‘红阳’猕猴桃cDNA文库构建及F3H基因的表达初探

Construction of cDNA library of ‘Hongyang’ kiwifruit and analysis of F3H expression

利用SMART（switching mechanism at 5’end of the RNA transcript）技术构建了红肉猕猴桃品种‘红阳’（Actinidia chinesis cv‘Hongyang’）内果皮组织的全长cDNA文库，此文库的构建有助于克隆与次生代谢相关的基因，特别是红肉猕猴桃花青素特异合成代谢的基因。文库滴度为6．7×10^4cfu／mL，库容为2．72×10^8cfu／mL，文库重组率99．8％，插入片段多数分布在700～1000bp。随机挑选1014个克隆进行测序，测序成功963个，经过序列拼接去除低质量序列后获得632个unigenes，包括92个contigs和540个singletons，获得已知功能unigenes共441个。从所测克隆中得到一个花青素途径的结构基因AcF3H，其cDNA序列长1369bp（GenBank登录号：FJ542819），CDS区为1101bp，编码366个氨基酸的多肽。与拟南芥、葡萄及龙胆已知乃日氨基酸序列比对，发现该基因十分保守。通过RT—PCR技术对苍溪栽培的不同发育时期‘红阳’猕猴桃果实AcF3H基因的表达进行了分析。结果表明，果肉转色前AcF3H基因表达量较高，而转色初期表达量降低，此后随着果实着色加深表达量维持在较高水平。

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 6.7×10^4 cfu/mL and 2.72×10^8 cfu/mL, respectively. The recombination 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, including 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 encoded 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.