黑果枸杞LrTTG1基因的克隆及表达分析
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摘要
以黑果枸杞为材料,利用RT-PCR和RACE技术克隆了花青素合成相关基因LrTTG1(GenBank登录号为MH633481)。序列分析表明,LrTTG1基因cDNA全长1 453bp,包含1 029bp开放阅读框,编码342个氨基酸,含有5个WD40重复基序。同源比对结果表明,LrTTG1与茄子SmTTG1的氨基酸序列相似性较高,达到83.73%。qRT-PCR分析显示,LrTTG1基因在茎、叶、花、青果、紫果和黑果中均有表达,且在青果中的表达水平(最高)约为黑果(最低)的4倍;紫外胁迫下LrTTG1基因的表达随胁迫时间的延长呈先降低后升高的变化趋势。花青素含量分析表明,黑果的花青素含量最高(11.3mg/g),分别约为紫果(1.2mg/g)和青果(0.53mg/g)含量的9.4倍和21.3倍。研究表明,随着黑果枸杞果实的发育,LrTTG1基因的表达量呈现下降趋势,而花青素的含量则呈上升趋势,两者呈负相关关系;推测LrTTG1基因在黑果枸杞花青素合成中可能具有重要的调节作用。
An anthocyanin synthesis related gene named LrTTG1(GenBank accession number MH633481)was cloned from Lycium ruthenicum using RT-PCR and RACE methods.The sequence analysis showed that the length of LrTTG1 gene was 1 453 bp,which contained a 1 029 bp ORF encoding 342 amino acids with five WD40 repeats.Homology amino acid sequences comparison indicated that LrTTG1 had higher similarity with Solanum melongena TTG1(83.73%).qRT-PCR analysis revealed that LrTTG1 gene was expressed in stems,leaves,flowers,green fruits,purple fruits and black fruits,and the expression level(the highest)in green fruits was about 4 times that of black fruits(the lowest).LrTTG1 gene expression showed a trend on firstly falling and then rising with UV irradiation time.Anthocyanin content analysis showed that the content in black fruits was the highest(11.3 mg/g),which were about 9.4 times and 21.3 times of purple fruits(1.2 mg/g)and green fruits(0.53 mg/g),respectively.This study showed that the expression of LrTTG1 gene tended to decrease with the development of fruits,while the content of anthocyanin tended to increase,showing a negative correlation between them.It is speculated that LrTTG1 gene may play an important role in the anthocyanin synthesis of L.ruthenicum.
An anthocyanin synthesis related gene named LrTTG1(GenBank accession number MH633481)was cloned from Lycium ruthenicum using RT-PCR and RACE methods.The sequence analysis showed that the length of LrTTG1 gene was 1 453 bp,which contained a 1 029 bp ORF encoding 342 amino acids with five WD40 repeats.Homology amino acid sequences comparison indicated that LrTTG1 had higher similarity with Solanum melongena TTG1(83.73%).qRT-PCR analysis revealed that LrTTG1 gene was expressed in stems,leaves,flowers,green fruits,purple fruits and black fruits,and the expression level(the highest)in green fruits was about 4 times that of black fruits(the lowest).LrTTG1 gene expression showed a trend on firstly falling and then rising with UV irradiation time.Anthocyanin content analysis showed that the content in black fruits was the highest(11.3 mg/g),which were about 9.4 times and 21.3 times of purple fruits(1.2 mg/g)and green fruits(0.53 mg/g),respectively.This study showed that the expression of LrTTG1 gene tended to decrease with the development of fruits,while the content of anthocyanin tended to increase,showing a negative correlation between them.It is speculated that LrTTG1 gene may play an important role in the anthocyanin synthesis of L.ruthenicum.
引文
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[24] TAKADA H,ISHIMARU K,MINAMISAWA K,et al.Expression of a mutated melon ethylene receptor gene affects stamen development in Nicotiana tabacum[J].Plant Science,2005,169:935-942.
[25]高树林,张超,杜丹妮,等.乙烯和葡萄糖处理对'洛阳红’牡丹切花花色和花青素合成的影响[J].园艺学报,2015,42(7):1 356-1 366.GAO S L,ZHANG C,DU D N,et al.Effect of glucose and ethylene on flower color and anthocyanin biosynthesis in tree peony Luoyanghong'cut flower[J].Acta Horticulturae Sinica,2015,42(7):1 356-1 366.
[26]姚攀锋.苦荞WD40转录因子的基因克隆及其对花青素合成的影响[D].成都:四川农业大学,2016.
[27]强维亚,蔡龙华,韩瑜,等.增强UV-B辐射对高山植物歪头菜生长及内源激素变化的影响[J].西北植物学报,2013,33(11):2 241-2 248.QIANG W Y,CAI L H,HAN Y,et al.Changes of endogenous hormones in the stress-response of alpine plant Vicia unijuga A.Br.to enhanceed UV-B radiation[J].Acta Botanica Boreali-Occident Sinica,2013,33(11):2 241-2 248.
[28] MA T R,ZHANG J W,LIANG H G,et al.Plant anthocyanin synthesis and gene regulation[J].Agricultural Science&Technology,2012,9(5):454-458.
[2] KONG J M,CHIA L S,GOH N K,et al.Analysis and biological activities of anthocyanins[J].Phytochemistry,2003,64(5):923-933.
[3] EZEKIEL R,SINGH N,SHARMA S,et al.Beneficial phytochemicals in potato——a review[J].Food Research International,2013,50(2):487-496.
[4]黄正明.玫瑰茄花水提取物和分得的花青素苷对对乙酰氨基酚诱发的大鼠肝毒性的作用[J].现代药物与临床,2004,19(5):210.HUANG Z M.Effects of water extract from roselle and anthocyanin on hepatotoxicity induced by acetaminophen in rats[J].Drugs&Clinic,2004,19(5):210.
[5] MATSUI T,EBUCHI S,KOBAYASHI M,et al.Anti-hyperglycemic effect of diacylated anthocyanin derived fromIpomoea batatas cultivar Ayamurasaki can be achieved through the alpha-glucosidase inhibitory action[J].Journal of Agricultural and Food Chemistry,2002,50(25):7 244-7 248.
[6] WANG L S,STONER G D.Anthocyanins and their role in cancer prevention[J].Cancer Letters,2008,269(2):281-290.
[7] HOU D X,YANAGITA T,UTO T,et al.Anthocyanidins inhibit protein 1activity and cell transformation:structure-activity relationship and molecular mechanisms[J].Carcinoacncsis,2004,25:29-36.
[8] DUAN Y,CHEN F,YAO X,et al.Protective effect of Lycium ruthenicum Murr.against radiation injury in mice[J].International Journal of Environmental Research&Public Health,2015,12(7):8 332-8 347.
[9]陈玮,凌文华,李茂全,等.黑米花青素在大鼠视网膜光化学损伤中的抗氧化作用研究[J].营养学报,2010,32(4):341-344.CHEN W,LING W H,LI M Q,et al.The antioxidative effects of anthocyanidins from black rice on retina photochemical damage in rats[J].Acta Nutrimenta Sinica,2010,32(4):341-344.
[10] XU W J,GRAIN D,BOBET S,et al.Complexity and robustness of the flavonoid transcriptional regulatory network revealed by comprehensive analyses of MYBbHLH-WDR complexes and their targets in Arabidopsis seed[J].New Phytol,2014,202:132-144.
[11] KOORNNEEF M.The complex syndrome of ttg mutants[J].Arabidopsis Information Service,1981,18:45-51.
[12] CHEN M X,ZHANG B,LI C X,et al.TRANSPARENT TESTA GLABRA1regulates the accumulation of seed storage reserves in Arabidopsis[J].Plant Physiology,2015,169:391-402.
[13] DEBEAUJON I,LEON-KLOOSTERZIEL KM,KOORNNEEF M.Influence of the testa on seed dormancy,germination,and longevity in Arabidopsis[J].Plant Physiology,2000,122:403-414.
[14] WALKER A R,DAVISON P A,BOLOGNESI-WINFIELD AC,et al.The TRANSPARENT TESTA GLABRA1locus,which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis,encodes a WD40repeat protein[J].Plant Cell,1999,11:1 337-1 350.
[15] HUMPHRIES J A,WALKER A R,TIMMIS J N,et al.Two WD-repeat genes from cotton are functional homologues of the Arabidopsis thaliana TRANSPARENT TESTA GLABRA1(TTG1)gene[J].Plant Molecular Biology,2005,57:67-81.
[16] LU J,LI J N,WANG S G,et al.Molecular cloning of two ortholog genes of Arabidopsis thaliana TTG1from oilseed rape(Brassica napus L.)[J].Biotechnology,2007,34(2):170-172.
[17] DRESSEL A,HEMLEBEN V.Transparent Testa Glabra 1(TTG1)and TTG1-like genes in Matthiola incana R.Br.and related Brassicaceae and mutation in the WD-40motif[J]Plant Biology,2009,11:204-212.
[18]刘新宇,韩洪强,葛海燕,等.茄子花青素合成中SmTTG1、SmGL3和SmTT8的表达及其蛋白质间的相互作用[J].园艺学报,2014,41(11):2 241-2 249.LIU X Y,HAN H Q,GE H Y,et al.Cloning,expression and interaction of anthocyanin-related transcription factors SmTTG1,SmGL3and SmTT8in eggplant[J].Acta Horticulturae Sinica,2014,41(11):2 241-2 249.
[19]师生波,韩发.紫外光源及太阳UV-B辐射的模拟实验[J].生物技术通报,200,6(s1):161-166.SHI S B,HAN F.Light sources for ultraviolet research and simulation of solar ultraviolet-B radiation[J].Biotechnology Bulletin,2006,(s1):161-166.
[20]邵文婷.茄子花青素合成相关基因SmMYB的克隆与功能研究[D].上海:上海交通大学,2013.
[21] CAREY C C,STRAHLE J T,SELINGER D A,et al.Mutations in the pale aleurone color1regulatory gene of the Zea mays anthocyanin pathway have distinct phenotypes relative to the functionally similar TRANSPARENT TESTA GLABRA1gene in Arabidopsis thaliana[J].Plant Cell,2004,16(2):450-464.
[22] BROUN P.Transcriptional control of flavonoid biosynthesis:a complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis[J].Current Opinion Plant Biology,2005,8:272-279.
[23]刘凯歌,齐双慧,段绍伟,等.甘蓝型油菜BnTTG1-1基因的功能分析[J].植物学报,2017,52(6):713-722.LIU K G,QI S H,DUAN S W,et al.Functional analysis of Brassica napus BnTTG1-1gene[J].Bulletin of Botany,2017,52(6):713-722.
[24] TAKADA H,ISHIMARU K,MINAMISAWA K,et al.Expression of a mutated melon ethylene receptor gene affects stamen development in Nicotiana tabacum[J].Plant Science,2005,169:935-942.
[25]高树林,张超,杜丹妮,等.乙烯和葡萄糖处理对'洛阳红’牡丹切花花色和花青素合成的影响[J].园艺学报,2015,42(7):1 356-1 366.GAO S L,ZHANG C,DU D N,et al.Effect of glucose and ethylene on flower color and anthocyanin biosynthesis in tree peony Luoyanghong'cut flower[J].Acta Horticulturae Sinica,2015,42(7):1 356-1 366.
[26]姚攀锋.苦荞WD40转录因子的基因克隆及其对花青素合成的影响[D].成都:四川农业大学,2016.
[27]强维亚,蔡龙华,韩瑜,等.增强UV-B辐射对高山植物歪头菜生长及内源激素变化的影响[J].西北植物学报,2013,33(11):2 241-2 248.QIANG W Y,CAI L H,HAN Y,et al.Changes of endogenous hormones in the stress-response of alpine plant Vicia unijuga A.Br.to enhanceed UV-B radiation[J].Acta Botanica Boreali-Occident Sinica,2013,33(11):2 241-2 248.
[28] MA T R,ZHANG J W,LIANG H G,et al.Plant anthocyanin synthesis and gene regulation[J].Agricultural Science&Technology,2012,9(5):454-458.