苹果ACO1转录因子MdHB1基因的克隆及其真核表达载体的构建

摘要

【Objective】By bioinformatics analysis,we defined preliminarily the putative sequences of apple expressed sequence tag(EST)as MdHB-1 gene.Cloning and eukaryotic expression vector construction of MdHB-1 gene in apple were conducted to further explore the molecular biology function of the gene.【Method】A Blast search of apple EST databases was performed using the LeHB-1 sequence of tomato LeACO1,and the EST sequence was identified as the closest match to LeHB-1,referred to here as MdHB-1.We designed a pair of specific primers corresponding to the identified EST sequence,and extracted total RNA from the floral organ of Royal Gala.A cDNA for about 1 000 bp was amplified by reverse transcription PCR(RTPCR).MdHB-1 and Eukaryotic vectors pPICZA were ligated.The recombinant expression vector was constructed and transformed into Pichia pastoris GS115,and then indicated by PCR.【Result】Sequence analysis showed that the fragment contains a full coding region of 1 011 bp encoding 336 amino acid residues.Its GenBank accession number is JQ678788.The results of sequence alignment analysed by Blastn and Blastp demonstrated that this MdHB-1 sequence had 50%-60% identities to the nucleotide sequence with the HB-1 genes of other plants.The amino acid sequence to MdHB-1 exhibited typical features of the conserved HD-Zip ,and the conserved amino acid residues presented more than 85% homologous traits with HD-Zip domain of LeHB-1 and AtHB-1.【Conclusion】MdHB-1 was cloned successfully from apple and eukaryotic expression vector was constructed exactly.%【目的】通过生物信息学分析，将苹果的一段EST序列初步定性为转录因子MdHB-1基因，对该基因进行克隆和真核表达载体构建，为进一步研究其分子生物学功能奠定基础。【方法】根据番茄LeACO-1转录因子LeHB-1的基因序列，对苹果EST库进行Blast，获得一段EST序列，根据该EST序列设计1对特异性引物，以“皇家嘎啦”苹果花器总RNA为材料，经RT-PCR得到1条长约1 000 bp的cDNA片段，T/A 克隆后进行序列测定及分析。将基因MdHB-1与真核表达载体pPICZA连接，构建真核表达载体pPICZA-MdHB-1，并导入毕赤酵母菌种GS115,通过 PCR鉴定该载体的导入情况。【结果】克隆到苹果MdHB-1基因（GenBank登录号为JQ678788），其开放阅读框为1 011 bp,编码336个氨基酸。Blastn和Blastp序列比对分析发现，MdHB-1与已登录的其他物种的HB-1基因相似性达50%～60%。该基因编码的氨基酸中存在保守序列HD-Zip结构域，该保守序列与已经报道的番茄LeHB-1、拟南芥AtHB-1的保守序列的同源性均大于85%。重组表达载体PICZA-MdHB-1构建正确并已成功导入酵母基因组中。【结论】成功克隆了苹果转录因子MdHB-1基因全长，并正确构建了其真核表达载体。