Screening of differentially expressed immune-related genes from spleen of broilers fed with probiotic Bacillus cereus PAS38 based on suppression subtractive hybridization

Abstract

The aim of this study was to construct the spleen differential genes library of broilers fed with probiotic Bacillus cereus PAS38 by suppression subtractive hybridization (SSH) and screen the immune-related genes. Sixty seven-day-old broilers were randomly divided into two groups. The control group was fed with basal diet, and the treated group was fed with basal diet containing Bacillus cereus PAS38 1×106 CFU/g. Spleen tissues were taken and extracted its total RNA at 42 days old, then SSH was used to construct differential gene library and screen immune-related genes. A total of 119 differentially expressed sequence tags (ESTs) were isolated by SSH and 9 immune-related genes were screened out by Gene ontology analysis. Nine differentially expressed genes were identified by qRT-PCR. JCHAIN, FTH1, P2RX7, TLR7, IGF1R, SMAD7, and SLC7A6 were found to be significantly up-regulated in the treated group. Which was consistent with the results of SSH. These findings imply that probiotic Bacillus cereus PAS38-induced differentially expressed genes in spleen might play an important role in the improvement of immunity for broilers, which provided useful information for further understanding of the molecular mechanism of probiotics responsible to affect the poultry immunity.

Screening of immune-related differentially expressed genes from primary lymphatic organs of broilers fed with probiotic bacillus cereus PAS38 based on suppression subtractive hybridization

“…Among them, JCHAIN , PRDX1 and CDK6 were significantly up-regulated in both organs, while IPO7 and RPS3 were significantly down-regulated. In our previous study, JCHAIN was also significantly up-regulated in the spleen [ 16 ]. JCHAIN protein (Joining chain of multimeric IgA and IgM) is a glycoprotein that can initiate polymerization by linking to homocysteine in the tail of IgM and IgA and bind to poly IgM and IgA stably by isulfide bonds, and JCHAIN plays an important role in the secretion and transport of immunoglobulin, and the activation of complement [ 17 , 18 ].…”

“…Finally, differential ESTs were connected to pBM16A T vector and transformed into competent Escherichia coli, which were coated on LB solid medium containing ampicillin antibiotics, IPTG and x-gal, and cultured overnight in a constant temperature incubator. The detailed reaction systems and reaction procedures are described by Li et al [16].…”

“…The results of qPCR were analyzed by Bio-Rad CFX manager 3.1 software, and the standard curve was obtained to calculate the copies of differential expressed genes. The detailed reaction systems and reaction procedures were described by Li et al [16]. The primers of differential expressed gene and their optimum annealing temperatures are shown in Table 1.…”

“…Considering the relationship between primary lymphoid organs and secondary lymphoid organs, and the results of our study on spleen differential genes [16], we selected these seven differential genes: JCHAIN, FTH1, IGF1R, PRDX1, CD3E, TLR7 and CDK6 to further analyze their gene functions. Shanghai GenePharma Co., Ltd. was entrusted to design and synthesize corresponding siRNA and negative control siRNA, and the siRNA sequence is shown in Table 5.…”

“…Suppression subtractive hybridization (SSH) technique combines the advantages of suppression PCR and subtractive hybridization, which can enrich rare transcripts with differences and is very suitable for samples with highly similar genetic backgrounds, so it is often used to screen differentially expressed genes in the field of zoology [ 13 – 15 ]. Our group has used SSH to construct the splenic differential gene library of broilers fed Bacillus cerus PAS38, and screened 9 immune-related differential genes including JCHAIN , FTH1 , IGF1R , TLR7 and others [ 16 ].…”