烟曲霉Af293 chsD基因缺失突变株的构建

Construction of Aspergillus Fumigatus Af293 chsD Deletion Mutant Strains

目的构建Af293 chs D缺失突变株,并对chs D的功能进行初步的研究。方法 PCR扩增chs D两侧部分序列并连接至p PK2质粒的潮霉素表达盒两端,从而构建重组质粒p PK2/chs D-(L+R)。酶切p PK2/chs D-(L+R)并胶回收含chs D左侧、hph表达盒及chs D右侧的片段,连接至p DHt/SK而构建二元质粒p DHt/chs D::hph。经ATMT方法将p DHt/chs D::hph转化至Af293,经潮霉素抗性筛选从而获得烟曲霉△chs D突变株,并测量Af293,△chs D突变株生长直径及扫描电镜下观察分生孢子形态。结果经PCR初步鉴定,筛选出的突变株中,其染色体中的chs D序列已被hph置换,初步形态研究表明,△chs D突变株的生长速度显著低于Af293野生型,且分生孢子凹陷呈干瘪状。结论成功获得Af293△chs D突变株。chs D基因功能初步研究表明,chs D可能与烟曲霉的生长相关。

Objective To construct Aspergilhu fumigatus Af293 chsD deletion mutant strains and investigate the func- tion of chsD. Methods Firstly, pPK2/chsD-（ L ＋ R） was constructed by cloning the left and right flanks of chsD sequence and inserting into the 5＇ and 3＇ end of hph cassette in pPK2, respectively. Then, a 5.6kb frag- ment, which includes the left, right flanks of chsD and the hph cassette was ligated into pDHt/sk to construct a recombinant plasmid, pDHt/chsD： ： hph. Further, pDHt/chsD： ： hph was transformed into Af293 by ATMT method. M293 LXchsD mutants were screened by hph resistance. The colonies diamer of Af293 and AchsD mutants were measured, and spore morphology of wild and LkchsD mutant strains were examined by SEM. Re- suits The chsD sequence in Af293 genome was replaced by hph,which was comfirmed by PCR assays. Pre- liminary morphology study suggest that the growth velocity of AchsD mutant was obviously slower than wild Af293 （P 〈 0.05 ）, and the spore of A chsD mutant was collapsed in the middle, presenting withered shape. Conclusion Af293 AchsD mutant strains were constructed,and chsD may play an important role in Asper- gillus fumigatus growth.