CAJ | 학술논문

[目的]克隆蝴蝶兰ACC合成酶cDNA片段,构建其反义表达载体.[方法]根据已报道的蝴蝶兰ACC合成酶的基因序列,设计并合成了1对引物,通过RT-PCR以蝴蝶兰总RNA中扩增出ACC合成酶的cDNA片段,将其连接到MD19-T质粒载体上进行测序.用Xba Ⅰ和Sac Ⅰ对重组质粒和pBI221酶切后连接,构建蝴蝶兰ACC合成酶的反义基因表达载体.[结果]获得了蝴蝶兰ACC合成酶的cDNA片段,测序结果显示,该片段与参考的已报道序列具有98.92%和76.36%的同源性.通过引入Xba Ⅰ和Sac Ⅰ酶切位点,进行载体构建,构建了蝴蝶兰ACC合成酶的反义基因表达载体.[结论]成功构建了蝴蝶兰ACC合成酶的反义基因植物表达载体,为进一步研究其对蝴蝶兰花期和生长的影响打下了基础.
[목적]극륭호접란ACC합성매cDNA편단,구건기반의표체재체.[방법]근거이보도적호접란ACC합성매적기인서렬,설계병합성료1대인물,통과RT-PCR이호접란총RNA중확증출ACC합성매적cDNA편단,장기련접도MD19-T질립재체상진행측서.용Xba Ⅰ화Sac Ⅰ대중조질립화pBI221매절후련접,구건호접란ACC합성매적반의기인표체재체.[결과]획득료호접란ACC합성매적cDNA편단,측서결과현시,해편단여삼고적이보도서렬구유98.92%화76.36%적동원성.통과인입Xba Ⅰ화Sac Ⅰ매절위점,진행재체구건,구건료호접란ACC합성매적반의기인표체재체.[결론]성공구건료호접란ACC합성매적반의기인식물표체재체,위진일보연구기대호접란화기화생장적영향타하료기출.
[Objective] The purpose of this reseach was to clone the cDNA fragment of ACC synthase (1-aminocyclopropane-1-carboxylate synthase,ACS)of phalaenopsis and to construct its antisense expression vector.[Method] A pair of specific primers were designed and synthesized according to the reported cDNA sequences of phalanopsis ACC synthase gene (GenBank:Z77854.1; AF004663.1).The partial cDNA of ACS was obtained from total RNA of phalaenopsis by RT-PCR.Then the cDNA fragment was linked to T-tailing pMD19-T vector for cloning and sequencing.After the recombinant plasmid and plant expression vector pBI221 both being digested with Sac I and Xba I,the fragment of ACS was ligated to the CaMV35S promoter downstream of pBI221 in antisense orientation to construct the antisense expression vector.[Result] The cDNA fragment of phalaenopsis ACS was cloned.The sequencing results showed that the obtained sequence was made up of 461 bp and the homologous rate was 98.92% and 76.36% compared with reported sequences.Using appropriated site of restriction endonuclease,the antisense expression vector of phalaenopsis ACS was successfully constructed.[Conclusion] The antisense plant expression vector of phalaenopsis ACS was constructed,which laid foundation for further study of the effects on florescene and growth of phalanopsis.