三种不同抗冻性葡萄中CBF2基因的生物信息学分析及植物表达载体构建

Bioinformatics analysis of CBF2 in three different chilling resistance grapes and construction of plant expression vector

关键词:  葡萄  CBF2转录激活因子  生物信息学分析  克隆  表达载体构建

Key words:  grapes  CBF2 transcriptional activator  bioinformatic analysis  cloning  expression vector construction

摘要:

从赤霞珠、贝达和山葡萄三种不同抗冻性葡萄的基因组DNA中分别克隆了CBF2全长基因，并利用生物信息学的方法进行了分析。结果显示：三种葡萄的CBF2基因长均为969 bp，编码253 个氨基酸，与GeneBank公布的的序列相比对，核苷酸相似性在96.7%～97.94%之间。生物信息学分析显示3种不同抗冻葡萄的CBF2基因具有连续的开放阅读框，推倒的氨基酸序列具有AP2结合域，初步判断克隆的CBF2具有诱导抗寒性产生的作用。但氨基酸序列、进化树、理化性质、疏水性/亲水性等在抗寒性较强的山葡萄、贝达与抗寒性较差的赤霞珠之间存在一些差异，与GenBank上的原始序列同源性最低的是赤霞珠，而且其CBF2 N端7个氨基酸残基序列与山葡萄和贝达的氨基酸序列存在较大差异，且具有较强的疏水性，这些差异可能与赤霞珠较弱的抗寒特性有关。以带有35S启动子的载体pBI121为基础，成功构建了植物表达载体pBI121 CaMV35S CBF2，为深入研究CBF2基因在葡萄抗寒性中的作用提供了基础资料。

Abstract:

The full length gene of C repeat binding factor 2 was amplified from the genomic DNA in three different chilling resistance grapes:Cabernet sauvignon，Beta and Vitis amurensis. The methods of bioinformatics were also applied to analyze the sequencing results. Sequencing results showed that the cloned CBF2 gene in three different chilling resistance grapes had 969 bp and encoded 253 amino acids，and had 96.7%-97.94% identity with the sequence published on GeneBank. The bioinformatics analysis showed thatCBF2 from three different chilling resistance grapes had continuous opening reading frame，and the deduced amino acid sequence also had AP2 binding domain，so we should have preliminary judegement of cold resistance effect of cloned CBF2. But the analysis from the homology of amino acid sequences，phylogenetic tree，physico chemical property and hydrophobicity and hydrophilia showed that there were some differences among the high chilling resistance grape Vitis amurensis and Beta，and the low chilling resistance grape Cabernet sauvignon.The comparison of sequence homology with the sequence published on GeneBank showed that the worst one was C.sauvignon；and the 7 amino acids from N terminal in C.sauvignon were totally different with Vitis amurensis and Beta；the difference might be related to the higher hydrophobicity in the head of amino acid residues from Cabernet sauvignon. Based on the vector pBI121 with promoter 35S，the plant expression vector pBI121 CaMV35S CBF2 was constructed successfully，which could offer the basic researching data to improve the cold tolerance ability of grape.