| 摘要: |
| 为了解黄酮醇合酶在甜樱桃类黄酮生物合成途径中的作用,根据其他物种已知的FLS cDNA保守序列设计简并引物,采用RT-PCR和RACE的技术,从甜樱桃(Prunus avium.L)嫩叶中扩增获得FLS cDNA全长,命名为PaFLS(GenBank登录号:JQ289290) 。该基因cDNA全长为1 077 bp,开放阅读框为1 014 bp,编码337个氨基酸,分子质量为38 ku,等电点pI为5.58。生物信息学分析表明PaFLS属于依赖2-酮戊二酸的双加氧酶家族(2OG-FeII_Oxy),该基因所编码的氨基酸序列与苹果、梨和草莓的同源性分别为99%、97%和77%。将该基因重组到表达载体pGEX4T-1中进行原核表达,电泳检测到一条约为65 ku的融合蛋白(含27 ku的GST标签)。组织时空表达分析表明,PaFLS在甜樱桃的花中表达量最高,这可能和黄酮醇参与花粉萌发及利于授粉有关。 |
| 关键词: 甜樱桃 黄酮醇合酶基因 基因克隆 原核表达 |
| DOI：10.11841/j.issn.1007-4333.2013.02.009 |
| 投稿时间：2012-09-03 |
| 基金项目:国家自然科学基金(31171938); 公益性行业(农业)科研专项经费(201003021) |
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| Cloning and expression analysis of PacFLS in sweet cherry (Prunus avium L.) |
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LIU Ai-ling1, SHEN Xin-jie1, LIU Yun1, YUAN Hua-zhao1, ZHANG Xiao-ming2, LI Tian-hong1
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| (1.Department of Fruit Science,College of Agriculture and Biotechnology,China Agricultural University,Beijing 100193,China;2.Institute of Forestry and Pomology,Beijing Academy of Agriculture and Forestry Science,Beijing 100093,China) |
| Abstract: |
| The flavonol synthase gene is an important gene involved in the flavonoid biosynthesis pathway.A full-length cDNA sequence of FLS gene,PaFLS (GenBank accessionn number:JQ289290),was cloned from the young leaves of Prunus avium L.by using RT-PCR and RACE method.The full-length cDNA of PaFLS was 1 077 bp with an open reading frame of 1 014 bp,which encoded a putative protein of 337 amino acids with a molecular weight of about 38 kDa and an isoelectric point of 5.58.GenBank conserved domain search and function analysis revealed that PaFLS belonged to the 2-oxoglutarate iron-dependent oxygenase (2OG-FeII_Oxy) family.Sequence analysis showed that the homologies of FLS protein from P.avium with Malus domestica,Pyrus communis and Fragaria ananassa were 99%,97% and 77%,respectively.The prokaryotic expression system of pGEX-4T-1/PaFLS was constructed and transformed into E.coli BL21.The protein expression was induced by 0.5 mmol/L IPTG.A 65 kDa fusion protein was detected by SDS-PAGE.The expression levels of PaFLS in different tissues of plants were analyzed by quantitative real-time PCR.Higher level of transcription was observed in the flowers than that in the fruits,pholems and leaves,which might be associated with pollinator attraction and pollen germination. |
| Key words: Prunus avium L. flavonol synthase gene gene cloning prokaryotic expression |
