【作者】 王娟；

【导师】 杨秀荣；

【作者基本信息】 广西大学 ， 农业硕士畜牧领域（专业学位）， 2020， 硕士

【摘要】 过度提高生长速度和饲料转化效率给地方鸡育种带来的一个问题是脂肪过度沉积。脂肪过度沉积不仅会造成饲料浪费、屠宰率降低,而且多余的脂肪还会造成环境污染,给肉鸡产业造成经济损失。脂肪沉积是受多因素共同调控的复杂过程,其调控的分子机理仍不清晰。南丹瑶鸡是广西优良的地方品种,其特点是皮薄肉脆,皮下脂肪少。为了探讨南丹瑶鸡腹脂沉积相关的基因和调控通路,本试验以南丹瑶鸡为研究对象,利用RNA-seq分析了高低腹脂转录组差异基因,对筛选出来的关键候选基因进行了克隆、生物信息学分析、组织表达谱分析、真核表达载体的构建及验证。具体研究结果如下:1.高低腹脂组RNA-seq分析及qPCR验证。利用Trizol提取120日龄南丹瑶鸡高低腹脂组中的腹脂总RNA,进行RNA-seq转录组测序。转录组数据发析筛选到1222个差异表达基因,对这些差异表达基因进行GO富集和KEGG通路分析,显著富集的通路主要参与机体生长发育过程以及与脂肪代谢相关的泛酸和辅酶合成(P<0.05)。为了验证转录组的准确性,利用q PCR检测了DGKD(diacylglycerol kinase delta)、PLA2G4A(phospholipase A2 group IVA)、SREBF1(sterol regulatory element binding transcription factor1)、DDX5(DEAD-box helicase 5)和RFC2(replication factor C subunit 2)等5个候选基因在高低腹脂组中的表达量。结果显示,q PCR的表达趋势与RNA-seq转录组的趋势一致,说明转录组的结果可信。其中,DDX5和RFC2两个基因在高腹脂组的腹脂中的表达量极显著低于低腹脂组(P<0.01)。2.DDX5和RFC2基因的克隆、生物信息学及组织表达谱分析。通过对DDX5和RFC2基因进行克隆和序列分析发现,DDX5基因和RFC2基因CDS区全长分别为1788bp和1080bp,各编码595和359个氨基酸;DDX5基因编码区存在2处同义突变(c.504C>T和c.507A>G)和1处错义突变(c.1060T>C处突变导致第354位氨基酸由色氨酸突变为精氨酸),RFC2基因编码区存在1处同义突变(c.336C>T)。利用q PCR检测了DDX5和RFC2基因在皮脂、腹脂、十二指肠、肝、肾、睾丸和卵巢等组织中的表达量,发现DDX5和RFC2基因在母鸡腹脂中的表达量显著高于公鸡中的表达量(P<0.01),而在母鸡的十二指肠中的表达量显著低于公鸡中的表达量(P<0.01),RFC2在睾丸中的表达量显著高于在卵巢中的表达量(P<0.01)。3.DDX5和RFC2基因真核表达载体的构建和验证。以DDX5和RFC2克隆载体为模板PCR扩增DDX5和RFC2,将其与p EGFP-N1真核表达载体连接,经PCR、双酶切和测序鉴定载体的准确性。将p EGFP-N1-DDX5和pEGFP-N1-RFC2重组质粒转入鸡DF1细胞系中,24小时后,在荧光显微镜下观察到绿色荧光,说明本研究成功构建了南丹瑶鸡p EGFP-N1-DDX5和p EGFP-N1-RFC2真核表达载体。综上所述,通过RNA-seq转录组分析,基因克隆序列分析、组织表达谱分析及定量分析初步确定DDX5和RFC2可能为南丹瑶鸡脂肪沉积的重要候选基因,构建了两基因的真核表达载体和在细胞水平验证了载体的功能,为进一步确定这两个基因的功能奠定了基础。更多还原

【Abstract】 One problem caused by excessively increasing growth rate and feed conversion efficiency for local chicken breeding is excessive fat deposition.Excessive fat deposition will not only cause waste of feed and lower slaughter rate,but also cause environmental pollution and economic losses to the broiler industry.Fat deposition is a complex process regulated by multiple factors,and the molecular mechanism of its regulation is still unclear.Nandan-Yao chicken is an excellent local breed in Guangxi.It is characterized by thin skin and crispy meat with less subcutaneous fat.In order to explore the genes and regulatory pathways related to abdominal fat deposition in Nandan-Yao chickens,this experiment took Nandan-Yao chickens as the research object and analyzed the differentially expressed genes of high and low abdominal fat birds using RNA-seq.We selected several candidate genes for further analysis.The results are as follows:1.RNA-seq analysis and qPCR verification.The total RNA of abdominal fat in the Nandan-Yao chickens aged 120-day-old were extracted with Trizol,and RNA-seq transcriptome sequencing was performed.Transcriptome analysis detected 1222 differentially expressed genes.GO enrichment and KEGG pathway analysis were performed on these differentially expressed genes.The significantly enriched pathways are mainly involved in the developmental growth and the synthesis of pantothenic acid and coenzymes related to fat metabolism(P <0.05).In order to verify the accuracy of the transcriptome,q PCR was used to detect the expression levels of 5 candidate genes in the high and low abdominal fat groups,including DGKD(diacylglycerol kinase delta),PLA2G4A(phospholipase A2 group IVA),SREBF1(sterol regulatory element binding transcription factor 1),DDX5(DEAD-box helicase 5)and RFC2(replication factor C subunit 2).The results showed that the trend of q PCR is consistent with that of RNA-seq,indicating that the results of the transcriptome are credible.The expression levels of DDX5 and RFC2 genes in abdominal fat in the high abdominal fat group were significantly lower than those in the low abdominal fat group(P <0.01).2.Cloning,bioinformatics and tissue expression profiling of DDX5 and RFC2 genes.Through cloning and sequence analysis of the DDX5 and RFC2 gene,we found that the full length of the CDS region of the DDX5 gene and RFC2 gene was 1788 bp and 1080 bp,encoding 595 and 359 amino acids,respectively;there were two synonymous mutations(c.504C> T and c.507A> G)and one missense mutation(c.1060T> C,the 354 th amino acid mutated from tryptophan to arginine acid)in the coding region of DDX5 gene.There is one synonymous mutation in the coding region of RFC2 gene(c.336C> T).Using q PCR to detect the expression level of DDX5 and RFC2 gene in different tissues such as sebum,abdominal fat,duodenum,liver,kidney,testis and ovary.It was found that the expression levels of DDX5 and RFC2 genes in abdominal fat of females were significantly higher than those in males(P <0.01),while the expression level in duodenum of females were significantly lower than that in males(P <0.01),the expression level of RFC2 in testis was significantly higher than that in ovary(P <0.01).3.Construction and verification of DDX5 and RFC2 genes eukaryotic expression vectors.The DDX5 and RFC2 cloning vectors were used as templates for PCR amplification of DDX5 and RFC2,which were connected to the p EGFP-N1 eukaryotic expression vector,and the accuracy of the vector was verified by PCR,double digestion and sequencing.The p EGFP-N1-DDX5 and p EGFP-N1-RFC2 recombinant plasmids were transferred into chicken DF1 cell line.After 24 hours,green fluorescence was observed under a fluorescence microscope,indicating that this study successfully constructed p EGFP-N1-DDX5 and p EGFP-N1-RFC2 eukaryotic expression vectors.In conclusion,through RNA-seq transcriptome analysis,gene cloning sequence analysis,tissue expression profiling analysis and quantitative analysis,it was confirmed that DDX5 and RFC2 may be important candidate genes for fat deposition in Nandan-Yao chicken.The eukaryotic expression vector of the two genes was constructed and the function of the vector was verified at the cellular level,which laid the foundation for the further determination of the functions of the two genes.更多还原