Screening Of High Effective Gossypol-degraded Flora And High-Throughput Transcriptome Sequencing Of Aspergillus Niger AN-1

Cottonseed meal is rich in crude protein,however,because of the existence of toxic substances-Gossypol,its application in animal husbandry has been limited.There are many effective detoxification methods for cottonseed meal,such as chemical method,physical detoxification method,microbial fermentation detoxification method and so on.There are many researches on microbial fermentation currently.It can be detoxicated while the nutritional value of the feed improved,but so far,the detoxification mechanism of microbial fermentation is not clear.In this experiment,we use gossypol acetic acid as the only carbon source and select a series of bacteria having highly efficient degradation to gossypol from soil of cotton plantations,picking out the best degrading bacteria to analyze its culture condition and growth character,and using Transcriptome sequencing technology to make comparative analysis.The main findings are as follows:Experiment 1:Screening and analysis of gossypol-degrading flora in environmentUsing gossypol acetate as the only carbon source to select many strains of degrading gossypol from soil of cotton plantations and rumen fluid of sheep,based on the Gene sequence information of 16S rRNA and 18S rRNA,the composition and structure of gossypol degrading bacteria community in soil environment and rumen fluid of sheep were systematically investigated.The results showed that the flora consists of three fungus and five species bacterias,they were respectively Aspergillus niger AN-1,Penicillium P-1,Pichia guilliermondii M-1,Bacillus subtilis BS-1,Bacillus licheniformis BL-1,Harbin Lactobacillus LH-1,Lactobacillus casei LC-1,Rhodococcus RE-1.These eight kinds of microbes are classified as three gates in the classification:Ascomycota,Firmicutes,and Actinobacteria.Observed the structure change by electron microscope and transmission electron,the results showed that gossypol caused varying degrees of damage to bacteria external morphology and internal structure,and reflected the difference of different strains of tolerance to gossypol.The fermentation tests of cottonseed meal shows that Aspergillus niger AN-1 and Rhodococcus RE-1 have higher tolerance rate of gossypol.Fermented cottonseed meal using single bacteria under the same condition,the results showed that bacteria selected have good degradation to gossypol in cottonseed meal,one of the best bacteria of the degradation effect are RE-1 and AN-1,The degradation rate of it upto 81.0%and 76.08%respectively,which consistent with SEM and TEM results.Exprement 2:Study on the culture conditions of gossypol degrading strain AN-1Study on the culture conditions of higher degradation rate of strain AN-1 to obtain its optimal growth state on gossypol culture medium and lay the foundation for follow research of transcriptome sequencing.Determine the optimum pH:formulated PDA and 0.20%acetic acid gossypol liquid medium and solid medium which pH were 4.5,5.0,5.5,6.0,6.5,7.0,7.5,inoculate gossypol degrading strain AN-1,cultured 72 h in an incubator of 30?.The result shows that AN-l's optimum growth pH was 6.0 in PDA and gossypol acetic acid medium,and the state of growth in the PDA medium is superior to gossypol acetate.Effects of different carbon sources on the AN-1:Inoculate AN-1 at the liquid medium which has the optimum pH of PDA and 0.20%gossypol acetate,culture it in 30°C,measure its growth curve and formulate the corresponding solid medium,then observe the growth state.The results shows that AN-1's growth state in PDA medium is superior to medium of gossypol,AN-1 growed in PDA medium reached the stationary phase at 48 h,the growth rate of AN-1 in gossypol acetic acid medium is slower,it reached the stationary phase at 60 h.Determination of the optimum temperature:formulated PDA and 0.20%acetic acid gossypol liquid medium and solid medium which pH were 6.0,arrange respectively at 22?,26 °C,30?,34 ?,38 ?,42 ?,46? thermostatic incubator.The result shows that bacteria AN-1's optimum growth temperature was 34? in PDA and gossypol acetic acid medium,and the state of growth in PDA medium is better than gossypol acetate.Determination of the optimum gossypol acetic acid concentration:formulated liquid and solid medium which PH at 6.0,concentration respectively is 0.05%,0.10%,0.15%,0.20%,0.25%,0.30%,place it in a thermostatic incubator under the optimum temperature conditions.The result shows that the growth state of AN-1 significantly decreased when gossypol acetate's added amount exceeds 0.2%,the optimal concentration of gossypol acetate is 0.2%.Experiment three:High-throughput transcriptome sequencing of gossypol degrading strain aspergillus niger AN-1High-throughput transcriptome sequencing AN-1 which growth in PDA medium and gossypol medium for 72 h,analyze by GO and KEGG,and speculate the process of Aspergillus niger AN-1 degrade gossypol,then randomly selected five genes for quantitative PCR to validated the transcriptome sequencing results.GO analysis obtained 25112 unigene annotated,analyze clustering from biological processes,cellular components and molecular functions,the respective proportion were 40.11%,29.48%and 30.42%.KEGG analysis obtained 10424 results annotated,there were 4697 pathways participate in metabolism,1894 pathways involved in biosynthesis,928 pathways joined signal information.The most pathway were metabolic pathway which genes have significant differentially expressed.Differences expression of genes obtained 421 genes whose expression upregulated and 470 genes whose expression decreased.Gene's node was ANI₁₄08124?Aldehyde dehydrogenase?.By quantitative PCR validation,the results show the expression of selected five gene consistent with transcriptome sequencing analysis,the transcriptome sequencing results reliable.