[Construction of cDNA library of 'Hongyang' kiwifruit and analysis of F3H expression].

ABSTRACT

The SMART switching mechanism at 5' end of the RNA transcript technique was used to construct a cDNA library from inner pericarp of the red flesh kiwifruit Actinidia chinesis cv 'Hongyang'. Construction of cDNA library facilitated cloning of the genes associated with the secondary metabolism, the specific genes in the course of anthocyanin biosynthesis. The titers of the primary library and the amplified library were 6.7x104 cfu/mL and 2.72x108 cfu/mL, respectively. The recom-bination rate was over 99.8%. The lengths of most cDNAs in the library ranged from 700 bp to 1 000 bp. A total of 1 014 clones randomly chosen from the cDNA library were sequenced and these expressed sequence tags (ESTs) were analyzed. A set of 963 sequences were obtained. Clustering and assembly of these cDNA sequences resulted in 632 unigenes, includ-ing 92 contigs and 540 singletons. Among them, 441 EST unigenes were predicted to have known functions. Gene AcF3H, which participated in anthocyanin biosynthesis from sequencing, was obtained. The length of the AcF3H cDNA was 1 369 bp (GenBank accession No FJ542819). Bioinformatic analysis showed that AcF3H ORF region was 1 101 bp, which en-coded a peptide with 366 amino acids. The amino acid sequences of this gene shared extensive homology to Arabidopsis, Vitis, and Eustoma. The expression of AcF3H was investigated in inner pericarp of 'Hongyang' at six stages during fruit development using RT-PCR. The expression level was high before colour-changed stage, and then decreased at the primary stage of pigmentation.