Primery Research Of B.melitensis DhbC Gene Affecting Gene Expression Of Sheep Leydig Cell

Brucella is a facultative intracellular parasitic bacterium that infects human and livestock and causes disease--Brucellosis.Tere still are more than incident 500,000 cases of human brucellosis around the world.It's difficult to cure the infected individual;furthermore,it may stay with patients for the whole life,which causes huge loss in both economy and health.The key factor of brucella pathogenicity is its intracellular parasitic ability.A certain amount of pathogens are required for the showing of clinical symptoms for the reason that brucella is dormant.When the pathogens proliferate and spread to the whole body,clinical signs,such as miscarriage and fever joint pain,appear.The replication of brucella requires the essential element iron.The dhbC gene is one of the genes required for the synthesis of the iron transporter 2,3-Dihydroxybenzoic acid.The research has shown that brucella abortus dhbC gene-deficient strains infect pregnant cows.Its virulence is significantly weaker than the parent strain.However,there is no related report on the changes of host gene expression after the dhbC gene-deficient strain brucella infection.In this experiment,the homologous recombination plasmid pGEM-7Zf?+?-? dhbC was constructed by enzyme digestion ligation method and PCR method,and it was transformed into competent cells of brucella melitensis M5-90 strain by electrotransformation.Screening of resistant plates,and 20 consecutive passages for stable inheritance of mutant strains,and finally construction of M5-90 ?dhbC strain.The M5-90-?dhbC deficient strain and the M5-90 strain were respectively infected with sheep Ley dig cell,and total RNA were extracted after 4 hours of incubation.The mRNAs were sequenced using Illumina Novaseq TM 6000,and 255 mRNA differentially expressed genes were generated through data processing and analysis;the miRNAs were sequenced using Illumina Hiseq2000/2500,and 90 miRNA differentially expressed genes were generated through data processing and analysis.Combined analysis of differentially expressed mRNAs and miRNAs.There were 17 pairs of mRNA differentially expressed genes and 36 miRNA differentially expressed genes corresponding to 74 pairs.There are some correspondences between up-regulated IDO1 gene and down-regulated miRNAs including chi-miR-19a,PC-3p-102014₂6,PC-3p-141193₁5,PC-3p-16050₃94,PC-3p-18023₃37,PC-3p-52141₇7,PC-3p-59431₆3 and PC-3p-71424₄8 were shown in results.GO enrichment analyses indicated the IDO1 gene was invovled in immune and inflammatory;There is a correspondence between down-regulated SLC2A5 gene and down-regulated miRNA PC-3p-38194₁20,and the SLC2A5 gene is invovled in Carbohydrate digestion and absorption pathways by KEGG enrichment analyzing;the other two up-regulated differential genes CCL5 and CXCL10 significantly enrich the Chemokine signaling pathway.