黑莓RuMYB10基因的克隆和表达Cloning and expression analysis of RuMYB10 in blackberry(Rubus spp.)
陈清;余昊唯;汤浩茹;王小蓉;刘泽静;
摘要(Abstract):
【目的】从黑莓中克隆RuMYB10的编码区全长序列,并分析其在黑莓果实发育过程中的表达情况与果实花青素苷积累的关系。【方法】以黑莓基因组DNA为模板,通过同源克隆和SON-PCR技术获得了RuMYB10基因全长编码序列(Accession No.:JQ359611);并以黑莓果实mRNA为模板进行验证。采用实时定量PCR技术检测该基因在果实不同发育阶段的表达水平。【结果】结果表明,该基因编码区全长共1 837 bp,编码216个氨基酸,推导蛋白分子质量为24 863 Da。具有MYB转录因子家族特有的R2R3保守序列。含有两个内含子,第2个内含子长度为750 bp,占全长40.8%;在R3重复单元中存在与bHLH因子互作的‘[DE]Lx2[RK]x3Lx6Lx3R’序列;而促进花青素苷合成的MYB转录因子3个特征氨基酸残基中的丙氨酸(A)被丝氨酸(S)取代。RuMYB10基因在黑莓果实发育后期,即果实变红到最后变黑的过程中大量表达,与花青素苷的积累相一致。【结论】从黑莓中克隆到包含完整ORF的转录因子基因RuMYB10,具有MYB因子家族结构特征和bHLH因子结合域,且在果实花青素苷积累高峰期表达量最高。
关键词(KeyWords): 黑莓;RuMYB10;全长克隆;序列分析;表达模式
基金项目(Foundation): 国家自然科学基金(No.30971990)
作者(Authors): 陈清;余昊唯;汤浩茹;王小蓉;刘泽静;
DOI: 10.13925/j.cnki.gsxb.2012.05.008
参考文献(References):
- [1]WANG Xiao-rong,TANG Hao-ru,DENG Qun-xian.Advancement in research of genetic diversity of bramble(Rubus L.)and its breed-ing in China[J].Acta Horticulturae Sinica,2006,33(1):190-196.王小蓉,汤浩茹,邓群仙.中国树莓属植物多样性及品种选育研究进展[J].园艺学报,2006,33(1):190-196.
- [2]ELISIA I,HU C,POPOVICH D G,KITTS D D.Antioxidant assess-ment of an anthocyanin-enriched blackberry extract[J].Food Chem-istry,2007,101(3):1052-1058.
- [3]STRIK B C,CLARK J R,FINN C E,BANADOS M P.Worldwide blackberry production[J].Horttechnology,2007,17(5):205-213.
- [4]CHEN Q,ZHANG X L,YU H W,WANG Y,TANG H R.Changes of total anthocyanins and proanthocyanidins in the developing black-berry fruits[J].International Journal of Chemtech Research,2012,4(1):129-137.
- [5]DONG Y H,BEUNING L,DAVIES K,MITRA D,MORRIS B,KOOTSTRA A.Expression of pigmentation genes and photo-regula-tion of anthocyanin biosynthesis in developing Royal Gala apple flowers[J].Australian Journal of Plant Physiology,1998,25:245-252.
- [6]ALMEIDA J R M,D’AMICO E,PREUSS A,CARBONE F,de vos RIC C H,DEIML B,MOURGUES F,PERROTTA G,FISCHER T C,BOVY A G,MARTENS S,ROSATI C.Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry(Fragaria×ananassa)[J].Archives of Biochemistry and Biophysics,2007,465(1):61-71.
- [7]FENG S,WANG Y,YANG S,XU Y,CHEN X.Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor PyMYB10[J].Planta,2010,232(1):245-255.
- [8]HICHRI I,BARRIEU F,BOGS J,KAPPEL C,DELROT S,LAU-VERGEAT V.Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway[J].Journal of Experimental Botany,2011,62(8):2465-2483.
- [9]TAKOS A M,JAFFE F W,JACOB S R,BOGS J,ROBINSON S P,WALKER A R.Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples[J].Plant Physiology,2006,142(3):1216-1232.
- [10]LOPIERO A R,PUGLISI I,RAPISARDA P,PETRONE G.Antho-cyanins accumulation and related gene expression in red orange fruit induced by low temperature storage[J].Journal of Agricultural and Food Chemistry,2005,53(23):9083-9088.
- [11]ALLAN A C,HELLENS R P,LAING W A.MYB transcription fac-tors that colour our fruit[J].Trends in Plant Science,2008,13(3):99-102.
- [12]LIN-WANG K,BOLITHO K,GRAFTON K,KORTSTEE A,KARUNAIRETNAM S,MCGHIE T K,ESPLEY R V,HELLENS R P,ALLAN A C.An R2R3MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae[J].BMC Plant Biology,2010,10:50.
- [13]BAN Y,HONDA C,HATSUYAMA Y,IGARASHI M,BESSHO H,MORIGUCHI T.Isolation and functional analysis of a MYB tran-scription factor gene that is a key regulator for the development of red coloration in apple skin[J].Plant Cell Physiology,2007,48(7):958-970.
- [14]ESPLEY R V,HELLENS R P,PUTTERILL J,STEVENSON D E,KUTTY-AMMA S,ALLAN A C.Red coloration in apple fruit is due to the activity of the MYB transcription factor,MdMYB10[J].Plant Journal,2007,49(3):414-427.
- [15]WANG Xiao-rong,TANG Hao-ru,FU Hua-qing,ZHONG Bi-feng,XIA Wu-feng.Karyotypes of15introduced bramble cultivars(Rubus L)[J].Scientia Silvae Science,2008,44(12):147-150.王小蓉,汤浩茹,付华清,钟必凤,夏武峰.15个引进树莓品种的核型[J].林业科学,2008,44(12):147-150.
- [16]PERKINS-VEAZIE P,CLARK J R,HUBER D J,BALDWIN E A.Ripening physiology in‘Navaho’thornless blackberries:color,res-piration,ethylene production,softening,and compositional changes[J].Journal of American Society for Horticultural Science,2000,125(3):357-363.
- [17]DONG Xiao-li,TANG Hao-ru,CHEN Qing,WANG Xiao-rong,HOU Yan-xia.Extraction of genomic DNA and optimization of ISSR reaction system with orthogonal design in bramble[J].Chinese Agri-cultural Science Bulletin,2009,15(14):27-31.董晓莉,汤浩茹,陈清,王小蓉,侯艳霞.树莓基因组DNA的提取及ISSR反应体系的正交优化[J].中国农学通报,2009,15(14):27-31.
- [18]GHANGAL R,RAGHUVANSHI S,CHAND-SHARMA P.Isolation of good quality RNA from a medicinal plant seabuckthorn,rich in secondary metabolites[J].Plant Physiology and Biochemistry,2009,47(11-12):1113-1115.
- [19]ANTAL Z,RASCLE C,FEVRE M,BRUEL C.Single oligonucleotide nested PCR:a rapid method for the isolation of genes and their flanking regions from expressed sequence tags[J].Current Genetics,2004,46(4):240-246.
- [20]MING-WEI S K,FLOYD M W.Phenolic content and antioxidant ca-pacities of Alabama-grown thornless blackberries[J].International Journal of Fruit Science,2007,7(4):33-45.
- [21]GIUSTI M M,WROSTAD R E.Characterization and Measurement ofAnthocyanins by UV-Visible Spectroscopy[M]//in Current Protocols in Food Analytical Chemistry,John Wiley&Sons,New York,2001.
- [22]TAMURA K,PETERSON D,PETERSON N,STECHER G,NEI M,KUMAR S.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology Evolution,2011,28(10):2731-2739.
- [23]ZIMMERMANN I M,HEIM M A,WEISSHAAR B,UHRIG J F.Comprehensive identification of Arabidopsis thaliana MYB tran-scription factors interacting with R/B-like BHLH proteins[J].Plant Journal,2004,40(1):22-34,107-119.
- [24]PAZ-ARES J,GHOSAL D,WIENAND U,PETERSON P A,SAEDLER H.The regulatory c1locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators[J].EMBO Journal,1987,6(12):3553-3558.
- [25]BOGS J,JAFFE F W,TAKOS A M,WALKER A R,ROBINSON S P.The grapevine transcription factor VvMYBPA1regulates proan-thocyanidin synthesis during fruit development[J].Plant Physiology,2007,143(3):1347-1361.
- [26]FOURNIER-LEVEL A,LACOMBE T,LE-CUNFF L,BOUR-SIQUOT J M,THIS P.Evolution of the VvMybA gene family,the major determinant of berry colour in cultivated grapevine(Vitis vinifera L.)[J].Heredity,2009,104:351-362.
- [27]TERRIER N,TORREGROSA L,AGEORGES A,VIALET S,VER-RIES C,CHEYNIER V,ROMIEU C.Ectopic expression of VvMyb-PA2promotes proanthocyanidin biosynthesis in grapevine and sug-gests additional targets in the pathway[J].Plant Physiology,2009,149(2):1028-1041.
- [28]AHARONI A,DE-VOS C H,WEIN M,SUN Z,GRECO R,KROON A,MOL J N,O'CONNELL A P.The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumula-tion in transgenic tobacco[J].Plant Journal,2001,28:319-332.
- [29]STRACKE R,WERBER M,WEISSHAAR B.The R2R3-MYB gene family in Arabidopsis thaliana[J].Current Opinion in Plant Biology,2001,4:447-456.
- [30]PAOLOCCI F,ROBBINS M P,PASSERI V,HAUCK B,MORRIS P,RUBINI A,ARCIONI S,DAMIANI F.The strawberry transcrip-tion factor FaMYB1inhibits the biosynthesis of proanthocyanidins in Lotus corniculatus leaves[J].Journal of Experimental Botany,2011,62(3):1189-1200.
- [31]YOSHIDA K,IWASAKA R,KANEKO T,SATO S,TABATA S,SAKUTA M.Functional differentiation of Lotus japonicus TT2s,R2R3-MYB transcription factors comprising a multigene family[J].Plant Cell Physiology,2008,49(2):157-169.
- [32]RAY H,YU M,AUSER P,BLAHUT-BEATTY L,MCKERSIE B,BOWLEY S,WESTCOTT N,COULMAN B,LLOYD A,GRUBER M Y.Expression of anthocyanins and proanthocyanidins after trans-formation of alfalfa with maize Lc[J].Plant Physiology,2003,132(3):1448-1463.