CAJ | 학술논문

试验首先对质粒pGSA1285进行改造,将pFGC5941质粒的查尔酮合酶的Intron片段取代pGSA1285的GUS片段,构建含有内含子并具有卡那抗性的pGSA2285植物沉默表达载体.同时设计引物、PCR扩增、在BI-1基因两端各加上两个酶切位点,将BI-1基因分别反向、正向插入改造后的质粒pGSA2285 Intron片段两端的多克隆位点中,构建得到烟草BI-1基因沉默载体pGSA4285.此沉默质粒经PCR鉴定、限制性酶切分析以及回复动员试验证明已正确构建并成功转入农杆菌,为获得含有BI-1基因沉默烟草植株及其在植物程序性死亡中调控作用的研究奠定试验基础.
시험수선대질립pGSA1285진행개조,장pFGC5941질립적사이동합매적Intron편단취대pGSA1285적GUS편단,구건함유내함자병구유잡나항성적pGSA2285식물침묵표체재체.동시설계인물、PCR확증、재BI-1기인량단각가상량개매절위점,장BI-1기인분별반향、정향삽입개조후적질립pGSA2285 Intron편단량단적다극륭위점중,구건득도연초BI-1기인침묵재체pGSA4285.차침묵질립경PCR감정、한제성매절분석이급회복동원시험증명이정학구건병성공전입농간균,위획득함유BI-1기인침묵연초식주급기재식물정서성사망중조공작용적연구전정시험기출.
The plasmid pGSA1285 was modified by substituting its GUS sequence with the intron sequence contained in vector pFGC5941. Using PCR-based amplification, two different restriction sites in each end of tobacco BI-1 gene were created, which made the construction of ihpRNA gene silencing vector more efficient. Then BI-1 gene were ligated into Multiple Cloning Site (MCS) besides the intron sequence of modified vector pGSA2285, respectively to form B/-1 ihpRNA gene silencing vector, named pGSA4285, containing sense and antisense B/-1 sequence. Through the identification of PCR and enzyme restriction analysis, the results showed that BI-1 ihpRNA gene silencing vector had been constructed and introduced into Agrobacterium tumefaciens EHA105 successfully, which laid a foundation for the further study on the function of BI-1 in PCD regulation in plants.