丹参均一化酵母双杂交文库的构建及与SmJAZ8互作蛋白的筛选
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摘要
为构建高质量的丹参c DNA文库以及通过酵母双杂交获得丹参Sm JAZ8互作蛋白基因。研究以丹参的根、茎、叶、花、毛状根为材料,利用SMART(switching mechanism at 5'end of RNA transcript)方式合成全长c DNA,并结合基于双链的特异性核酸酶(duplex-specific nuclease,DSN)均一化技术,构建丹参的均一化全长c DNA文库。通过文库鉴定,检测到c DNA文库库容为1.45×106,重组率为100%,插入片段平均大小为500~2 000 bp。构建p DEST-p GADT7-Sm JAZ8重组载体,将该载体转化Y2HGold菌种,通过酵母双杂交进行互作蛋白的筛选,最终筛选到了丹参Dna J蛋白和UBQ蛋白。该研究既成功构建了丹参均一化全长c DNA文库,又为后续丹参功能基因筛选和基因功能的研究奠定了基础。
The study is aimed to construct high quality Salvia miltiorrhiza c DNA library and obtain the Sm JAZ8 gene of S. miltiorrhiza by yeast two-hybrid system. In this study,full-length cDNA was synthesized from roots,stems,leaves,flowers and hairy roots of S. miltiorrhiza. The full-length c DNA library was synthesized by SMART method and constructed with DSN homogenization technique.The results showed that the library capacity was 1. 45 × 10~6,the recombination rate was 100%,and the average size of the insert was500-2 000 bp. The recombinant vector of p DEST-p GADT7-Sm JAZ8 was constructed and transformed into Y2 HGold strain. The interaction protein was screened by yeast two-hybrid system. The Dna J protein and UBQ protein were screened by yeast two-hybrid system.This study has successfully constructed a full-length c DNA library of S. miltiorrhiza,and laid the foundation for the follow-up study on functional gene screening and gene function of S. miltiorrhiza.
The study is aimed to construct high quality Salvia miltiorrhiza c DNA library and obtain the Sm JAZ8 gene of S. miltiorrhiza by yeast two-hybrid system. In this study,full-length cDNA was synthesized from roots,stems,leaves,flowers and hairy roots of S. miltiorrhiza. The full-length c DNA library was synthesized by SMART method and constructed with DSN homogenization technique.The results showed that the library capacity was 1. 45 × 10~6,the recombination rate was 100%,and the average size of the insert was500-2 000 bp. The recombinant vector of p DEST-p GADT7-Sm JAZ8 was constructed and transformed into Y2 HGold strain. The interaction protein was screened by yeast two-hybrid system. The Dna J protein and UBQ protein were screened by yeast two-hybrid system.This study has successfully constructed a full-length c DNA library of S. miltiorrhiza,and laid the foundation for the follow-up study on functional gene screening and gene function of S. miltiorrhiza.
引文
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[30]Yang K Z,Xia C X,Dou X Y,et al.A mutation in,thermosensitive male sterile 1,encoding a heat shock protein with Dna J and PDI domains,leads to thermosensitive gametophytic male sterility in Arabidopsis[J].Plant J,2009,57(5):870.
[31]Vitha S,Froehlich J E,Koksharova O,et al.ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2[J].Plant Cell,2003,15(8):1918.
[32]Raonaik C,Delacruz W,Laplaza J M,et al.The rub family of ubiquitin-like proteins.Crystal structure of Arabidopsis rub1 and expression of multiple rubs in Arabidopsis[J].J Biol Chem,1998,273(52):34976.
[33]Schwartz D C,Hochstrasser M.A superfamily of protein tags:ubiquitin,SUMO and related modifiers[J].Trends Biochem Sci,2003,28(6):321.
[34]Cao S,Yan L.Construction of a high-quality yeast two-hybrid(Y2H)library and its application in identification of interacting proteins with key vernalization regulator Ta VRN-A1 in wheat[J].BMC Res Notes,2013,6(1):81.
[2]Wu Y B,Ni Z Y,Shi Q W,et al.Constituents from Salvia species and their biological activities[J].Chem Rev,2012,112(11):5967.
[3]陈鹏,郑佳,程江涛,等.丹参酮ⅡA对NSTEACS患者PCI术后肾功能及中医症候疗效的影响[J].世界科学技术——中医药现代化,2016(3):453.
[4]Kai G,Liao P,Xu H,et al.Molecular mechanism of elicitor-induced tanshinone accumulation in Salvia miltiorrhiza,hairy root cultures[J].Acta Physiol Plant,2012,34(4):1421.
[5]Cui G,Duan L,Jin B,et al.Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza[J].Plant Physiol,2015,169(3):1607.
[6]Wasternack C,Hause B.Jasmonates:biosynthesis,perception,signal transduction and action in plant stress response,growth and development.An update to the 2007 review in Annals of Botany[J].Ann Bot,2013,111(6):1021.
[7]孙程,周晓今,陈茹梅,等.植物JAZ蛋白的功能概述[J].生物技术通报,2014(6):1.
[8]Kazan K,Manners J M.JAZ repressors and the orchestration of phytohormone crosstalk[J].Trends Plant Sci,2012,17(1):22.
[9]蒋敏,王江,文国松,等.铁皮石斛均一化全长c DNA文库的构建与序列分析[J].中国中药杂志,2013,38(4):504.
[10]王怀琴,郭晓荣,杨新兵,等.利用酵母双杂交筛选与丹参R2R3-MYB类转录因子Sm PAP1互作的蛋白[J].基因组学与应用生物学,2016,35(10):2819.
[11]曾日中,段翠芳,黎瑜,等.茉莉酸刺激的橡胶树胶乳c DNA消减文库的构建及其序列分析[J].热带作物学报,2003,24(3):1.
[12]段翠芳,曾日中,聂智毅,等.橡胶树胶乳c DNA文库的构建及其EST序列的功能分析[J].热带作物学报,2007,28(3):46.
[13]储昭晖,彭开蔓.水稻全生育期均一化c DNA文库的构建和鉴定[J].科学通报,2002,47(21):1656.
[14]姜翠翠,陈桂信,潘东明,等.[木奈]褐变果实均一化全长c DNA文库的构建及部分ESTs序列分析[J].热带作物学报,2013,34(2):292.
[15]郭蓉.丹参PPO基因酵母双杂交诱饵质粒与c DNA文库构建[D].杨凌:西北农林科技大学,2015.
[16]崔光红,黄璐琦,唐晓晶,等.丹参功能基因组学研究Ⅰ——c DNA芯片的构建[J].中国中药杂志,2007,32(12):1137.
[17]马艺沔,袁丽钗.丹参根部RNA的提取与全长均一化c DNA的制备[J].中药材,2014,37(5):770.
[18]张成,王喆之.丹参钙调蛋白c DNA的克隆及其反义表达载体的构建(英文)[J].分子植物育种,2006,4(3):339.
[19]刘德泉,郭文云,何则铭,等.大豆胚发育期酵母双杂文库的构建及与b HLH转录因子互作蛋白的筛选[J].大豆科学,2015,34(5):789.
[20]李晨,闫晓红,周新安,等.大豆种子不同发育时期全长均一化c DNA文库的构建[J].中国农业科学,2010,43(3):462.
[21]Patanjali S R,Parimoo S,Weissman S M.Construction of a uniform-abundance(normalized)c DNA library[J].Proc Natl Acad Sci USA,1991,88(5):1943.
[22]Zhulidov P A,Bogdanova E A,Shcheglov A S,et al.A method for the preparation of normalized c DNA libraries enriched with full-length sequences[J].Bioorg Khim,2005,31(2):170.
[23]Nelson R T,Shoemaker R.Identification and analysis of gene families from the duplicated genome of soybean using EST sequences[J].BMC Genomics,2006,7(1):204.
[24]余海洋,张宇,王萌,等.巴西橡胶树胶乳均一化酵母双杂交c DNA文库构建[J].植物生理学报,2016(3):312.
[25]Grebe M,Gadea J,Steinmann T,et al.A conserved domain of the arabidopsis GNOM protein mediates subunit interaction and cyclophilin 5 binding[J].Plant Cell,2000,12(3):343.
[26]Jia J,Fu J,Zheng J,et al.Annotation and expression profile analysis of 2073 full-length c DNAs from stress-induced maize(Zea mays L.)seedlings[J].Plant J,2006,48(5):710.
[27]Chen K M,Holmstr9m M,Raksajit W,et al.Small chloroplasttargeted Dna J proteins are involved in optimization of photosynthetic reactions in Arabidopsis thaliana[J].BMC Plant Biol,2010,10(1):43.
[28]Georgopoulos C P,Lundquist-Heil A,Yochem J,et al.Identification of the E.coli Dna J,gene product[J].Mol Genet Genomics,1980,178(3):583.
[29]Kong F,Deng Y,Zhou B,et al.A chloroplast-targeted Dna J protein contributes to maintenance of photosystemⅡunder chilling stress[J].J Exp Bot,2014,65(1):143.
[30]Yang K Z,Xia C X,Dou X Y,et al.A mutation in,thermosensitive male sterile 1,encoding a heat shock protein with Dna J and PDI domains,leads to thermosensitive gametophytic male sterility in Arabidopsis[J].Plant J,2009,57(5):870.
[31]Vitha S,Froehlich J E,Koksharova O,et al.ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2[J].Plant Cell,2003,15(8):1918.
[32]Raonaik C,Delacruz W,Laplaza J M,et al.The rub family of ubiquitin-like proteins.Crystal structure of Arabidopsis rub1 and expression of multiple rubs in Arabidopsis[J].J Biol Chem,1998,273(52):34976.
[33]Schwartz D C,Hochstrasser M.A superfamily of protein tags:ubiquitin,SUMO and related modifiers[J].Trends Biochem Sci,2003,28(6):321.
[34]Cao S,Yan L.Construction of a high-quality yeast two-hybrid(Y2H)library and its application in identification of interacting proteins with key vernalization regulator Ta VRN-A1 in wheat[J].BMC Res Notes,2013,6(1):81.