The mutant pel - d92l was expressed in pichia pastoris gs115 , sds - page detection showed that the expression product pel - d92l - gs is different from pel - gs , and its " yield decreased dramatically , the themostability of pel - d92l - gs is also different from the pel - gs , but their optimum temperatures are same . 3 . directed evolution of pel through random mutagenesis mutagenesis pcr carried out in error - prone conditions was used on the vector psk - pel , using the oligos " beginning " and " end " , homologous to the 5 ' and to the 3 " ends of the gene of pel respectively 三、 pel基因的随机诱变用易错pcr方法对pel基因进行随机诱变， pcr产物与ppic3 . 5k连接，转化大肠杆菌，获得的混合质粒电转化毕赤酵母gs115 ， omm平板筛选适于低温或对热稳定的重组子，筛选获得一株最活反应温度、热稳定性、发酵酶活均有提高的突变体pel - ep5 - gs ，其最活反应温度为45 ，比野生型高出5 ； 40处理30min残留活性为56 ，大大高于野生型的6 ；初始ph7 . 3528条件下培养72h ，发酵上清酶活为325u / ml 。
The 38mda plasmid containing cryla ( b ) and cryii genes from ybt - 791 was purified and transformed by electroporation into the acrystalliferous mutants 4d10 ( serotype h3ab ) and bti ips 78 / 11 ( serotype h14 ) . the two different serotype transformants , mbl - 1 and mbl - 2 harboring cryl a ( b ) and cryii were obtained , respectively . sds - page analysis showed that the transformants could express 130kda and 65kda proteins 并将携带有cry a ( b )和cry基因的38mda质粒分别电转化到不同血清型苏云金芽胞杆菌无晶体突变株4d10 ( h _ ( 3ab ) )和btiips ? 78 11 ( h _ ( 14 ) )中，证明了相同基因型在不同血清型受体中有不同的杀虫活性。
In this article , the misgurin gene and adaptor are synthesized according to the amino acid sequence reported in the genebank and the need of construction and expression . adopted a new strategy , multiple copy gene is ligated in the same direction . and then it is cloned into expression vector plasmid ppic9k 本文根据genebank登录的氨基酸序列，同时考虑构建和表达的需要，化学合成了misgurin基因和接头，采用一种新的策略，在体外将基因多拷贝同向串连，并将其克隆于表达质粒ppic9k中，通过鉴定并测序正确后，电转化真核表达宿主? ?毕赤酵母菌株gs115 ，通过营养缺陷型培养基筛选重组子，再利用g418抗性筛选出整合有多拷贝外源基因的重组子。
Kustaki : ori44 , oriw30 and ori2062 were chosen to construct the gfp resolution vector . there were 9 kinds of gfp resolution vector . all of the recombinant plasmids were introduced into crystal negative b . thuringiensis 8mb 171 . the fluorescence of the 9 kinds engineered strain can be detected by the fluorescent microscope 将得到的9种gfp解离载体电转化到苏云金芽胞杆菌无晶体突变株bmb171中，荧光显微镜镜检显示9株含gfp的苏云金芽胞杆菌重组菌在波长为300nm - 510nm的激发光下可发射绿色荧光。
