| Gossypol is the main factor limiting the application of cottonseed meal(CSM)in breeding industry.Screening high efficient gossypol degrading bacteria is an effective way to solve the problem of CSM utilization.Previous studies in our laboratory showed that the expression of catalase(CAT)gene of gossypol degrading microorganisms was significantly up-regulated after being induced by gossypol.Therefore,based on the previous work,we cloned CAT gene,and used gene knockout technology to study the role of CAT gene in the degradation of gossypol by microorganisms.In this experiment,gossypol acetate was used as the sole carbon source.A strain which could utilize gossypol was screened from cotton planting soil.It was identified as Rhodococcus erythropolis by 16 S r RNA,and named RE.The content of free gossypol(FG)and total gossypol(TG)decreased by 87.46% and 53.46% in fermented CSM,respectively.The CAT gene deletion mutant of AKD95758 ΔRE was obtained by CRISPR/cas9 gene knockout of RE.The amplified fragment size of CAT gene of ΔRE was 197 bp,and that of RE was 1 462 bp.The results of growth curve test showed that there was no significant difference in24 h biomass between RE and ΔRE under different carbon and nitrogen source medium,temperature and p H conditions(P> 0.05).When the inoculation amount was 1%,2%and 3%,the biomass of RE and ΔRE had no significant difference at 24 h(P> 0.05);However,the 24 h biomass of wild strain RE was significantly higher than that of mutant ΔRE when the inoculation amount was 4% and 5%(P< 0.05).The CAT activity of RE cultured in LB medium was 14.50,and that of ΔRE was 8.45.The CAT activity decreased significantly(P< 0.05).The CAT activity of RE cultured in gossypol medium was 16.73,which was significantly higher than that in LB medium(P< 0.05);the CAT activity of ΔRE was 9.61,which was significantly higher than that of ΔRE cultured in LB medium(P< 0.05).The results of CAT gene expression assay showed that the relative expression levels of AKD95758 and AKE00039 genes of RE cultured in gossypol medium were significantly higher than those of RE cultured in LB medium(P< 0.05);moreover,the relative expression of AKE00039 gene of ΔRE in gossypol medium was significantly higher than that of ΔRE mutant in LB medium(P< 0.05).However,there was no significant difference in the relative expression of AKE00039 gene between RE andΔRE in LB medium(P> 0.05);there was no significant difference in the relative expression of AKE00039 gene between RE and ΔRE cultured in gossypol medium(P>0.05).In the experiment of CSM fermentation,group A(1% RE),group B(1% ΔRE),group C(1% RE + 333 U/100 g CAT),group D(1% RE + 666 U/100 g CAT),group E(1%ΔRE + 333 U/100 g CAT),group F(1% ΔRE + 666 U/100 g CAT),group G(333 U/100 g CAT),and group H(666 U/100 g)were set.The results showed that the degradation rates of FG and TG in group A(87.82% and 54.37%)were significantly higher than those in group B(78.58% and 47.53%)(P< 0.05).The degradation rates of FG and TG in groups C and D,E and F were higher than those in groups A and B,respectively.Compared with unfermented CSM,the crude protein content of group A and group B increased by 19.30% and 18.13%,respectively;the content of acid detergent fiber decreased by 12.79% in group A and 11.16% in group B;the content of neutral detergent fiber decreased by 17.33% in group A and 16.30% in group B.In conclusion,CAT gene plays an important role in the degradation of gossypol by Rhodococcus erythropolis,but the mechanism remains to be further studied. |
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