| Our lab has a very good research foundation of Penicillium red pigments, and the red pigments can be obtained not only by co-culture, but also by pure-culture. However, these researchers focused on the optimization of fermentation conditions and increasing yield, so pigment synthesis mechanism and pigment compositions are still not clear. In previous work, we found that the Penicillium novae-zeelandiae HSD07 B had great potential in red pigment production. In this paper, investigations about red pigment form P. novae-zeelandiae HSD07 B, including the red pigment biofilm cultivation techniques and transcriptome sequencing analysis were conducted to detect the red pigment producing mechanism.Red pigment can’t be produced by P. novae-zeelandiae. HSD07 B in liquid shake-flask fermentation, and it only appeared when the biofilm was formed during static treatment. In the study, an optimal culture method was obtained: 1 m L of inoculation quantity(spore concentration, 2 x 107 / m L); inoculating in 300 m L PD medium with initial p H value of 6; shake-flask culture at 30 ℃ and 150 r/min for 40 h; and static culture at 30 ℃ for biofilm culture. After static culture for 24 h, red biofilm was formed, and the initial red pigment productivity was high during 7-day cultivation. After day 7, the curve of red pigment productivity had a decreasing trend. A maximum yield of red pigment was obtained on day 9.A new biofilm culture method with solid supporter was established in order to suit sampling for the transcriptome sequencing. This method is on the basis of the cultivation previously mentioned. After 40 h shake-flask culture, a sterile filtration membrane was placed on the fermented liquid surface, and flasks were cultivated statically at 30℃ to make hyphae in the fermented liquid form red biofilm by adhering to the sterile filtration membrane. By this method, the achievement of sequencing samples becomes easier.Taking the red hyphae in the early period of red pigment production as T1 and booming period as T2, as well as the hyphae in the corresponding period in shake-flask culture were named as Ck1 and Ck2. After Illumina platform transcriptome sequencing, 16 G sequencing data were obtained. There are 21204700934 reads in total and about 5.3 billion reads per sample. By statistical analysis of these data, we got 10621 pieces of unigene. There are a total of 772 SNPs and 6475 SSRs in the unigenes, meanwhile the unigenes with GO annotation are 5496 pieces and the unigenes with KEGG annotation are 3086 pieces. These data will present the basis for the following differential expression analysis.By analysis of sequencing data, 1148 differential expression genes containing 823 pieces of up regulated genes and 325 pieces of down regulated genes were found between Ck1 and T1,and 1789 differential expression genes containing 1257 pieces of up regulated genes and 532 pieces of down regulated genes were obtained between Ck2 and T2. By annotation and analysis for these differential expression genes, the results show that several genes involved in oxidation-reduction process have changed between T1 and Ck1, and genes related to membranes function has up-regulated. These genes were found at the early phase of red pigment production, which maybe have relation to the forming biofilm and the change of fermentation style. By comparison analysis between T2 and Ck2, genes involved in erythronolide synthase activity, transition metal ion binding, tetrapyrrole binding functions, heme binding functions, monooxygenase activity and iron ion binding functions have change significantly. Because T2 is in the booming phase of red pigment production, so these changes possibly involved in red pigment production. Through KEGG pathways variance analysis, the polycyclic aromatic hydrocarbon degradation pathway has significant differences not only between Ck1 and T1 but also between T2 and Ck2, and the benzene series in pathways may be related to the structures of the red pigments. |
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