CAJ | 학술논문

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2.72×10~8scfu/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1369 bp(GenBank登录号:FJ54 2819),CDS区为1101 bp,编码366个氨基酸的多肽.与拟南芥、葡萄及龙胆已知F3H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.
이용SMART(switching mechanism at 5' end of the RNA transcript)기술구건료홍육미후도품충‘홍양'(Actinidia chinesis CV‘Hongyang')내과피조직적전장cDNA문고,차문고적구건유조우극륭여차생대사상관적기인,특별시홍육미후도화청소특이합성대사적기인.문고적도위6.7×10~4cfu/mL,고용위2.72×10~8scfu/mL,문고중조솔99.8%,삽입편단다수분포재700～1000bp.수궤도선1014개극륭진행측서,측서성공963개,경과서렬병접거제저질량서렬후획득632개unigenes,포괄92개contigs화540개singletons,획득이지공능unigenes공441개.종소측극륭중득도일개화청소도경적결구기인AcF3H,기cDNA서렬장1369 bp(GenBank등록호:FJ54 2819),CDS구위1101 bp,편마366개안기산적다태.여의남개、포도급룡담이지F3H안기산서렬비대,발현해기인십분보수.통과RT-PCR기술대창계재배적불동발육시기‘홍양'미후도과실AcF3H기인적표체진행료분석.결과표명,과육전색전AcF3H기인표체량교고,이전색초기표체량강저,차후수착과실착색가심표체량유지재교고수평.
The SMART switching mechanism at 5'end of the RNA transcript technique was used to construct a cDNA tated 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 1000 bp.A total of 1014 clones randomly chosen from the cDNA library were sequenced and these expressed seqnence 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 1369 bp (GenBank accession No:FJ542819).Bioinformatic analysis showed that AcF3H ORF region was 1101 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.