雞與鵪鶉屬間雜交種胚胎早期死亡相關(guān)基因的功能研究
發(fā)布時(shí)間：2018-11-28 18:39
【摘要】：雞和鵪鶉為家禽生產(chǎn)中的常見物種,雞與鵪鶉同種不同屬,其間雜交屬于禽類中的典型屬間遠(yuǎn)緣雜交,是基因功能研究和比較基因組學(xué)的良好資源;而雞與鵪鶉雜交種在孵化過程中大量死亡,已有研究表明雜交種胚胎的死亡與胚胎組織器官發(fā)育不完全和性分化相關(guān),而在胚胎發(fā)育、性別分化過程中參與的基因和信號通路眾多。本研究對雌、雄雞和雌、雄雜交種數(shù)字基因表達(dá)譜進(jìn)行生物信息學(xué)分析篩選出與胚胎發(fā)育、性別分化相關(guān)基因,構(gòu)建基因過表達(dá)載體和RNA干擾載體,以雞胚細(xì)胞為實(shí)驗(yàn)材料,調(diào)控目標(biāo)基因表達(dá)量,利用實(shí)時(shí)熒光定量技術(shù)檢測相關(guān)基因表達(dá)變化,探索目標(biāo)基因在表達(dá)異常時(shí)對胚胎發(fā)育的影響,為進(jìn)一步研究雌性雜交種胚胎早期死亡的原因奠定基礎(chǔ)。1.對雌雄雞與雌雄雜交種DGE庫通過生物信息學(xué)分析篩選與胚胎發(fā)育、性分化相關(guān)顯著富集的信號通路,如MPKA、Hedgehog、Wnt、Notch等;表達(dá)差異顯著的相關(guān)基因FGF8、DACH、LFNG、SOX1、SOX2、SHH、FOXL2、GPR149、CCND1。2.差異基因FGF8、SHH、FOXL2過表達(dá)載體和干擾載體的構(gòu)建,參照NCBI上公布的原雞FGF8、SHH基因m RNA序列,在基因編碼區(qū)分別設(shè)計(jì)擴(kuò)增全長引物,以p EGFP-C2為載體,構(gòu)建雞FGF8和SHH基因過表達(dá)載體;經(jīng)菌液PCR、酶切鑒定和測序驗(yàn)證,過表達(dá)載體構(gòu)建成功。根據(jù)NCBI上公布的原雞FGF8、SHH和Foxl2基因的m RNA序列,分別依據(jù)基因編碼區(qū)特點(diǎn)設(shè)計(jì)3對sh RNA干擾引物,以pmi RZip為載體,構(gòu)建靶向FGF8、SHH、Foxl2基因干擾載體;經(jīng)測序驗(yàn)證,干擾載體構(gòu)建成功。構(gòu)建成功的FGF8、SHH過表達(dá)載體和FGF8、SHH、Foxl2干擾載體轉(zhuǎn)染雞胚細(xì)胞,驗(yàn)證表達(dá)載體和干擾載體效果。3.分析FGF8、SHH、Foxl2基因?qū)ε咛グl(fā)育的影響,分別將FGF8、SHH、FOXL2基因表達(dá)和干擾載體轉(zhuǎn)染雞胚細(xì)胞,檢測相關(guān)基因表達(dá),實(shí)驗(yàn)結(jié)果得出:當(dāng)FGF8基因表達(dá)量發(fā)生變化時(shí),在MAPK信號通路中,細(xì)胞間的鏈接蛋白Grb2表達(dá)水平降低,進(jìn)而影響后續(xù)一系列信號傳遞與通路的激活,使胚胎不能正常生長發(fā)育。在Hedgehog信號通路中,干擾SHH基因的表達(dá)時(shí)Hedgehog信號通路不能夠被激活而處于抑制狀態(tài),使胚胎早期不能夠正常發(fā)育,當(dāng)SHH基因過表達(dá)時(shí),能夠成功激活Hedgehog信號通路,并且過度激活,同樣對胚胎產(chǎn)生負(fù)面影響,例如胚胎出現(xiàn)畸形。干擾Foxl2基因使其表達(dá)量降低,造成胚胎發(fā)育早期參與性別分化的DMRT1、SOX9、CYP19基因表達(dá)發(fā)生紊亂,而導(dǎo)致在發(fā)育性分化過程中出現(xiàn)異常,使胚胎不能正常生長發(fā)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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S813.22
本文編號：2363905
[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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S813.22
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