CAJ | 학술논문

利用荧光定量PCR方法检测毕赤酵母外源β甘露聚糖酶基因（ man）拷贝数。以毕赤酵母中高度保守基因三磷酸甘油醛脱氢酶基因（ gap）为内参基因，分别构建含有gap和man的克隆质粒，进行RT PCR反应构建gap和man双标准曲线；获得的双标准曲线具有良好的重复性，其相关系数（ R2）均为0?999，其扩增效率分别为105?1％和102?2％。提取含有外源man基因的毕赤酵母基因组进行RT PCR检测，通过双标准曲线计算出外源man基因的拷贝数。结果显示：预先经过博莱霉素（ Zeoicn）抗性筛选得到的10株毕赤酵母重组菌株中含有1、2、3、4、5和7个不等拷贝的β甘露聚糖酶基因。结果表明该方法能够高效快速筛选和鉴定出含有不同外源β甘露聚糖酶基因拷贝数的毕赤酵母重组菌株。
이용형광정량PCR방법검측필적효모외원β감로취당매기인（ man）고패수。이필적효모중고도보수기인삼린산감유철탈경매기인（ gap）위내삼기인，분별구건함유gap화man적극륭질립，진행RT PCR반응구건gap화man쌍표준곡선；획득적쌍표준곡선구유량호적중복성，기상관계수（ R2）균위0?999，기확증효솔분별위105?1％화102?2％。제취함유외원man기인적필적효모기인조진행RT PCR검측，통과쌍표준곡선계산출외원man기인적고패수。결과현시：예선경과박래매소（ Zeoicn）항성사선득도적10주필적효모중조균주중함유1、2、3、4、5화7개불등고패적β감로취당매기인。결과표명해방법능구고효쾌속사선화감정출함유불동외원β감로취당매기인고패수적필적효모중조균주。
The copy number of heterologousβ-mannanase gene ( man) in Pichia pastoris were detected by real-time fluorescent quantitative PCR. The gap gene highly conserved in Pichia pastoris was chosen as the reference gene. The standard curves of gap and Man were generated with the standard plasmids containing gap and man, respectively. Both standard curves had good repeatability with the correlation coefficients ( R2 ) of 0?999,and their amplification efficiencies were 105?1% and 102?2%,respectively. The copy number of man gene in Pichia genome was determined by RT-PCR and calculated according to the double standard curves. Ten recombinant P?pastoris strains screened by Zeocin resistance harbored 1, 2,3,4,5 and 7 copies of man. The above method could be used for efficiently and quickly screening Pichia pastoris recombinants with multi-copy Man and determining copy numbers of man.