猕猴桃果实L-半乳糖内酯脱氢酶和脱氢抗坏血酸还原酶cDNA片段的克隆与序列分析

Cloning and sequence analysis of cDNA fragments of L-galactono-1,4-lactone dehydrogenase and dehydroascorbate reductase from fruit of Actinidia deliciosa

克隆猕猴桃果实L-半乳糖内酯脱氢酶(GalLDH)和脱氢抗坏血酸还原酶(DHAR)基因,可为揭示猕猴桃高抗坏血酸(AsA)含量的分子机制奠定基础。研究以猕猴桃果实为材料提取总RNA,采用RT-PCR法,克隆出L-GalLDH基因的cDNA片段857 bp和DHAR基因的cDNA片段594 bp并测序,根据测序结果推测其氨基酸序列,并与刺梨、拟南芥、花椰菜、甘薯和草莓等植物的GalLDH基因和DHAR基因氨基酸序列进行亲缘分析。结果表明,猕猴桃GalLDH基因氨基酸序列和核苷酸序列与刺梨的相似性高达88%,其在同源基因进化关系树中单独聚为一类;DHAR基因氨基酸序列和核苷酸序列与苹果的相似性高达98%和99%,两者在进化关系上聚为一类。可知克隆到的2个核苷酸片段确为猕猴桃L-GalLDH和DHAR基因cDNA片段,同源基因植物亲缘关系聚类与传统的形态分类有一定的差异。

In order to research the molecular mechanism of Vc accumulation,857 bp and 594 bp cDNA encoding L-galactono-l,4-1actone dehydrogenase（GalLDH）and dehydroascorbate reductase（DHAR） frag ment were cloned from fruit of Actinidia deliciosa by the method of RT-PCR, and then translated into ami- no acid sequences. In GenBank compared with Rosa roa：burghii Trat,Arabidopsis thaliana, Ipomoea bata tas. et. homologous genes plants, the nucleotide sequence and the amino acid sequence of GalLDH gene showed 88 % of identity with those of Rosa ro3cburghii Tratt. The nucleotide sequence and the amino acid sequence of DHAR gene showed 98% and 99% of identity with those apples, respectively, and they both were classified the same family. These results suggest that the both cDNA fragments are from DHAR and GalLDH gene of Actinidia deliciosa.