‘变叶海棠’幼叶cDNA文库构建及CHS基因克隆

摘要

为了理解苹果类黄酮代谢的分子机制,以富含类黄酮‘变叶海棠,苹果幼叶为材料,利用SMART法构建一个苹果叶片全长cDNA文库.初级文库滴度为2.28×104 cfu/mL,库容为3.30×106个独立克隆,重组率100％,插入片段平均长度大于1.5 kb.并对随机取16个重组子进行测序,经分析完整全长cDNA为7条.并从测序中获得一个查尔酮合酶(Chalcone sythase,CHS)基因,该基因cDNA全长1548 bp,有一个1170 bp的开放阅读框,编码含389个氨基酸残基的蛋白,蛋白的理论分子质量为42.44 KDa,等电点5.65,该基因与已知苹果CHS基因高度同源.并构建该基因植物表达载体,用冻融法将其导入根癌农杆菌EHA105.%To understand the molecular mechanisms of apple flavonoids metabolism, we established a full-length cDNA library from the young leaf of Malus toringoides (Rehd. ) by the SMART technique. The titers of the primary library and the capacity were 2. 28 104 cfu/mL and 3. 30 106 clones, respectively. The average size of cDNA inserts was above 1500 bp with a recombination rate of 100%. 16 clones were randomly sequenced and of 7 of which contained full length cDNA sequences, including a chalcone synthase gene. The length of the MdCHS cDNA was 1 548 bp. Bioinformatic analysis showed that MdCHS ORF region was 1170 bp, which encoded a peptide 389 amino acids with molecular weight 42. 44 kD and pi 5. 65. The ami no acid sequences of this gene shared high homology to the known apple CHS gene. Plant expression vectors for genetic transformation was successfully constructed and transformed into Agrobacterium tumefaciens strain EHA105 by a freeze-thaw method. These studies lay the foundation for Agrobacterium-mediated transformation of CHS gene and cloning of other flavonoids-related gene.