發(fā)布時間：2018-06-16 13:50

  本文選題：銀黑狐 + 藍狐　； 參考：《中國農(nóng)業(yè)科學院》2016年博士論文

【摘要】：銀黑狐和藍狐雜交屬于屬間遠緣雜交,雜交一代銀霜狐(藍狐♂×銀黑狐♀,HBS)和藍霜狐(銀黑狐♂×藍狐♀,HSB)公狐表現(xiàn)為完全或部分不育,不能產(chǎn)生精子。近年來,轉(zhuǎn)錄組學技術(shù)被應用于馬和驢雜交、黃牛和牦牛遠緣雜交雄性不育,篩選得到調(diào)控精子發(fā)生的關(guān)鍵基因。本研究利用準備配種期和配種期銀黑狐、藍狐和銀霜狐、藍霜狐公狐作為試驗材料,通過比較分析正�？捎你y黑狐、藍狐,以及不育的雜交狐精液品質(zhì)、血清激素水平和睪丸、附睪的組織形態(tài)學差異,尋找雜交狐雄性不育的可能原因。以準備配種期和配種期銀黑狐、藍狐、雜交狐的睪丸組織為研究對象,采用轉(zhuǎn)錄組測序技術(shù),建立銀黑狐、藍狐、雜交狐的轉(zhuǎn)錄組數(shù)據(jù)庫,針對4種狐貍2個不同發(fā)育時期、同一發(fā)育時期4種不同的狐貍之間進行差異基因分析,篩選雜交狐和銀黑狐、藍狐調(diào)控精子發(fā)生過程的差異表達基因,確定精子發(fā)生關(guān)鍵基因,揭示雜交狐雄性不育的生理學差異和分子調(diào)控機制。結(jié)果表明配種期銀黑狐、藍狐、銀霜狐和藍霜狐的單次射精量之間差異不顯著(P0.05)。銀黑狐和藍狐精液中存在大量精子,兩種雜交狐銀霜狐和藍霜狐精液中沒有精子存在,雜交狐精子發(fā)生過程停滯。銀霜狐和藍霜狐生精細胞由初級精母細胞階段向次級精母細胞階段發(fā)育的過程發(fā)生了障礙,精子發(fā)生過程停滯在次級精母細胞階段。準備配種期銀霜狐和藍霜狐低濃度的睪酮和雌二醇,高濃度的FSH、LH和PRL引起睪丸和生精小管發(fā)育不完全。配種期銀霜狐和藍霜狐低濃度的睪酮,高濃度的LH和PRL引起睪丸和生精小管發(fā)育不完全,導致雜交狐生精過程障礙,進而導致不育。對4種狐貍準備配種期和配種期睪丸組織進行高通量測序及序列組裝,共獲得Clean bases175.36G,1142703819條Transcripts,673146243條Unigenes,建立了較為全面的狐貍睪丸轉(zhuǎn)錄組數(shù)據(jù)庫,與NR、NT、SwissProt數(shù)據(jù)庫注釋比對,分別注釋了76243、276899、56851條Unigenes。對轉(zhuǎn)錄組測序獲得的差異基因進行分析,將4種狐貍同一個發(fā)育時期以及同一種狐貍準備配種期和配種期比較發(fā)現(xiàn),篩選到調(diào)控精子發(fā)生的差異基因有151個。進一步分析發(fā)現(xiàn),有26個基因主要在第一次減數(shù)分裂由初級精母細胞向次級精母細胞發(fā)育階段發(fā)揮作用,這26個基因分別是ASPM,BAG6,BRDT,CCNA1,DDX4,GGN,GGNBP2,GMCL1,HSF2,HSF2BP,KHDRBS3,MAEL,MAK,MEIG1,MLH1,MNS1,NDRG3,PACRG,PAFAH1B1,PGAM2,RAD51C,SCMH1,SOX30,SUN5,TDRD9和TEX15。還有兩個基因?qū)τ诰影l(fā)生很重要,KATNAL1基因于睪丸支持細胞特異表達,ACSBG2基因于睪丸間質(zhì)細胞特異表達。對18個差異表達基因進行熒光定量PCR驗證發(fā)現(xiàn),差異基因熒光定量PCR表達趨勢與轉(zhuǎn)錄組測序結(jié)果基本一致,說明轉(zhuǎn)錄組測序結(jié)果基本反映了雜交狐和銀黑狐、藍狐睪丸中基因真實的表達水平。綜上所述,銀黑狐和藍狐雜交一代銀霜狐和藍霜狐精子發(fā)生過程停滯在初級精母細胞向次級精母細胞發(fā)育階段,篩選得到的28個基因可以作為后續(xù)研究銀黑狐和藍狐雜交雄性不育機制的候選關(guān)鍵基因。而這28個基因在銀霜狐和藍霜狐精子發(fā)生過程中的具體功能和相互作用關(guān)系有待于進一步驗證。本研究獲得的研究結(jié)果對系統(tǒng)研究雜交狐雄性不育的分子機制奠定了基礎。
[Abstract]:The crossbreeding of Silver Black Fox and blue fox belongs to the Intergenera distant hybridization. The cross generation silver fox (blue fox, silver and black fox, HBS) and blue fox (Silver Black Fox, HSB) are infertile and can not produce sperm. In recent years, the technique of transcriptional technology has been applied to the hybridization of horse and donkey, the distant hybridization male sterility of yellow cattle and yaks, and the screening of male sterility. The key genes regulating spermatogenesis were obtained. In this study, Silver Black foxes, blue foxes, and blue fox foxes were used as experimental materials to compare and analyze the semen quality of normal fertile Silver Black Fox, blue fox, and sterile hybrid fox, and the histomorphological differences between the serum irritable level and the testis and epididymis. The possible cause of male sterility of the hybrid fox. In order to prepare the testicular tissue of the Silver Black Fox, blue fox and the hybrid fox, the transcriptional database of the Silver Black Fox, blue fox and the hybrid fox was set up by the transcriptional sequence technique, and the difference between the 4 foxes at the 2 different developmental periods and the 4 different foxes at the same development period was carried out. The differential gene analysis was used to screen the differentially expressed genes in the spermatogenesis of hybrid Fox and silver fox and blue fox. The key genes of spermatogenesis were determined and the physiological and molecular mechanisms of male sterility of hybrid fox were revealed. The results showed that there was no significant difference in the single ejaculation of Silver Black Fox, blue fox, silver frosting Fox and blue fox (P0.05). There are a large number of sperm in the semen of silver and black fox and blue fox. There is no sperm in the semen of two kinds of Fox and blue fox. The process of spermatogenesis of hybrid fox is stagnant. The development of silver frosting Fox and blue fox spermatogonial cells from the primary spermatocyte stage to secondary spermatocyte stage, the process of spermatogenesis stagnates in secondary spermatogenesis. The mother cell stage. Prepare the low concentration of testosterone and estradiol in the silvery frosting Fox and blue fox. High concentrations of FSH, LH and PRL cause incomplete development of testicles and seminiferous tubules. The low concentration of testosterone in the silvery Fox and blue fox in the mating period, the high concentration of LH and PRL causes the testicles and seminiferous tubules incomplete, resulting in the barrier of the hybrid fox spermatogenesis. Clean bases175.36G, 1142703819 Transcripts and 673146243 Unigenes were obtained for 4 kinds of foxes in the preparation and mating stage of the testis. The database of the fox testicular transcriptional group was set up, compared with the NR, NT, SwissProt database annotated, and 762432768 were annotated respectively. 9956851 Unigenes. analyzed the differential genes obtained by the sequencing of the transcriptional group, and found that there were 151 different genes regulating spermatogenesis in 4 foxes at the same period of development and the same kind of fox, and 151. Further analysis found that there were 26 genes mainly in the first meiosis from primary spermatogenesis. The 26 genes are ASPM, BAG6, BRDT, CCNA1, DDX4, GGN, GGNBP2, GMCL1, HSF2, HSF2BP, KHDRBS3, two genes are important for spermatogenesis. The specific expression of the ACSBG2 gene in the Leydig cells was supported. The fluorescence quantitative PCR verification of the 18 differentially expressed genes showed that the expression trend of the differential gene fluorescence quantitative PCR was basically consistent with the sequence of the transcriptional sequence, indicating that the sequencing results of the transcriptional group basically reflected the cross Fox and the Silver Black Fox, and the gene in the blue fox testis was true. In summary, the spermatogenesis of Silver Black Fox and blue fox is stagnant in the development stage of primary spermatocyte to secondary spermatocyte. The 28 genes selected can be used as a candidate key gene for subsequent study of the male sterile mechanism of Silver Black Fox and blue fox. The 28 genes are in silver frost. The specific functions and interactions in the spermatogenesis of Fox and blue frosting fox need further verification. The results obtained in this study have laid a foundation for the systematic study of the molecular mechanism of male sterility of hybrid foxes.
【學位授予單位】：中國農(nóng)業(yè)科學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S865.23

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本文編號：2026916