桂花β-胡萝卜素羟化酶基因的克隆与序列分析
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摘要
为揭示桂花不同品种β-胡萝卜素羟化酶(β-carotene hydroxylase,HYB)基因的序列差异,利用已构建的桂花花瓣转录组数据库中Unigene序列信息,结合RT-PCR技术对桂花品种玉玲珑、金球桂、堰虹桂和日香桂中2个β-胡萝卜素羟化酶基因(HYB1和HYB2)的最大阅读框进行扩增,并测序验证。生物信息学分析结果表明,4个桂花品种HYB1基因均含有长为909 bp的开放阅读框,编码302个氨基酸残基; HYB2基因均含有长为915 bp的开放阅读框,编码304个氨基酸残基。4个桂花品种HYB1和HYB2推导的氨基酸序列均具备保守的组氨酸结构域。HYB1基因核苷酸序列存在27个变异位点,其氨基酸序列存在8个变异位点; HYB2基因核苷酸序列存在12个变异位点,其氨基酸序列存在3个变异位点。进化树分析结果发现,4个桂花品种的HYB1和HYB2蛋白与茄科植物番茄和辣椒HYB蛋白亲缘性最高。
To reveal the sequence difference of β-carotene hydroxylase( HYB) genes in different cultivars of Osmanthus fragrans,based on the Unigene sequence information from petal transcriptome database of O. fragrans,the open reading frames( ORFs) of two β-carotene hydroxylase genes HYB1 and HYB2 in four cultivars Yulinglong,Jinqiugui,Yanhonggui and Rixianggui,were amplified with RT-PCR technique and verificated by sequencing method in this study. Bioinformatics analysis revealed that ORF sequences of all HYB1 genes in four cultivars were 909 bp,encoding 302 amino acid residues,while ORF sequences of all HYB2 genes in four cultivars were 915 bp,encoding 304 amino acid residues. Conserved histidine structure domains were found present in the predicted amino acid residues of HYB1 and HYB2 in four cultivars. There were 27 nucleotide mutation sites in the HYB1 gene sequences and 8 amino acid residues mutation sites in its predicted protein sequences in four cultivars. Accordingly,there were 12 nucleotide mutation sites in the HYB2 gene sequences and 3 amino acid residues mutation sites in its predicted protein sequences. Phylogenetic analysis indicated that HYB1 and HYB2 were much closer to HYB proteins from Lycopersicon esculentum and Capsicum annuum of Solanaceae plants.
To reveal the sequence difference of β-carotene hydroxylase( HYB) genes in different cultivars of Osmanthus fragrans,based on the Unigene sequence information from petal transcriptome database of O. fragrans,the open reading frames( ORFs) of two β-carotene hydroxylase genes HYB1 and HYB2 in four cultivars Yulinglong,Jinqiugui,Yanhonggui and Rixianggui,were amplified with RT-PCR technique and verificated by sequencing method in this study. Bioinformatics analysis revealed that ORF sequences of all HYB1 genes in four cultivars were 909 bp,encoding 302 amino acid residues,while ORF sequences of all HYB2 genes in four cultivars were 915 bp,encoding 304 amino acid residues. Conserved histidine structure domains were found present in the predicted amino acid residues of HYB1 and HYB2 in four cultivars. There were 27 nucleotide mutation sites in the HYB1 gene sequences and 8 amino acid residues mutation sites in its predicted protein sequences in four cultivars. Accordingly,there were 12 nucleotide mutation sites in the HYB2 gene sequences and 3 amino acid residues mutation sites in its predicted protein sequences. Phylogenetic analysis indicated that HYB1 and HYB2 were much closer to HYB proteins from Lycopersicon esculentum and Capsicum annuum of Solanaceae plants.
引文
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[12] BOUVIER F,KELLER Y,D’HARLINGUE A,et al.Xanthophyll biosynthesis:Molecular and functional characterization of carotenoid hydroxylases from pepper fruits(Capsicum annuum L.)[J]. Biochimica Et Biophysica Acta,1998,1391(3):320-328.
[13]张超,刘玉成,王艺光,等.桂花类胡萝卜素异构酶基因的克隆及表达分析[J].生物技术通报,2017,33(6):89-96.
[14]张超,罗云,王艺光,等.桂花15-顺式-ζ-胡萝卜素异构酶基因(Z-ISO)的克隆及表达分析[J].农业生物技术学报,2016,24(10):1512-1521.
[2] WANG Y,ZHANG C,DONG B,et al. Carotenoid accumulation and its contribution to flower coloration of Osmanthus fragrans[J]. Frontier in Plant Science,2018,9:1499.
[3] HAN Y,WANG X,CHEN W,et al. Differential expression of carotenoid-related genes determines diversified carotenoid coloration in flower petal of Osmanthus fragrans[J].Tree Genetics&Genomes,2014,10(2):329-338.
[4] ZHU C F,BAI C,SANAHUJA G,et al. The regulation of carotenoid pigmentation in flowers[J]. Archives of Biochemistry&Biophysics,2010,504(1):132-141.
[5]廖正平,郑益平,罗鹏,等.中国水仙Nt BCH基因的克隆及LCYE RNAi与反义BCH双价植物表达载体的构建[J].热带作物学报,2013,34(12):2382-2390.
[6]周兴文,周银慧,赵英,等.金花茶Cn BCH基因c DNA全长克隆及生物信息学分析[J].分子植物育种,2017,15(1):77-83.
[7] WANG H M,TO K Y,LAI H M,et al. Modification of flower colour by suppressingβ-ring carotene hydroxylase genes in Oncidium[J]. Plant Biology,2016,18(2):220-229.
[8] ZHANG C,WANG Y,FU J,et al. Transcriptomic analysis and carotenogenic gene expression related to petal coloration in Osmanthus fragrans ‘Yanhong Gui’[J]. TreesStructure and Function,2016,30(4):1207-1223.
[9]冯紫洲,李平,张继星,等.蓖麻BCH基因的克隆与序列分析[J].内蒙古民族大学学报(自然科学版),2016,31(5):408-415.
[10]孙化雨,陈颖,赵韩生,等.毛竹β-胡萝卜素羟化酶基因的分子特征及其功能[J].林业科学,2015,51(10):53-59.
[11]冯唐锴,李思光,汪艳璐,等.南丰蜜橘β-胡萝卜素羟化酶基因的克隆和序列分析[J].西北植物学报,2007,27(2):238-243.
[12] BOUVIER F,KELLER Y,D’HARLINGUE A,et al.Xanthophyll biosynthesis:Molecular and functional characterization of carotenoid hydroxylases from pepper fruits(Capsicum annuum L.)[J]. Biochimica Et Biophysica Acta,1998,1391(3):320-328.
[13]张超,刘玉成,王艺光,等.桂花类胡萝卜素异构酶基因的克隆及表达分析[J].生物技术通报,2017,33(6):89-96.
[14]张超,罗云,王艺光,等.桂花15-顺式-ζ-胡萝卜素异构酶基因(Z-ISO)的克隆及表达分析[J].农业生物技术学报,2016,24(10):1512-1521.