兔眼蓝莓组培红色突变株CHS基因的克隆与分析
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摘要
蓝莓Vaccinium spp.果实含有丰富的花青素,但目前对蓝莓花青素合成相关基因的研究还较少。为分析蓝莓花青素合成相关基因,也为今后高花青素含量品种的育种工作提供一定的生物信息学基础,以红色突变兔眼蓝莓Vaccinium ashei为试验材料,采用同源克隆方法 ,克隆到查尔酮合成酶基因(CHS)的cDNA全长。生物信息学分析表明:该基因cDNA全长1 398 bp,有1个1 170 bp的完整开放阅读框(ORF),编码389个氨基酸。与杜鹃Rhododendron simsii,山茶Camellia japonica,红花油茶Camellia chekiangoleosa,中华猕猴桃Actinidia chinensis,高丛蓝莓Vaccinium corymbosum,苹果Malus domestica的CHS基因同源性分别为90%,83%,83%,83%,83%与82%。兔眼蓝莓CHS基因编码的蛋白不存在信号肽,无跨膜结构域,定位于细胞质中,肽链表现为疏水性,α-螺旋和随机卷曲是其蛋白质二级结构中量最大的结构元件,β-转角和β-折叠散布于整个蛋白质中,对编码的蛋白的结构域预测显示该蛋白含有类似查尔酮合成酶超家族(CHS-like超家族)结构域。由此推测:兔眼蓝莓CHS基因是高度保守的,表达的蛋白为疏水性蛋白,是一种结构和性质都相对稳定的蛋白。
Blueberries(Vaccinium spp.) have attracted much attention because of their abundant anthocyanins;however,there have been few studies on the genes related to anthocyanin biosynthesis in blueberries.To analyze related genes of anthocyanin biosynthesis in blueberries and also to provide some bioinformatics basis for breeding of plants with a high anthocyanin content,a red mutant Vaccinium ashei was selected as the experimental material,and the full-length cDNA of chalcone synthase gene(CHS) was cloned by the homologous cloning method.The bioinformatics analysis showed that the full-length cDNA was 1 398 bp and had an openreading frame(ORF) which was 1 170 bp encoding 389 amino acids.The homology on the nucleotide level of the CHS gene with the CHS genes of several species were 90% for Rhododendron simsii;83% for Camellia japonica,Camellia chekiangoleosa,Actinidia chinensis,and Vaccinium corymbosum;and 82% for Malus domestica.The CHS protein of Vaccinium ashei had no signal peptide and no transmembrane domain.It was located in the cytoplasm,the peptide chain was hydrophobic,the α-helix and random coil were the largest constructional elements in the protein secondary structure,and β-turns and β-sheets were interspersed throughout the protein.The domain prediction of the encoded protein showed that the protein contained the chalcone synthase superfamily(CHS-like superfamily) domain.Thus inferred that the CHS gene of rabbit-eye blueberry was highly conserved,the expressed protein was hydrophobic,and it was a kind of protein with a relatively stable structure and properties.
Blueberries(Vaccinium spp.) have attracted much attention because of their abundant anthocyanins;however,there have been few studies on the genes related to anthocyanin biosynthesis in blueberries.To analyze related genes of anthocyanin biosynthesis in blueberries and also to provide some bioinformatics basis for breeding of plants with a high anthocyanin content,a red mutant Vaccinium ashei was selected as the experimental material,and the full-length cDNA of chalcone synthase gene(CHS) was cloned by the homologous cloning method.The bioinformatics analysis showed that the full-length cDNA was 1 398 bp and had an openreading frame(ORF) which was 1 170 bp encoding 389 amino acids.The homology on the nucleotide level of the CHS gene with the CHS genes of several species were 90% for Rhododendron simsii;83% for Camellia japonica,Camellia chekiangoleosa,Actinidia chinensis,and Vaccinium corymbosum;and 82% for Malus domestica.The CHS protein of Vaccinium ashei had no signal peptide and no transmembrane domain.It was located in the cytoplasm,the peptide chain was hydrophobic,the α-helix and random coil were the largest constructional elements in the protein secondary structure,and β-turns and β-sheets were interspersed throughout the protein.The domain prediction of the encoded protein showed that the protein contained the chalcone synthase superfamily(CHS-like superfamily) domain.Thus inferred that the CHS gene of rabbit-eye blueberry was highly conserved,the expressed protein was hydrophobic,and it was a kind of protein with a relatively stable structure and properties.
引文
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[14]WANG Yong,LI Jing,XIA Renxue.Expression of chalcone synthase and chalcone isomerase genes and accumulation of corresponding flavonoids during fruit maturation of Guoqing No.4 satsuma mandarin(Citrus unshiu Marcow)[J].Sci Hortic,2010,125(2):110-116.
[15]BERNARDI J,LICCIARDELLO C,RUSSO M P,et al.Use of a custom array to study differentially expressed genes during blood orange(Citrus sinensis L.Osbeck)ripening[J].J Plant Physiol,2010,167(4):301-310.
[16]ZHANG Xiaodong,ALLAN A C,YI Qiong,et al.Differential gene expression analysis of Yunnan red pear,Pyrus pyrifolia,during fruit skin coloration[J].Plant Mol Biol Rep,2011,29(2):305-314.
[17]FENG Shouqian,WANG Yanling,YANG Song,et al.Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor Py MYB10[J].Planta,2010,232(1):245-255.
[18]CRIFO T,PUGLISI I,PETRONE G,et al.Expression analysis in response to low temperature stress in blood oranges:implication of the flavonoid biosynthetic pathway[J].Gene,2011,476(1/2):1-9.
[19]杨丽,刘雅莉,王跃进,等.百合查尔酮合成酶(CHS)基因的克隆与分析[J].西北植物学报,2006,26(5):933-936.YANG Li,LIU Yali,WANG Yuejin,et al.Cloning and analysis of chalcone synthase genes in Lilium[J].Acta Bot Boreal-Occident Sin,2006,26(5):933-936.
[20]KATIYAR A,SMITA S,LENKA S K,et al.Genomewide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis[J].BMC Genom,2012,13(1):544.doi:10.1186/1471-2164-13-544.
[2]Al-TAMEMI S,Al-ZADJALI S,Al-GHAFRI F,et al.Chediak-higashi syndrome:novel mutation of the CHS1/LYS T gene in 3 Omani patients[J].J Pediatr Hematol Oncol,2014,36(4):248-250.
[3]WARD D M,SHIFLETT S L,HUYNH D,et al.Use of expression constructs to dissect the functional domains of the CHS/beige protein:identification of multiple phenotypes[J].Traffic,2003,4(6):403-415.
[4]JEONG S T,GOTO-YAMAMOTO N,HASHIZUME K,et al.Expression of multi-copy flavonoid pathway genes coincides with anthocyanin,flavonol and flavan-3-ol accumulation of grapevine[J].Vitis,2008,47(3):135-140.
[5]GOTO-YAMAMOTO N,WAN G H,MASAKI K,et al.Structure and transcription of three chalcone synthase genes of grapevine(Vitis vinifera)[J].Plant Sci,2002,162(6):867-872.
[6]MORIGUCHI T,KITA M,TOMONO Y,et al.One type of chalcone synthase gene expressed during em bryogenesis regulates the flavonoid accumulation in citrus cell cultures[J].Plant Cell Physiol,1999,40(6):651-655.
[7]LU Xu,ZHOU Wei,GAO Feng.Cloning,characterization and localization of CHS gene from blood orange,Cit rus sinensis(L.)Osbeck cv.Ruby[J].Mol Biol Rep,2009,36(7):1983-1990.
[8]FERRER J L,JEZ J M,BOWMAN M E,et al.Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis[J].Nat Struct Biol,1999,6(8):775-784.
[9]JEZ J M,NOEL J P.Mechanism of chalcone synthase:p Ka of the catalytic cysteine and the role of the conserved histidine in a plant polyketide synthase[J].J Biol Chem,2001,275(50):39640-39646.
[10]ELOMAA P,HONKANEN J,PUSKA R,et al.Agrobacterium-mediated transfer of antisense chalcone synthase c DNA to Gerbera hybrida inhibits flower pigmentation[J].Nat Biotechnol,1993,11(4):508-511.
[11]KAMIISHI Y,OTANI M,TAKAGI H,et al.Flower color alteration in the liliaceous ornamental Tricyrtis sp.by RNA interference-mediated suppression of the chalcone synthase gene[J].Mol Breed,2012,30(2):671-680.
[12]MORITA Y,SAITO R,BAN Y,et al.Tandemly arranged chalcone synthase a genes contribute to the spatially regulated expression of si RNA and the natural bicolor floral phenotype in Petunia hybrid[J].Plant J Cell Mol Biol,2012,70(5):739-749.
[13]HONDA C,KOTODA N,WADA M,et al.Anthocyanin biosynthetic genes are coordinately expressed during red coloration in apple skin[J].Plant Physiol Biochem,2002,40(11):955-962.
[14]WANG Yong,LI Jing,XIA Renxue.Expression of chalcone synthase and chalcone isomerase genes and accumulation of corresponding flavonoids during fruit maturation of Guoqing No.4 satsuma mandarin(Citrus unshiu Marcow)[J].Sci Hortic,2010,125(2):110-116.
[15]BERNARDI J,LICCIARDELLO C,RUSSO M P,et al.Use of a custom array to study differentially expressed genes during blood orange(Citrus sinensis L.Osbeck)ripening[J].J Plant Physiol,2010,167(4):301-310.
[16]ZHANG Xiaodong,ALLAN A C,YI Qiong,et al.Differential gene expression analysis of Yunnan red pear,Pyrus pyrifolia,during fruit skin coloration[J].Plant Mol Biol Rep,2011,29(2):305-314.
[17]FENG Shouqian,WANG Yanling,YANG Song,et al.Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor Py MYB10[J].Planta,2010,232(1):245-255.
[18]CRIFO T,PUGLISI I,PETRONE G,et al.Expression analysis in response to low temperature stress in blood oranges:implication of the flavonoid biosynthetic pathway[J].Gene,2011,476(1/2):1-9.
[19]杨丽,刘雅莉,王跃进,等.百合查尔酮合成酶(CHS)基因的克隆与分析[J].西北植物学报,2006,26(5):933-936.YANG Li,LIU Yali,WANG Yuejin,et al.Cloning and analysis of chalcone synthase genes in Lilium[J].Acta Bot Boreal-Occident Sin,2006,26(5):933-936.
[20]KATIYAR A,SMITA S,LENKA S K,et al.Genomewide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis[J].BMC Genom,2012,13(1):544.doi:10.1186/1471-2164-13-544.