Aspergillus oryzae Transcriptome or Gene expression

In order to evaluate the role of HacA in coping with ER stress and as possible regulator of theunfolded protein response inA.oryzae, we proceeded to characterize hacAknockout (HacA-DE) and hacA in constitutively activeexpression (HacA-CA) strains. To construction hacAknockout strain, we generated oneby replacing thehacA(AO090124000074)coding region with a pyrG cassette. Correct integration of thecassette and replacement of thehacA sequence was verified by PCR andthe absence of hacA was confirmed in theHacA-DE strain.To obtain astrain with a constitutively activated HacA transcription factor, we integrated the spliced form ofhacAthatlacks the 20 nucleotide intron intoHacA-DE strain hacA loci. Transformants withthe correct integration pattern wereselected after PCR analysis and the hacA gene in constitutively active was successfully expressed in theHacA-CA (?hacA:: hacA-i)strain.The isolated mRNAs of niaD300, HacA-DE and HacA-CAwere subjected to RNAsequencing.

为评估HacA在应对内质网应激（ER stress）中的作用，以及其作为米曲霉（Aspergillus oryzae，简称A.oryzae）中未折叠蛋白反应潜在调控因子的功能，我们对hacA基因敲除（HacA-DE）菌株与组成型激活表达hacA的（HacA-CA）菌株开展了表征分析。
为构建hacA基因敲除菌株，我们通过将hacA（AO090124000074）的编码区替换为pyrG盒（pyrG cassette），获得了目标重组菌株。经聚合酶链式反应（PCR）验证，该盒式元件已正确整合且hacA序列被成功替换，并在HacA-DE菌株中确认了hacA基因的完全缺失。
为获得组成型激活的HacA转录因子菌株，我们将缺失20个核苷酸内含子的hacA剪接变体整合至HacA-DE菌株的hacA基因座。经PCR分析筛选得到整合模式正确的转化子，并在HacA-CA（?hacA:: hacA-i）菌株中成功实现了组成型激活型hacA基因的表达。
我们提取了niaD300、HacA-DE与HacA-CA菌株的总mRNA，随后进行了RNA测序。