利用抑制消减杂交技术筛选鹅就巢行为相关基因

旨在利用抑制消减杂交技术构建鹅就巢与产蛋cDNA基因文库,筛选就巢母鹅上调与下调表达的基因.以就巢期和产蛋期鹅卵巢组织互为检测子和驱动子,进行正反双向消减杂交,获得鹅卵巢差异表达基因文库.从双向文库随机挑选单菌落进行PCR验证,结果表明文库质量良好.选取654个阳性克隆进行测序分析,获得641条表达序列标签(ESTs).对ESTs进行去除载体序列、质量检测、聚类及拼接,经BLASTn比对,有166条ESTs找到与之匹配的同源序列,其中92个是功能基因.比较就巢与产蛋SSH文库,发现就巢SSH库中参与细胞凋亡、信号转导及细胞结构的基因出现频率较高;产蛋特异性基因文库中参与物质能量代谢、细胞防御的基因较多;尚有许多差异片段属于未知功能蛋白或理论推定蛋白.结果提示,催乳素受体、抗苗勒管激素以及雌激素受体基因在就巢期上调表达可能与鹅就巢行为的启动与维持有关.该结果为进一步研究鹅就巢行为分子调控机制奠定基础.     

伊犁鹅产蛋前后各期卵巢转录谱的构建及卵泡发育相关基因的分析

【目的】构建产蛋前后各期伊犁鹅卵巢转录组文库,结合生物信息学分析揭示不同产蛋期伊犁鹅卵巢组织差异表达基因,并鉴定出影响鹅卵巢发育的关键基因,为伊犁鹅的繁殖调控提供理论参考。【方法】选择处于开产期（KL组）、产蛋期（CL组）及休产期（XL组）的伊犁鹅各4只,屠宰后取其卵巢组织,用于构建卵巢转录组文库。通过新基因挖掘、基因差异表达、基因注释以及蛋白互作网络分析筛选出与鹅卵泡发育相关的候选基因;随机挑选8个差异表达基因,应用实时荧光定量PCR验证其表达情况。【结果】伊犁鹅卵巢组织学结果表明,在开产期时,伊犁鹅卵巢表面有大量初级卵泡,而产蛋期卵巢则显示出卵泡的层级性,在休产期卵巢中可观察到卵泡出现向内凹陷、闭锁的现象。通过转录组测序（RNA-Seq）,从构建的12个伊犁鹅卵巢cDNA文库中获得有效读数57 811 186~85 328 377条,Q30值均>93.38%,每个样品所产的测序读数于鹅参考基因组上的比对率在82.79%~89.24%。在卵巢中注释的新基因共有1 112个,单核苷酸多态性（SNP）位点总数为1 642 273~2 425 069个;SNP和插入缺失（InDel）均主要注释于内含子区域;各时期的可变剪切类型主要集中为最后1个外显子可变剪切（TSS）及第1个外显子可变剪切（TTS）。在KL vs CL、XL vs CL及XL vs KL组中分别有337、1 136和525个差异表达基因,共有差异表达基因为α2A肾上腺素能受体（ADRA2A）、表皮蛋白（CP）、非转移性黑色素瘤糖蛋白B （GPNMB）及α-1-抗胰蛋白酶样（LOC106033756）。GO功能富集分析发现,差异表达基因主要富集在肽基酪氨酸磷酸化的正调控、细胞黏附及质膜外侧等过程。KEGG通路富集分析发现,差异表达基因主要显著富集于神经活性配体-受体相互作用、ECM-受体相互作用及类固醇生物合成等。结合蛋白互作网络分析,筛选到与鹅卵巢发育相关的潜在调控因子Bruton’s酪氨酸激酶（BTK）、血小板源性生长因子受体α（PDGFRA）、整合素β3（ITGB3）等。实时荧光定量PCR结果显示,RNA-Seq结果准确可靠。【结论】本研究揭示了产蛋前后各期伊犁鹅卵巢组织中的基因表达差异,筛选到影响伊犁鹅卵巢发育的神经活性配体-受体相互作用、ECM-受体相互作用、类固醇生物合成等重要通路与BTK、PDGFRA和ITGB3等关键候选基因,为了解鹅卵巢组织调控产蛋性能的分子机制提供了理论支撑。     

Characterization of ovarian morphology and reproductive hormones in Zhedong white geese (Anser cygnoides domesticus) during the reproductive cycle

Zhedong white goose (Anser cygnoides domesticus) is a native Chinese breed with strong broodiness and low egg production, which is related to the physiology of reproduction. However, thus far, the physiology of goose reproduction has not been well elucidated. In the present study, the ovarian morphology and reproductive hormones of Zhedong white geese were investigated during the reproductive cycle (the laying and brooding periods). The results showed that the surface of the ovary was atrophied and follicular atresia appeared to some extent in the brooding period compared with the laying period. The concentrations of follicle-stimulating hormone, progesterone and luteinizing hormone were significantly higher than those in the brooding period (p < 0.05). In contrast, the concentrations of prolactin (PRL) and anti-Müllerian hormone (AMH) in the laying period were significantly lower than those in the brooding period (p < 0.05). In addition, the mRNA expression levels of PRL, AMH, dopamine-β-hydroxylase (DβH) and cytochrome P450 side-chain cleavage enzyme (P450scc) were detected in the hypothalamus, pituitary and ovaries by using real-time polymerase chain reaction. The results showed that AMH mRNA was expressed specifically in ovary tissue. The expression levels of DβH and PRL in the brooding period was significantly higher than those in the laying period in the three tissues, especially in the early and middle stages of the brooding period. Moreover, AMH mRNA expression in the ovaries presented the same trend. In addition, P450scc mRNA was highly expressed in both the ovary and pituitary in the laying period. These results revealed the remarkable features of ovarian morphology and characterized the hormonal pattern and expression profile during the reproductive cycle, all of which contribute to understanding the differences in reproductive physiology between the laying and brooding periods in Zhedong white geese.     

我国地方鹅种就巢性状相关转录组研究进展

随着转录组测序技术的发展,利用组学技术解析我国地方鹅就巢性的研究逐渐深入.在不同品种鹅的各个繁殖阶段,围绕下丘脑-垂体-卵巢轴的不同水平进行转录组分析,可以深入解析鹅就巢的遗传机制.文章针对转录组解析我国地方鹅繁殖性能的研究进行综述,分析不同品种鹅在各产蛋阶段的基因表达图谱,汇总与鹅繁殖性状显著相关的差异表达基因,总结...     

Study on the Changes of Reproductive Hormone and Regulation of Follicle Development During the Reproductive Cycle of Yili Goose in Xinjiang

This experiment was aimed to study the changes of reproductive hormone and regulation of follicle development during the reproductive cycle of Yili goose in Xinjiang. 2 years old Yili goose were chosen which were in good health and similar weight. 32 samples were randomly selected to take blood samples for hormone detection during the laying, broodiness and ceased periods, and 15 samples were randomly selected to gather ovarian tissue for the observation of the development of ovary and follicular. The results showed that during the laying period, the GnRH of the Yili goose was extremely significantly higher than the broodiness and ceased periods of 16.78% and 58.29% (P<0.01);PRL was significantly lower than the broodiness period of 13.00% (P<0.05), and was extremely significantly higher than the ceased period of 28.94% (P<0.01); FSH was higher than the broodiness period of 6.98% (P>0.05),and was extremely significantly higher than the ceased period of 21.12% (P<0.01); LH was extremely significantly lower than the broodiness period of 8.38% (P<0.01),and was extremely significantly higher than the ceased period of 16.84% (P<0.01); E₂ were extremely significantly higher than that the broodiness and ceased periods of 29.80% and 112.40% (P<0.01); P₄ levels were extremely significantly higher than the broodiness and ceased periods of 28.89% and 30.34% (P<0.01). The ovary volume and weight of Yili goose were extremely significantly higher than that of broodiness and ceased periods (P<0.01). In the laying period, the mature follicles of Yili goose were significantly larger than that of the broodiness and resting periods, the number of secondary follicles was more and the number of primary follicles was lesser. Ovarian growth and mature follicle atresia, granule layer shrinkage, inward depression, cytoplasmic cavity, follicular atrophy at all levels in the broodiness period. The follicle cytoplasmic cavity of the ovary was larger in the ceased period than broodiness period, and inward concave further deeper, a large number of primordial follicles evenly arranged. Thus, during the laying period, the responses of GnRH, E₂ and LH of Yili goose were active and played a leading role on the development of ovarian and follicular. PRL and LH maintained a high level of secretion, cooperated to maintain the broodiness behavior during the broodiness period.     

新疆伊犁鹅繁殖周期生殖激素变化及卵巢卵泡发育规律的研究

为探究新疆伊犁鹅繁殖周期中血浆主要生殖激素、卵巢形态及卵泡发育的变化规律,试验以健康状况良好、体重相近的2岁伊犁鹅为试验对象,分别在产蛋期、就巢期及休产期各随机选取32只,采集1次血样,测定激素水平;在各时期分别选取5只采集卵巢组织,观测卵巢及卵泡发育情况。结果表明,在产蛋期,伊犁鹅的GnRH分别极显著高于就巢期和休产期16.78%和58.29%（P<0.01）;PRL显著低于就巢期13.00%（P<0.05）,极显著高于休产期28.94%（P<0.01）;FSH高于就巢期6.98%（P>0.05）,极显著高于休产期21.12%（P<0.01）;LH极显著低于就巢期8.38%（P<0.01）,极显著高于休产期16.84%（P<0.01）;E₂水平分别极显著高于就巢期和休产期29.80%和112.40%（P<0.01）;P₄水平分别极显著高于就巢期和休产期28.89%和30.34%（P<0.01）。产蛋期伊犁鹅的卵巢体积和重量均极显著大于就巢期和休产期（P<0.01）。产蛋期伊犁鹅成熟卵泡与就巢期和休产期相比明显较大,次级卵泡数量较多,初级卵泡数量较少;就巢期卵巢上生长及成熟卵泡发生闭锁,颗粒胞层收缩,向内凹陷,胞质出现空腔,各级卵泡出现萎缩;与就巢期相比,休产期卵巢上卵泡胞质空腔变大,向内凹陷程度进一步加深,大量原始卵泡均匀排列。由此可见,在产蛋期,伊犁鹅的GnRH、E₂、LH反应活跃,对卵巢卵泡发育起主导作用;在就巢期,PRL、LH维持较高的分泌水平,协同维持就巢。     

共轭亚油酸影响边鸡胸肌脂类代谢的相关差异基因的筛选

旨在通过转录组测序技术筛选出共轭亚油酸(conjugated linoleic acids,CLA)影响边鸡胸肌脂类代谢的相关基因,为日粮中CLA调控边鸡脂类代谢的作用机制提供理论依据。本试验选取90日龄健康边鸡180只,随机分为5组,每组3个重复,每组日粮中分别添加不同比例的CLA 0%(对照组)、0.5%、1.0%、1.5%和2.0%,预饲期1周,正饲期6周。饲养试验结束后采集胸肌组织进行转录组测序,使用DESeq2软件对测序数据进行差异表达分析,对差异表达基因进行GO功能和KEGG通路富集分析,筛选出与胸肌脂类代谢相关的差异表达基因,利用qPCR对差异表达基因进行验证。通过转录组测序数据分析,共筛选出1 229个差异表达基因,其中594个基因上调,635个基因下调。随机选取9个差异表达基因进行qPCR验证,其相对表达量变化趋势与测序结果一致。GO功能分析发现,差异表达基因主要集中在生物学过程中的细胞过程、单一生物过程、生物调节和代谢过程。KEGG通路富集分析发现,各试验组中差异表达基因显著富集在不同的信号通路中,主要富集在ECM-受体相互作用、黏着斑、吞噬体、细胞凋亡、肌动蛋白细胞骨架调节和细胞因子-细胞因子受体相互作用等通路中。通过GO和KEGG功能注释分析,共筛选出MCAT、APOA1、PTGDS、ALDH3A2、PLTP、FABP3、FOXO1、UCP3和FABP4等18个主要的参与脂类代谢相关的差异表达基因,可能在调控边鸡胸肌脂类代谢中发挥重要的作用。本研究通过转录组测序筛选出CLA影响边鸡胸肌脂类代谢过程中的主要调控基因,发现了日粮中CLA影响差异表达基因作用的主要生物学过程和信号通路,为今后研究CLA影响边鸡胸肌脂类代谢的分子作用机制奠定了基础。     

Cloning,expression, and polymorphism at the 5ʹ-flanking region of the GnRH gene and their association with laying traits in Muscovy duck (Cairina moschata)

1. Gonadotropin-releasing hormone (GnRH), a neuropeptide, plays a vital role in the hypothalamus–pituitary–gonadal (HPG) axis. In vertebrates, GnRH is crucial for the onset of sexual development and the entire reproductive process. The purpose of this study was to identify genetic factors associated with egg-laying traits of Muscovy ducks.

2. The full-length cDNA (474 bp) of Muscovy duck GnRH was obtained and characterised. It encodes 92 amino acids containing a 1-amino acid signal peptide cleavage site. Phylogenetic analysis revealed that Muscovy duck GnRH has a close relationship with Anas platyrhynchos GnRH.

3. GnRH showed significantly different expression profiles between 4 developmental periods in the hypothalamus, pituitary, and ovary. The expression of GnRH in the laying period (36 weeks) was higher than at other periods in the three tissues. GnRH was widely expressed in 12 examined tissues of nesting and laying Muscovy ducks. In the hypothalamus, pituitary and gonads, the expression of GnRH was higher than in other tissues.

4. In laying Muscovy ducks, the expression of GnRH in the hypothalamus, pituitary, ovary, muscular stomach, pancreas, heart, duodenum and spleen was significantly higher than in nesting dusks. Differences were detected in the liver and glandular stomach between laying ducks and nesting ducks. Differences between the kidney and lung were not significant.

5. In the pituitary, the GnRH and GnRH receptor (GnRHR) genes shared the same expression profiles during 4 time points. Both genes had the highest expression at 36 weeks of age.

6. A mutation (g.206G > A) in the 5?-flanking region was associated with egg-laying performance. Individuals with genotype GG had better egg-laying performance than the individuals with genotype AA. GnRH may be used as a marker gene for laying performance in the Muscovy duck.

     

Cloning and expression analysis of PRL and PRLR genes in black Muscovy duck

ABSTRACT

1. Prolactin hormone (governed by the PRL gene) is secreted by the anterior pituitary of animals, which combines with its receptor (prolactin receptor, PRLR) to act on target cells. Both PRL and PRLR are mainly associated with reproductive performance. The genetic mechanism of nesting in poultry is not yet clear, and so the aim of the current study was to determine expression patterns of PRL and PRLR at different times across the breeding stages of black Muscovy ducks.

2. In this study, the CDS regions of PRL and PRLR were determined by RACE sequencing. The expression levels of PRL and PRLR in the pituitary, ovary and uterus from the black Muscovy duck were compared and analysed during the pre-laying, laying and nesting periods.

3. The results showed that PRL and PRLR are highly homologous in a variety of poultry species. The expression of the PRL gene in the pituitary was the highest, which was significantly higher than seen in the ovary and uterus. This trend ran through the entire prenatal period, i.e. the laying period and the nesting period. The expression level of the PRLR gene in the pituitary and ovary was generally low, and expression in the uterus was the highest. There was no significant difference in expression of the PRLR gene between pituitary and ovary during different periods, but the expression level of the PRLR gene in the uterus reached its highest level during the nesting stage, which was significantly higher than seen in the early laying period.     

日粮粗蛋白质水平对伊犁鹅产蛋规律、繁殖激素分泌及相关基因mRNA表达的影响

为探讨日粮粗蛋白质水平对伊犁鹅的产蛋规律、繁殖激素分泌及生殖轴相关基因mRNA表达量的影响,本研究随机选取200只3岁伊犁鹅（年龄、饲养管理水平一致及体重相近）,随机分为4个组,每组10个重复。对照组及公鹅饲喂鹅场自配料（粗蛋白质水平11.20%）,试验组母鹅分别饲喂粗蛋白质水平为13.86%、15.20%和16.48%的日粮,其余营养指标基本一致。预试期1周,正试期9周。结果表明：①试验期内伊犁鹅的产蛋率呈波动变化,对照组伊犁鹅的产蛋率在第1~2周逐渐上升,第4~5周出现小幅度增长,其余阶段表现为下滑趋势;各试验组伊犁鹅的产蛋率在第1~2、4~5周出现较大幅度增长,且在第5周时达到产蛋高峰,此后逐渐下滑。15.20%粗蛋白质组伊犁鹅就巢率显著低于11.20%、13.86%粗蛋白质组。②与对照组相比,13.86%粗蛋白质组血清促性腺激素释放激素（GnRH）浓度极显著升高（P<0.01）,且血清雌二醇（E₂）、促黄体素（LH）浓度均显著升高（P<0.05）;15.20%、16.48%粗蛋白质组血清GnRH、E₂及LH浓度均极显著升高（P<0.01）,且血清促卵泡素（FSH）浓度显著升高（P<0.05）。各试验组血清催乳素（PRL）浓度均显著降低（P<0.05）。③与对照组相比,15.20%粗蛋白质组下丘脑GnRH、LHR基因表达量显著上调（P<0.05）,PRL、PRLR基因表达量显著下调（P<0.05）;垂体中LH基因表达量显著上调（P<0.05）;卵巢中LH与ESR2基因表达量显著或极显著上调（P<0.05,P<0.01）。综上所述,基于日粮粗蛋白质水平对伊犁鹅的产蛋率、就巢率、血清生殖激素浓度及生殖轴相关基因mRNA相对表达量影响的综合评估,建议产蛋期伊犁鹅日粮粗蛋白质水平为15.20%。     

基于转录组测序挖掘广西麻鸡生长发育相关基因

【目的】通过对1和60日龄广西麻鸡腿肌进行转录组测序(RNA-Seq),筛选出广西麻鸡生长发育关键基因。【方法】选择1和60日龄健康母鸡各3只,分别采集腿肌组织,利用Illumina Novaseq^TM 6000平台进行mRNA转录组测序,利用DESeq2软件进行差异表达基因的分析,并对差异表达基因进行GO功能和KEGG通路富集分析,进一步筛选出生长发育相关基因,并用实时荧光定量PCR验证转录组测序结果。【结果】转录组测序结果显示,6个样本分别获得34 296 198~41 647 642条clean reads。与1日龄腿肌相比,60日龄腿肌共获得2 304个差异表达基因,其中998个基因上调,1 306个基因下调。GO功能富集分析发现,共获得富集条目572条,其中生物过程381条,细胞组分70条,分子功能121条。KEGG通路富集分析发现,共获得34个显著富集的信号通路,其中心肌收缩、MAPK信号通路、紧密连接、肌动蛋白细胞骨架信号通路、心肌细胞中的肾上腺素能信号传导等与生长发育相关。通过GO功能和KEGG通路富集分析获得5个生长发育相关基因,分别为肌球蛋白重链10(MYH10)、肌球蛋白链15(MYH15)、成纤维细胞生长因子10(FGF10)、成纤维细胞生长因子16(FGF16)、肌肉生长抑制素(GDF8)基因,其中MYH10、MYH15、FGF10为上调差异表达基因,FGF16和GDF8为下调差异表达基因。所选差异表达基因实时荧光定量PCR结果与转录组测序的基因表达水平一致,说明转录组测序结果可靠。【结论】本试验通过对广西麻鸡1和60日龄2个不同生长阶段进行转录组测序分析,获得MYH10、MYH15、FGF10、FGF16、GDF8共5个与生长发育相关的关键基因,为后续进一步探讨广西麻鸡生长发育的分子调控机制提供参考。     

牦牛泌乳期垂体组织比较转录组学分析

试验旨在了解不同产奶量牦牛泌乳期垂体组织间的转录组差异,为进一步阐述牦牛垂体组织调控泌乳的机制提供参考。提取牦牛垂体组织总RNA,并应用RNA-Seq技术对高产奶量牦牛和低产奶量牦牛泌乳期间垂体组织转录组进行高通量测序及分析。结果显示,通过比较分析高产奶量牦牛和低产奶量牦牛垂体组织转录组数据,筛选出114个差异表达基因,其中55个表现为上调,59个表现为下调。进一步功能分析表明,这些差异基因涉及多种GO分类及KEGG通路,其中GO分析显示,差异基因与许多和氨基酸代谢相关的生物学过程存在紧密关联;KEGG通路分析显示,最为富集的是细胞黏附分子通路,且金黄色葡萄球菌感染和利什曼病等大量与免疫或疾病相关的通路也出现显著富集。本研究对比了高产奶量牦牛和低产奶量牦牛泌乳期垂体组织转录组数据,筛选并分析了相关差异表达基因,为提高牦牛产奶量的研究提供新的思路。     

家禽就巢性的影响因素及其内分泌调控机理研究进展

就巢性是家禽进行繁殖必不可少的行为，对家禽的繁殖能力有很大影响。家禽就巢性作为一个低遗传力性状，其受环境、内分泌及遗传3种因素调控。其中环境因素中光照、温度、饲养管理模式等都对家禽就巢性具有显著影响。与禽类就巢性密切相关的繁殖内分泌激素主要包括：促性腺激素释放激素（GnRH）、催乳素（PRL）、促卵泡激素（FSH）、促黄体素（LH）、孕酮（P₄）和雌二醇（E₂）。遗传是家禽就巢性的决定因素，就巢性候选基因研究主要基于内分泌研究中的相关繁殖激素。随着基因组测序技术发展，大量就巢性相关新基因被证实，为就巢性分子遗传机制的研究提供了新的思路。目前，家禽就巢性的遗传调控机制仍不清楚，开展就巢性的分子调控机制研究，将极大地提高家禽产蛋性能，促进优良地方禽类的保护、开发和利用。作者对环境、内分泌和遗传控制就巢行为的研究进行综述，并对禽类就巢行为的发生和调控机制进行探讨。     

黄体期和卵泡期小尾寒羊DIO2与DIO3组织表达

为分析DIO2和DIO3基因在小尾寒羊不同繁殖时期(黄体期和卵泡期)下丘脑-垂体-性腺轴各组织中的表达差异,阐明这2个基因在绵羊发情转换中的表达模式,实验采用实时荧光定量PCR(Real-time PCR)技术对比分析DIO2和DIO3基因在黄体期和卵泡期小尾寒羊下丘脑-垂体-性腺轴各组织中的表达差异。结果表明:DIO2和DIO3基因在下丘脑、垂体、松果体、大脑、小脑、卵巢、子宫、输卵管、肾脏、肾上腺10种组织中均表达;DIO2基因在黄体期和卵泡期的垂体组织中表达量显著高于其他组织(P<0.05),其中黄体期垂体、子宫、下丘脑、松果体、输卵管和卵巢DIO2的表达量显著高于卵泡期(P<0.05);DIO3在卵泡期松果体、下丘脑、子宫、垂体和输卵管的表达量显著高于黄体期(P<0.05)。综上,DIO2能抑制绵羊发情,而DIO3促进发情。