G,DENG C L,LUO Z R,SUN N J,GRIERSON D,YIN X R,CHEN K S.Hypoxia-responsive ERFs involved in postdeastringency softening of persimmon fruit[J].Plant Biotechnology Journal,2017,15(11):1409-1419.
[10]REECE-HOYES J S,MARIAN WALHOUT A J.Yeast one-hybrid assays:a historical and technical perspective[J].Methods,2012,57(4):441-447.
[11]SHULAEV V,KORBAN S S,SOSINSKI B,ABBOTT A G,ALDWINCKLE H S,FOLTA K M,IEZZONI A,MAIN D,ARUS P,DANDEKAR A M,LEWERS K,BROWN S K,DAVIS T M,GARDINER S E,POTTER D,VEILLEUX R E.Multiple models for Rosaceae genomics[J].Plant Physiology,2008,147(3):985-1003.
[12]MIN T,FANG F,GE H,SHI Y N,LUO Z R,YAO Y C.Two novel anoxia-induced ethylene response factors that interact with promoters of deastringency-related genes from persimmon[J].PLo S One,2014,9(5):e97043.
[13]ZHANG B,CHEN K S,BOWEN J,ALLAN A,ESPLEY R,KARUNAIRETNAM S,FERGUSON I.Differential expression within the lox gene family in ripening kiwifruit[J].Journal of Experimental Botany,2006,57(14):3825-3836.
[14]SHULAEV V,SARGENT D J,CROWHURST R N,MOCKLER T C,FOLKERTS O,DELCHER A L,JAISWAL P,……,FOLTA K M.The genome of woodland strawberry(Fragaria vesca)[J].Nature Genetics,2011,43(2):109-118.
[15]DARWISH O,SLOVIN J P,KANG C,HOLLENDER C A,GERETZ A,HOUSTON S,LIU Z.SGR:an online genomic resource for the woodland strawberry[J].BMC Plant Biology,2013,13(1):223-223.
[16]HIRAKAWA H,SHIRASAWA K,KOSUGI S,TASHIRO K,NAKAYAMA S,YAMADA M,KOHARA M,WATANABE A,KISHIDA Y,FUJISHIRO T,TSURUOKA H,MINAMI C,SASAMOTO S,KATO M,NANRI K,KOMAKI A,YANAGI T,QIN G,MAEDA F,ISHIKAWA M,KUHARA S,SATO S,TABATA S,ISOBE S N.Dissection of the octoploid strawberry genome by deep sequencing of the genomes of Fragaria species[J].DNA Research,2014,21(2):169-181.
[17]DARWISH O,SHAHAN R,LIU Z,SLOVIN J P,ALKHAROUF N W.Re-annotation of the woodland strawberry(Fragaria vesca)genome[J].BMC Genomics,2015,16(1):29-37.
[18]MITUSDA N,IKEDA M,TAKADA S,TAKIGUCHI Y,KONDOU Y,YOSHIZUMI T,FUJITA M,SHINOZAKI K,MATSUI M,OHME-TAKAGI M.Efficient yeast one-/two-hybrid screening using a library composed only of transcription factors in Arabidopsis thaliana[J].Plant&Cell Physiology,2010,51(12):2145-2151.