越橘查耳酮合酶基因的克隆及表达分析

Molecular Cloning and Expression of Chalcone Synthase Gene in Blueberry

为研究越橘花色素苷合成的分子机制,利用RT-PCR和RACE技术从越橘(Vaccinium spp.)果实中克隆了查耳酮合酶基因(CHS)的全长cDNA,命名为VcCHS,GenBank登录号为JN654702。VcCHS全长1 438 bp,包含107 bp的5'非编码区、71 bp的3'非编码区和1个长度为1 260 bp编码419个氨基酸的开放阅读框。该基因编码的蛋白具有CHS家族普遍存在的功能活性位点:Cys(C)164、His(H)303、Asn(N)336和特征多肽序列(RLM-MYQQGCFAGGTVLR)。多重比对分析发现越橘VcCHS基因编码的氨基酸序列与葡萄(Vitis vinifera)CHS基因编码的氨基酸序列相似性达90.2%。系统进化分析表明,该序列与杜鹃花目的植物聚为一类。VcCHS基因在果实发育的整个过程均有不同程度的转录表达,花期和果实成熟期表达量较高,绿果期表达量最低。VcCHS相对表达量变化与花色素苷相对含量的变化趋势具有一致性,并均在果皮组织中达到最高。

A full-length cDNA sequence of ehalcone synthase （CHS） gene was obtained from skins of the blueberry fruits using RT- PER and RACE, named VcCHS （ GenBank accession No. JN654702）. Sequence analyses indicated that VcCHS was 1 438 bp in full length and contained a 5＇-untranslated region （5＇-UTR） of 107 bp, a 3＇-UTR of 71 bp, and an opening reading frame （ORF） of 1 260 bp encoding a protein of 419 amino acids, which possessed the conserved active sites for the CHS function （ Cys（C） t64, His（H） 3o3, Ash（N） 336） and the sequence of the family signature （RLMMYQQGCFAGGTVLR）. Sequence alignment and phylogenetic tree analyses showed that VcCHS had more than 90. 2% of consistency with Vitis vinifera, and had the closest phylogenetic relationship with the plant of Ericaceae. Relative real-time PCR analyses indica- ted that the expression of VcCHS gene was detected throughout the blueberry development, exhibiting stronger in the stage of flowers and blue fruits than in green fruits. The relative expression of VcCHS had significant consistency with the content of anthocyanins, and showed the highest transcript abundance in the skins.