胭脂萝卜RsUFGTs的克隆与表达分析
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摘要
为确定参与胭脂萝卜花青苷生物合成的类黄酮糖基转移酶基因(RsUFGTs),从萝卜基因组中筛选出3个候选的RsUFGTs基因,以胭脂萝卜为材料,克隆了3个RsUFGTs的开放阅读框(ORF)并进行序列及表达分析。结果表明:RsUFGT1和RsUFGT2的ORF长度分别为1 383和1 374 bp,分别编码460和457个氨基酸,而RsUFGT3提前终止;氨基酸序列比对和系统进化树分析表明RsUFGT1和RsUFGT2均含有PSPG保守特征结构域,且属于ClusterⅠ亚族成员;利用实时荧光定量PCR分析它们在不同着色萝卜品种中的表达发现,RsUFGT1在‘红心1号’胭脂萝卜皮和肉组织中表达量较高,在‘满堂红’萝卜皮和‘春不老’的萝卜皮与肉中均不表达,其表达量与花青苷含量呈正相关,而RsUFGT2的表达则无明显规律。因此,RsUFGT1可能是胭脂萝卜花青苷生物合成的重要结构基因之一。
To clarify the flavonoid glycosyltransferase gene(RsUFGTs) involved in the biosynthesis of anthocyanin in red radish,three candidate RsUFGTs genes were screened from the radish genome,and the open reading frames(ORFs) of three RsUFGTs were cloned and sequenced and expressed. Results showed that the ORF length of RsUFGT1 and RsUFGT2 was 1 383 bp and 1 374 bp,encoding 460 and 457 amino acids,respectively. However,RsUFGT3 was mutated with early termination. Protein sequence alignment and phylogenetic tree analyses indicated that RsUFGT1 and RsUFGT2 both contain PSPG conserved domain and belong to the same UFGT class. The results of real-time quantitative PCR analysis showed that the expression of RsUFGT1 in ‘Hongxin No.1' was relatively high and no expression was detected in skin of ‘Mantanghong' and in skin and flesh of ‘Chunbulao'. The expression of RsUFGT1 was positively correlated with anthocyanins content in tissues of different radish cultivars. However,no obvious rules of RsUFGT2 expression were detected. It is indicated that RsUFGT1 may be one of the important structural genes of anthocyanin biosynthesis in red radish.
To clarify the flavonoid glycosyltransferase gene(RsUFGTs) involved in the biosynthesis of anthocyanin in red radish,three candidate RsUFGTs genes were screened from the radish genome,and the open reading frames(ORFs) of three RsUFGTs were cloned and sequenced and expressed. Results showed that the ORF length of RsUFGT1 and RsUFGT2 was 1 383 bp and 1 374 bp,encoding 460 and 457 amino acids,respectively. However,RsUFGT3 was mutated with early termination. Protein sequence alignment and phylogenetic tree analyses indicated that RsUFGT1 and RsUFGT2 both contain PSPG conserved domain and belong to the same UFGT class. The results of real-time quantitative PCR analysis showed that the expression of RsUFGT1 in ‘Hongxin No.1' was relatively high and no expression was detected in skin of ‘Mantanghong' and in skin and flesh of ‘Chunbulao'. The expression of RsUFGT1 was positively correlated with anthocyanins content in tissues of different radish cultivars. However,no obvious rules of RsUFGT2 expression were detected. It is indicated that RsUFGT1 may be one of the important structural genes of anthocyanin biosynthesis in red radish.
引文
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[21] SUN W,MENG X,LIANG L,et al.Overexpression of a Freesia hybrida flavonoid 3-O-glycosyltransferase gene,Fh3GT1,enhances transcription of key anthocyanin genes and accumulation of anthocyanin and flavonol in transgenic petunia (Petunia hybrida)[J].In vitro cellular & developmental biology,2017,53(5):478-488.
[22] MORITA Y,ISHIGURO K,TANAKA Y,et al.Spontaneous mutations of the UDP-glucose:flavonoid 3-O-glucosyltransferase gene confers pale- and dull-colored flowers in the Japanese and common morning glories[J].Planta,2015,242(3):575-587.
[23] KNOCH E,SUGAWARA S,MORI T,et al.UGT79B31 is responsible for the final modification step of pollen-specific flavonoid biosynthesis in Petunia hybrida[J].Planta,2018,247(4):779-790.
[24] SONG C,ZHAO S,HONG X,et al.A UDP-glucosyltransferase functions in both acylphloroglucinol glucoside and anthocyanin biosynthesis in strawberry (Fragaria×ananassa) [J].Plant journal,2016,85(6):730-742.
[2] SPRINGOB K,NAKAJIMA J I,YAMAZAKI M,et al.Recent advances in the biosynthesis and accumulation of anthocyanins[J].Natural product reports,2003,20(3):288-303.
[3] BOWLES D.A multigene family of glycosyltransferases in a model plant,Arabidopsis thaliana[J].Biochemical society transactions,2002,30(2):301-306.
[4] LIM E K,BALDAUF I,LI Y,et al.Evolution of substrate recognition across a multigene family of glycosyltransferases in Arabidopsis[J].Glycobiology,2003,13(3):139-145.
[5] MDOLO L V,BLOUNT J W,ACHNINE L,et al.A functional genomics approach to (iso)flavonoid glycosylation in the model legume Medicago truncatula[J].Plant molecular biology,2007,64(5):499-518.
[6] KOBAYASHI S,ISHIMARU M,DING C K,et al.Comparison of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene sequences between white grapes (Vitis vinifera) and their sports with red skin[J].Plant science,2001,160(3):543-550.
[7] PEPPI M C,WALKER M A,FIDELIBUS M W.Application of abscisic acid rapidly upregulated UFGT gene expression and improved color of grape berries[J].VITIS,2008,47:11-14.
[8] HU C,GONG Y,JIN S,et al.Molecular analysis of a UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene from purple potato (Solanum tuberosum)[J].Molecular biology reports,2011,38(1):561-567.
[9] 招雪晴,李勃,苑兆和.石榴PgUFGT基因克隆及表达分析[J].西北植物学报,2017,37(4):646-653.
[10] 赵志常,张波,高爱平,等.芒果UFGT基因的克隆及表达分析[J].江苏农业科学,2016,44(5):31-34.
[11] 孟富宣,周军,王大玮,等.云南特有火把梨UFGT基因片段的克隆与序列分析[J].西南林业大学学报,2016,36(1):21-27.
[12] LI X J,ZHANG J Q,WU Z C,et al.Functional characterization of a glucosyltransferase gene,LcUFGT1,involved in the formation of cyanidin glucoside in the pericarp of Litchi chinensis[J].Physiol plantarum,2016,156(2):139-149.
[13] 张洪伟,梁毅,刘小义等.洋葱UFGT基因的克隆和表达分析[J].核农学报,2015,29(9):1677-1686.
[14] WROLSTAD R E,CULBERTSON J D,CORNWELL C J,et al.Detection of adulteration in blackberry juice concentrates and wines[J].Journal-association of official analytical chemists,1982,65(6):1417-1423.
[15] TAMURA K,PETERSON D,PETERSON D N,et al.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular biology and evolution,2011,28(10):2731-2739.
[16] WEI Y Z,HU F C,HU G B,et al.Differential expression of anthocyanin biosynthetic genes in relation to anthocyanin accumulation in the pericarp of Litchi chinensis Sonn[J/OL].PLoS One,2011,6(4):e19455[2018-10-26].https://www.taodocs.com/p-84045991.html.
[17] SCHMITTGEN T D,LIVAK K J.Analyzing real-time PCR data by the comparative C(T) method[J].Nature protocols,2008,3(6):1101-1108.
[18] LIM S,SONG J,KIM D,et al.Activation of anthocyanin biosynthesis by expression of the radish R2R3-MYB transcription factor gene RsMYB1[J].Plant cell reports,2016,35(3):641-653.
[19] PAQUETTE S,MΦLLER B L,BAK S.On the origin of family 1 plant glycosyltransferases[J].Phytochemistry,2003,62(3):399-413.
[20] 韩振云,宋婷婷,田佶,等.苹果属观赏海棠McUFGT的克隆及其在不同叶色品种间的表达差异分析[J].园艺学报,2014,41(2):301-310.
[21] SUN W,MENG X,LIANG L,et al.Overexpression of a Freesia hybrida flavonoid 3-O-glycosyltransferase gene,Fh3GT1,enhances transcription of key anthocyanin genes and accumulation of anthocyanin and flavonol in transgenic petunia (Petunia hybrida)[J].In vitro cellular & developmental biology,2017,53(5):478-488.
[22] MORITA Y,ISHIGURO K,TANAKA Y,et al.Spontaneous mutations of the UDP-glucose:flavonoid 3-O-glucosyltransferase gene confers pale- and dull-colored flowers in the Japanese and common morning glories[J].Planta,2015,242(3):575-587.
[23] KNOCH E,SUGAWARA S,MORI T,et al.UGT79B31 is responsible for the final modification step of pollen-specific flavonoid biosynthesis in Petunia hybrida[J].Planta,2018,247(4):779-790.
[24] SONG C,ZHAO S,HONG X,et al.A UDP-glucosyltransferase functions in both acylphloroglucinol glucoside and anthocyanin biosynthesis in strawberry (Fragaria×ananassa) [J].Plant journal,2016,85(6):730-742.