牡丹ACC氧化酶基因的克隆与反义载体的构建

根据报道的牡丹ACC氧化酶基因(DQ337251)cDNA序列,设计一对特异引物,以牡丹品种"洛阳红"基因组DNA为模板,用PCR扩增方法克隆出牡丹ACC氧化酶基因的部分片段,并将其连接到pMD18-T载体上进行测序.结果表明,克隆的序列全长为467 bp,其中包括一个长度为157 bp的序列,推测它可能是一个内含子,其它序列与已报道序列同源性为98.2%;用SacⅠ和XbaⅠ对重组质粒和载体pBI 121酶切、连接,构建牡丹ACC氧化酶基因的反义表达载体.

Cloning of ACC Oxidase Gene and Construction of Antisense Vector on Peony

As a respiration climacteric flower,the peony fresh (cut) flower release some endogenous ethylene which can impact aging strongly with the flower senescence.The ACC(1-aminocyclopropane-1-carboxylic acid) oxidase was one of the key rate-limiting enzymes for ethylene biosynthesis in higher plants.The studies on the expression and regulation-control of ACC oxidase have some important roles in prolonging the florescence.A pair of specific primers,P1 and P2,were designed and synthesized according to the reported cDNA sequence of the ACC oxidase gene of peony(the sequ-ence number in the GenBank was DQ337251) in this experiment.A DNA frag-ment was obtained from "luoyanghong"genomic DNA by PCR at first,and linked it to pMD18-T which was a cloning vector.Then sequencing the cloned fragment.The result shows that the whole length of the fragment was 467bp.A sequence,157bp among them,might be an intron.The homologous rate of the remaining sequence was 98.2% compared with the reported sequence.The recombined plasmid and the plant expression vector pBI 121 were digested with the Sac I and Xba I.The fragment of ACC oxidase gene from peony and pBI 121 were reclaimed and ligated,an antisense expression vector was constructed in the end.