杨梅果糖激酶基因MrFRK2的克隆及在成熟期果实中的表达分析

以‘荸荠’和‘东魁’杨梅果实为研究材料,利用RT-PCR和RACE技术克隆得到果糖激酶基因MrF RK2的全长c DNA序列。MrF RK2全长1 279 bp,ORF阅读框全长996 bp,3′端非编码区227bp,5′端非编码区56 bp,编码332个氨基酸残基。通过氨基酸序列分析发现Mr FRK2含有pfk B碳水化合物磷酸果糖激酶家族高度保守的1个ATP结合域和2个底物结合域;Mr FRK2与杨树Pt FRK2相似性最高,达到85%。利用实时荧光定量PCR技术分析基因表达的结果表明,Mr FRK2在‘东魁’杨梅果实的发育早期大量表达,在成熟后期表达量下降,果实成熟初期Mr FPK2表达量‘东魁’显著高于‘荸荠’。果实成熟期间果糖含量‘东魁’显著高于‘荸荠’,与果实成熟期间Mr FPK2的表达量相反。这表明,MrF PK2在杨梅果实成熟期间果糖降解过程中可能起一定作用。

Molecular Cloning and Expression Analysis of MrFRK2 in Chinese Bayberry During Fruit Ripening

The full length cDNA of MrFRK2 was obtained by using RT-PCR and RACE amplification from Chinese bayberry（Myrica rubra Sieb. and Zucc.）‘Biqi’and‘Dongkui’fruit. MrFRK2 was 1 279 bp in full length and encoded a predicted protein of 332 amino acids（ORF length 996 bp），and flanked by 56 nucleotides at the 5′-UTR and 227 nucleotides at the 3′-UTR. The sequence homology comparison showed that，MrFRK2 containedthree conserved regions inherent to the pfkB family of carbohydrate kinases，an ATP-binding domain，and two substrate recognition domains. Phylogenetic analysis indicated that MrFRK2 shared the highest similarity with PtFRK2 with 85% homology. Furthermore，the changes of MrFRK2 expression，fructose and sucrose content during fruit ripening were investigated by q-PCR and HPLC analysis. Meanwhile，Dongkui fruit also experienced higher levels of fructose and lower sucrose-to-fructose ratio than in Biqi fruit. Correlation analysis showed fructose content was significantly negative correlation with the expression of MrFRK2 during ripening. Therefore，our results suggested that the higher fructose content in Dongkui fruit might be associated with lower levels of MrFRK2 expression，thereby increased the sucrose-to-fructose ratio in Biqi fruit during ripening.