用Overlap-PCR法从Trichodermareesei QM9414基因组DNA中克隆并表达木聚糖酶Ⅲ

Cloning and Expression of xynⅢ from Genomic DNA of Trichoderma reesei QM9414 by Overlap-PCR

禾本科植物木聚糖酶在其成熟过程中需在细胞进入程序性死亡后 ,经蛋白水解酶多次剪切方显活性 ,常规蛋白质克隆表达系统无法表达这类酶。通过GenBank搜索获得一与之同族、结构相似的来源于T .reeseiQM94 1 4(ATCC2 6 92 1 )突变种PC 3 7菌株的xynⅢ。但该酶在T .reeseiQM94 1 4中不表达 ,而在基因组中存在。通过overlap PCR法将 4个外显子分别克隆、测序 ,再连接测序 ,最终获得该基因的全长cDNA序列。将该基因连接到表达载体pETBlue 2上 ,并转化到TunerDE3表达菌株中 ,常规条件下可表达并有木聚糖酶活性显示。低温 (1 5℃ ) 6

After the cell enters into its programmed cell death, xylanases from grass plants gradually matured through its N terminal and C terminal sequence been cut by acid proteases several times. They could not be expressed by conventional protein expression system. Search the GenBank database, xynIII from a mutant of T. reesei QM9414(ATCC26921)was found. It is similar to grass plants’ xylanase in their families and structures. It couldn’t express in T. reesei QM9414, but its gene exist in genomic DNA as one copy. Through overlap PCR method, 4 exons of xynIII were cloned, sequenced, spliced, and the whole cDNA of mature xynⅢ was acquired. The cDNA was inserted into pETBlue 2 vector and transformed into E. coli DE3 pLacI cell. XynⅢ could be expressed in the transformed cell under the conditions of 37℃,1mmol/L IPTG induced for 3h. Low temperature (15℃), long time(64h) induction(0 2 mmol/L IPTG) could enhance xynⅢ activity.