鸡与鹌鹑属间杂交种胚胎早期死亡相关基因的功能研究

发布时间：2018-11-28 18:39

【摘要】：鸡和鹌鹑为家禽生产中的常见物种,鸡与鹌鹑同种不同属,其间杂交属于禽类中的典型属间远缘杂交,是基因功能研究和比较基因组学的良好资源;而鸡与鹌鹑杂交种在孵化过程中大量死亡,已有研究表明杂交种胚胎的死亡与胚胎组织器官发育不完全和性分化相关,而在胚胎发育、性别分化过程中参与的基因和信号通路众多。本研究对雌、雄鸡和雌、雄杂交种数字基因表达谱进行生物信息学分析筛选出与胚胎发育、性别分化相关基因,构建基因过表达载体和RNA干扰载体,以鸡胚细胞为实验材料,调控目标基因表达量,利用实时荧光定量技术检测相关基因表达变化,探索目标基因在表达异常时对胚胎发育的影响,为进一步研究雌性杂交种胚胎早期死亡的原因奠定基础。1.对雌雄鸡与雌雄杂交种DGE库通过生物信息学分析筛选与胚胎发育、性分化相关显著富集的信号通路,如MPKA、Hedgehog、Wnt、Notch等;表达差异显著的相关基因FGF8、DACH、LFNG、SOX1、SOX2、SHH、FOXL2、GPR149、CCND1。2.差异基因FGF8、SHH、FOXL2过表达载体和干扰载体的构建,参照NCBI上公布的原鸡FGF8、SHH基因m RNA序列,在基因编码区分别设计扩增全长引物,以p EGFP-C2为载体,构建鸡FGF8和SHH基因过表达载体;经菌液PCR、酶切鉴定和测序验证,过表达载体构建成功。根据NCBI上公布的原鸡FGF8、SHH和Foxl2基因的m RNA序列,分别依据基因编码区特点设计3对sh RNA干扰引物,以pmi RZip为载体,构建靶向FGF8、SHH、Foxl2基因干扰载体;经测序验证,干扰载体构建成功。构建成功的FGF8、SHH过表达载体和FGF8、SHH、Foxl2干扰载体转染鸡胚细胞,验证表达载体和干扰载体效果。3.分析FGF8、SHH、Foxl2基因对胚胎发育的影响,分别将FGF8、SHH、FOXL2基因表达和干扰载体转染鸡胚细胞,检测相关基因表达,实验结果得出:当FGF8基因表达量发生变化时,在MAPK信号通路中,细胞间的链接蛋白Grb2表达水平降低,进而影响后续一系列信号传递与通路的激活,使胚胎不能正常生长发育。在Hedgehog信号通路中,干扰SHH基因的表达时Hedgehog信号通路不能够被激活而处于抑制状态,使胚胎早期不能够正常发育,当SHH基因过表达时,能够成功激活Hedgehog信号通路,并且过度激活,同样对胚胎产生负面影响,例如胚胎出现畸形。干扰Foxl2基因使其表达量降低,造成胚胎发育早期参与性别分化的DMRT1、SOX9、CYP19基因表达发生紊乱,而导致在发育性分化过程中出现异常,使胚胎不能正常生长发育。
[Abstract]:Chicken and quail are common species in poultry production. However, a large number of chickens and quail hybrids died during hatching. Some studies have shown that the death of hybrid embryos is related to the incomplete development of embryonic tissues and organs and sexual differentiation, but in embryo development. Many genes and signaling pathways are involved in the process of sex differentiation. In this study, the digital gene expression profiles of female, male, female and male hybrids were screened by bioinformatics analysis. The genes related to embryonic development and sex differentiation were selected, and gene overexpression vector and RNA interference vector were constructed. Chicken embryo cells were used as experimental materials. To regulate the expression of target gene and detect the change of related gene expression by real-time fluorescence quantitative technique, and to explore the effect of target gene on embryo development when the expression of target gene is abnormal. It lays a foundation for further study on the causes of early embryo death in female hybrids. 1. The DGE library of female and male hybrids was screened by bioinformatics analysis for the signal pathways, such as MPKA,Hedgehog,Wnt,Notch, which were significantly enriched in embryo development and sexual differentiation. Expression of significantly differentially expressed genes related to FGF8,DACH,LFNG,SOX1,SOX2,SHH,FOXL2,GPR149,CCND1.2. According to the m RNA sequence of FGF8,SHH gene published on NCBI, the full-length primers were designed and amplified in the gene coding region, using p EGFP-C2 as vector. The overexpression vectors of chicken FGF8 and SHH genes were constructed. The overexpression vector was successfully constructed by PCR, digestion and sequencing. According to the m RNA sequence of FGF8,SHH and Foxl2 gene published on NCBI, three pairs of sh RNA interference primers were designed according to the characteristics of gene coding region, and pmi RZip was used as vector to construct the target FGF8,SHH,Foxl2 gene interference vector. After sequencing, the interference vector was successfully constructed. The FGF8,SHH overexpression vector and FGF8,SHH,Foxl2 interference vector were successfully constructed and transfected into chicken embryo cells to verify the effect of expression vector and interference vector. 3. The effect of FGF8,SHH,Foxl2 gene on embryo development was analyzed. The FGF8,SHH,FOXL2 gene expression and interference vector were transfected into chicken embryo cells, and the expression of related genes was detected. The results showed that: when the expression of FGF8 gene changed, In the MAPK signaling pathway, the expression level of intercellular link protein Grb2 is decreased, which affects the subsequent signal transduction and activation of the pathway, which makes the embryo unable to develop normally. In the Hedgehog signaling pathway, the Hedgehog signaling pathway can not be activated and inhibited when interfering with the expression of SHH gene, so that the early embryonic development is not normal. When the SHH gene is overexpressed, the Hedgehog signaling pathway can be successfully activated and over-activated. It also has negative effects on embryos, such as deformities. Interfering with the Foxl2 gene reduced its expression, resulting in the disorder of DMRT1,SOX9,CYP19 gene expression in the early stage of embryonic development, and the abnormal expression of DMRT1,SOX9,CYP19 gene in the process of developmental differentiation, which resulted in the abnormal growth and development of the embryo.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S813.22

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