银黑狐和蓝狐杂交一代公狐不育机制的初步研究

发布时间：2018-06-16 13:50

本文选题：银黑狐 + 蓝狐　；参考：《中国农业科学院》2016年博士论文

【摘要】：银黑狐和蓝狐杂交属于属间远缘杂交,杂交一代银霜狐(蓝狐♂×银黑狐♀,HBS)和蓝霜狐(银黑狐♂×蓝狐♀,HSB)公狐表现为完全或部分不育,不能产生精子。近年来,转录组学技术被应用于马和驴杂交、黄牛和牦牛远缘杂交雄性不育,筛选得到调控精子发生的关键基因。本研究利用准备配种期和配种期银黑狐、蓝狐和银霜狐、蓝霜狐公狐作为试验材料,通过比较分析正常可育的银黑狐、蓝狐,以及不育的杂交狐精液品质、血清激素水平和睾丸、附睾的组织形态学差异,寻找杂交狐雄性不育的可能原因。以准备配种期和配种期银黑狐、蓝狐、杂交狐的睾丸组织为研究对象,采用转录组测序技术,建立银黑狐、蓝狐、杂交狐的转录组数据库,针对4种狐狸2个不同发育时期、同一发育时期4种不同的狐狸之间进行差异基因分析,筛选杂交狐和银黑狐、蓝狐调控精子发生过程的差异表达基因,确定精子发生关键基因,揭示杂交狐雄性不育的生理学差异和分子调控机制。结果表明配种期银黑狐、蓝狐、银霜狐和蓝霜狐的单次射精量之间差异不显著(P0.05)。银黑狐和蓝狐精液中存在大量精子,两种杂交狐银霜狐和蓝霜狐精液中没有精子存在,杂交狐精子发生过程停滞。银霜狐和蓝霜狐生精细胞由初级精母细胞阶段向次级精母细胞阶段发育的过程发生了障碍,精子发生过程停滞在次级精母细胞阶段。准备配种期银霜狐和蓝霜狐低浓度的睾酮和雌二醇,高浓度的FSH、LH和PRL引起睾丸和生精小管发育不完全。配种期银霜狐和蓝霜狐低浓度的睾酮,高浓度的LH和PRL引起睾丸和生精小管发育不完全,导致杂交狐生精过程障碍,进而导致不育。对4种狐狸准备配种期和配种期睾丸组织进行高通量测序及序列组装,共获得Clean bases175.36G,1142703819条Transcripts,673146243条Unigenes,建立了较为全面的狐狸睾丸转录组数据库,与NR、NT、SwissProt数据库注释比对,分别注释了76243、276899、56851条Unigenes。对转录组测序获得的差异基因进行分析,将4种狐狸同一个发育时期以及同一种狐狸准备配种期和配种期比较发现,筛选到调控精子发生的差异基因有151个。进一步分析发现,有26个基因主要在第一次减数分裂由初级精母细胞向次级精母细胞发育阶段发挥作用,这26个基因分别是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。还有两个基因对于精子发生很重要,KATNAL1基因于睾丸支持细胞特异表达,ACSBG2基因于睾丸间质细胞特异表达。对18个差异表达基因进行荧光定量PCR验证发现,差异基因荧光定量PCR表达趋势与转录组测序结果基本一致,说明转录组测序结果基本反映了杂交狐和银黑狐、蓝狐睾丸中基因真实的表达水平。综上所述,银黑狐和蓝狐杂交一代银霜狐和蓝霜狐精子发生过程停滞在初级精母细胞向次级精母细胞发育阶段,筛选得到的28个基因可以作为后续研究银黑狐和蓝狐杂交雄性不育机制的候选关键基因。而这28个基因在银霜狐和蓝霜狐精子发生过程中的具体功能和相互作用关系有待于进一步验证。本研究获得的研究结果对系统研究杂交狐雄性不育的分子机制奠定了基础。
[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.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S865.23

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