SSH文库技术结合DGGE技术筛选饲喂蜡样芽孢杆菌肉鸡脾脏免疫差异基因

doi:10.7668/hbnxb.20190909

摘要/Abstract

摘要： 为探究蜡样芽孢杆菌PAS38影响肉鸡免疫的分子机制，将60羽7日龄白羽肉鸡分为2组，对照组饲喂基础日粮，试验组饲喂含蜡样芽孢杆菌PAS38 1×10⁶ cfu/g的基础日粮。于42日龄时从每个组随机取6只肉鸡，解剖采取脾脏并提取总RNA、反转录为cDNA。然后分别采用SSH和ACSSH技术构建脾脏差异基因文库并筛选免疫相关基因。结果显示，SSH筛选得到119个差异基因，其中上调基因63个，下调基因56个，包括9个免疫调节基因；ACSSH筛选得到5个差异基因，都为下调基因，无免疫调节基因。结果表明，饲喂蜡样芽孢杆菌能影响JCHAIN、FTH1、P2RX7等肉鸡脾脏免疫基因的表达；SSH文库明显优于ACSSH文库，ACSSH技术存在一定缺陷。

关键词: 肉鸡, 蜡样芽孢杆菌, SSH技术, ACSSH技术, 差异基因

Abstract: To explore the molecular mechanism of the effect of Bacillus cereus PAS38 on the immunity in broilers. 60 7-day-old broilers were divided into two groups,the control group was fed with basal diet,and the treatment group was fed with basal diet containing Bacillus cereus PAS38 1×10⁶ cfu/g. Six broilers were randomly selected from each group at the age of 42 days,spleens were taken and total RNA was extracted,then,and reversed transcription to cDNA. SSH and ACSSH techniques were used to construct splenic differential gene library and screen immune-related genes respectively. The results showed that 119 differentially expressed genes were screened by SSH,including 63 up-regulated genes and 56 down-regulated genes,and including 9 immunoregulatory genes,while five differentially expressed genes were screened by ACSSH,all of which were down-regulated genes,and no immunoregulatory genes were found. The results indicated that feeding Bacillus cereus PAS38 could affect the expression of immune genes such as JCHAIN, FTH1 and P2RX7 in the spleen of broilers,SSH library was superior to ACSSH library,and ACSSH technology was defective.

Key words: Broilers, Bacillus cereus, SSH technology, ACSSH technology, Differential gene

中图分类号:

Q78
S432.4

李佳骏, 李建臻, 李万强, 王振华, 王君锐, 肖丹, 潘康成, 杨苗. SSH文库技术结合DGGE技术筛选饲喂蜡样芽孢杆菌肉鸡脾脏免疫差异基因[J]. 华北农学报, 2020, 35(S1): 392-398. doi: 10.7668/hbnxb.20190909.

LI Jiajun, LI Jianzhen, LI Wanqiang, WANG Zhenhua, WANG Junrui, XIAO Dan, PAN Kangcheng, YANG Miao. Immune-Related Differentially Expressed Genes of Spleen in Broilers Fed with Probiotic Bacillus cereus Screened by SSH Library and DGGE Technique[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(S1): 392-398. doi: 10.7668/hbnxb.20190909.

http://www.hbnxb.net/CN/Y2020/V35/IS1/392

参考文献

[1] Yan F,Polk D B. Probiotics:progress toward novel therapies for intestinal diseases[J]. Current Opinion in Gastroenterology,2010,26(2):95-101. doi:10.1097/MOG.0b013e328335239a.
[2] 张李荣. 三种益生菌对维鸡生长、小肠发育及盲肠微生物区系的影响[D]. 扬州:扬州大学,2018.
Zhang L R. Effects of three probiotics on growth,small intestine development and cecum microorganism of chicks[D]. Yangzhou:Yangzhou University,2018.
[3] Narayanan G,Baskaralingam V,Jiann C C,Rekha R,Vijayakumar S,Anjugam M,Iswarya A. Dietary supplementation of probiotic Bacillus licheniformis Dahb1 improves growth performance,mucus and serum immune parameters,antioxidant enzyme activity as well as resistance against Aeromonas hydrophila in tilapia Oreochromis mossambicus[J]. Fish & Shellfish Immunology,2018(74):501-508. doi:10.1016/j.fsi.2017.12.066.
[4] Madej J P,Stefaniak T,Bednarczyk M. Effect of in ovo-delivered prebiotics and synbiotics on lymphoid-organs' morphology in chickens[J]. Poultry Science,2015,94(6):1209-1219. doi:10.3382/ps/pev076.
[5] Zhang J C,Yu J F,Hong H L,Liu J,Lu H,Yan C. Identification of heavy metal pollutant tolerance-associated genes in Avicennia marina(Forsk.)by suppression subtractive hybridization[J]. Marine Pollution Bulletin,2017,119(1):81-91. doi:10.1016/j.marpolbul.2017.03.023.
[6] Yu Z B,Mu C K,Song W W,Li R H,Chen Y E,Wang C L. Screening of genes related to ovarian development in the swimming crab, Portunus trituberculatus,by suppression subtractive hybridization[J]. Genetics and Molecular Research,2015,14(4):18675-18686. doi:10.4238/2015.December.28.16.
[7] Kang X L,Liu Y F,Zhang J B,Xu Q,Liu C,Fang M. Characteristics and expression profile of KRT71 screened by suppression subtractive hybridization cDNA library in curly fleece Chinese Tan Sheep[J]. DNA and Cell Biology,2017,36(7):1-13. doi:10.1089/dna.2017.3718.
[8] Muyzer G,Smalla K. Application of denaturing gradient gelelectrophoresis(DGGE)and temperature gradient gelelectrophoresis(TGGE)in microbialecology[J]. Antonie Van Leeuwenhoek,1998,73(1):127-141. doi:10.1023/a:1000669317571.
[9] 王雪寒,夏冬双,武志华,任兴波,丁一秀,马强,刘惠荣. 内蒙古西部地区土壤放线菌的多样性分析[J]. 基因组学与应用生物学,2018,37(10):4340-4348. doi:10.13417/j.gab.037.004340.
Wang X H,Xia D S,Wu Z H,Ren X B,Ding Y X,Ma Q,Liu H R. Analysis on the diversity of soil actinomycetes in the western region of Inner Mongolia[J]. Genomics and Applied Biology,2018,37(10):4340-4348.
[10] Sevcan A,Aiyoub S E,Gozde O. Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinations[J]. Bioresource Technology,2015(192):735-740. doi:10.1016/j.biortech.2015.05.086.
[11] Garofalo C,Bancalari E,Milanović V,Cardinali F,Osimani A,Sardaro M L S,Bottari B,Bernini V,Aquilanti L,Clementi F,Neviani E,Gatti M. Study of the bacterial diversity of foods:PCR-DGGE versus LH-PCR[J]. International Journal of Food Microbiology,2017(242):24-36. doi:10.1016/j.ijfoodmicro.2016.11.008.
[12] Karina E M,Christian M,Marco V,Valencia F E,Ruas-Madiedo P,Delgado S. Bacterial diversity of the colombian fermented milk "Suero Costen o" assessed by culturing and high-throughput sequencing and DGGE analysis of 16S rRNA gene amplicons[J]. Food Microbiology,2017(68):129-136. doi:10.1016/j.fm.2017.07.011.
[13] Andrea O,Vesna M,Cristiana G,Cardinali F,Roncolini A,Sabbatini R,De Filippis F,Ercolini D,Gabucci C,Petruzzelli A,Tonucci F,Clementi F,Aquilanti L. Revealing the microbiota of marketed edible insects through PCR-DGGE,metagenomic sequencing and real-time PCR[J]. International Journal of Food Microbiology,2018(276):54-62. doi:10.1016/j.ijfoodmicro.2018.04.013.
[14] Gadde U,Oh S T,Lee Y S,Davis E,Zimmerman N,Rehberger T,Lillehoj H S. The effects of direct-fed microbial supplementation,as an alternative to antibiotics,on growth performance,intestinal immune status,and epithelial barrier gene expression in broiler chickens[J]. Probiotics & Antimicro,2017(9):397-405. doi:10.1007/s12602-017-9275-9.
[15] Gong Y,Li H,Li Y. Effects of Bacillus subtilis on epithelial tight junctions of mice with inflammatory bowel disease[J]. Journal of Interferon & Cytokine Research,2016,36(2):1-11. doi:10.1089/jir.2015.0030.
[16] 朱沛霁.枯草芽孢杆菌对雪山鸡生产性能、肠道健康和免疫机能的影响及机制[D].扬州:扬州大学,2017.
Zhu P Q. Effect of dietary supplementation of Bacillus Subtilis on growth performance,intestinal health and immune function responses of Xueshan Broiler[D]. Yangzhou:Yangzhou Universuty,2017.
[17] He Y,Wang A Q,Chen S,Wu Z,Zhang J Y,Wang M S,Jia R Y,Zhu D K,Liu M F,Yang Q,Wu Y,Sun K F,Chen X G,Cheng A C. Differential immune-related gene expression in the spleens of duck Tembusu virus-infected goslings[J]. Veterinary Microbiology,2017(212):39-47. doi:10.1016/j.vetmic.2017.08.002.
[18] Byadgi O,Chen Y C,Maekawa S,Wang P C,Chen S C. Immune-Related functional differential gene expression in Koi Carp(Cyprinus carpio)after challenge with Aeromonas sobria[J]. International Journal of Molecular Sciences,2018,19(7):2107. doi:10.3390/ijms19072107.
[19] Gao L,Yang Y,Yao Q,Chen K. Differentially expressed genes in the midguts of BmNPV-susceptible and resistant silkworm strains determined using suppression subtractive hybridization[J]. Invertebrate Survival Journal,2018,15(1):256-264.
[20] Liu Q N,Xin Z Z,Chai X Y,Jiang S H,Li C F,Zhang H B,Ge B M,Zhang D Z,Zhou C L,Tang B P. Characterization of immune-related genes in the yellow catfish Pelteobagrus fulvidraco in response to LPS challenge[J]. Fish & Shellfish Immunology,2016(56):248-254. doi:10.1016/j.fsi.2016.05.019.
[21] Fujita T,Kitaura F,Fujii H. A critical role of the Thy28-MYH9 axis in b cell-specific expression of the Pax5 gene in chicken B cells[J]. PLoS One,2015,10(1):e0116579. doi:10.1371/journal.pone.0116579.
[22] Jamrog L,Chemin G,Fregona V,Coster L,Pasquet M,Oudinet C,Rouqui N,Prade N,Lagarde S,Cresson C,H brard S,Nguyen H N S,Bousquet M,Quelen C,Brousset P,Mancini S J C,Delabesse E,Khamlichi A A,Gerby B,Broccardo C. PAX5-ELN oncoprotein promotes multistep B-cell acute lymphoblastic leukemia in mice[J]. PNAS,2018,115(41):10357-10362. doi:10.1073/pnas.1721678115.
[23] Danilo E. PCR-DGGE fingerprinting:novel strategies for detection of microbes in food[J]. Journal of Microbiological Methods,2004,56(3):297-314. doi:10.1016/j.mimet.2003.11.006.
[24] Giusy F,Giovina R. Detection of polymorphisms within the FAS cDNA gene sequence by GC-clamp denaturing gradient gel electrophoresis[J]. Immunogenetics,1994,39(6):437-439. doi:10.1007/bf00176163.
[25] Luo H F,Qi H Y,Xue K,Wang X Y,Wang C,Zhang H X. Influence of application of GC-clamp on study of soil microbial diversity by PCR-DGGE[J]. Acta Ecologica Sinica,2003,23(10):2170-2175.

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