3＇ Noncoding Region Construction of GHR Gene-luciferase Report Vector and Valuation

To analyze miR-139 target sites in 3＇ UTR of GHR gene in dairy cow mammary gland, a GHR 3＇ UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy...     

Investigating structural impact of a valine to isoleucine substitution on anti-Müllerian hormone in silico and genetic association of the variant and AMH expression with egg production in chickens

Anti-Müllerian hormone(AMH) acts in maintaining orderly cyclic recruitment of early follicles, suggesting that it is a promising candidate for influencing animal reproductive efficiency. This study aimed to elucidate the effect of a missense mutation of Val~(566) Ile on the structure of AMH protein and the genetic association of Val~(566) Ile and AMH expression with egg production in chickens. Structural perturbations of Val~(566) Ile were predicted by homology modeling. The association of the variant with the number of eggs was tested using a quantitative trait transmission disequilibrium test model. AMH expression in granulosa cells in Lueyang black-boned chickens was compared with that in Nick chickens. The Val~(566) of AMH is a nonconservative amino acid among mammals and birds, but its hydrophobicity is completely conservative. The substitution of Val~(566) for Ile~(566) potentially disrupted hydrogen bonds and solvent accessibility of 22 residues and created a short α-helix in the C terminus of AMH. Despite having striking structure-disrupting potential, the variant was not statistically associated with the number of eggs(P0.05) in the Lueyang black-boned chickens. We did not detect differential expression of AMH between Lueyang black-boned chickens and Nick chickens(P0.05). These results confirmed the structural impact of Val~(566) Ile, but suggested that Val~(566) Ile and AMH expression might not be the major genetic determinants for egg production in Lueyang black-boned chickens.     

Expression of Chicken Toll-Like Receptors and Signal Adaptors in Spleen and Cecum of Young Chickens Infected with Eimeria tenella

Toll-like receptors （TLRs） are a group of highly conserved molecules which initiate the innate immune response to pathogens by recognizing structural motifs of microbes. Understanding the changes in chicken Toll-like receptors （ChTLRs） and signal adaptors expression that occur with Eimeria tenella infection will help to elucidate the molecular basis of immune control of coccidiosis caused by Eimeria. The present study detected the dynamic changes in the expression of ChTLRs and associated signal adaptors in the spleen and cecum ofE. tenella-infected chickens during the early stage of infection. The results showed that the expression peak for ChTLRs, MyD88 and TRIF occurred at 12 h post-infection （hpi）, ChTLR3, ChTLRI 5 and MyD88 mRNA expression in the spleen ofE. tenella infected chickens were significantly higher （P〈0.05） than that of negative control chickens, and there were similar tendencies of these molecules expression in the cecum and spleen of E. tenella-infected chickens. The expression of MyD88 was upregnlated at four time points in the cecum of E. tenella-infected chickens. The results of this study indicate that ChTLR3, ChTLR15 and MyD88 play a role in young chickens infected with E. tenella.     

百宜黑鸡与长顺绿壳蛋鸡线粒体DNA控制区全序列分析

采用PCR和直接测序的方法测定百宜黑鸡和长顺绿壳蛋鸡线粒体DNA控制区全序列,并分析mtDNA D-loop序列3个区的变异。结果表明,百宜黑鸡和长顺绿壳蛋鸡mtDNA控制区全序列长度分别为1 231 bp、1 230 bp,2种贵州地方鸡之间,mtDNA D-loop序列Ⅰ区序列变异率为4.43%,变异速率最快,其他两区的变异率分别为:Ⅱ区0.64%,Ⅲ区1.56%。百宜黑鸡mtDNA控制区的碱基含量分别为A 27.10%、T 33.63%、G 13.12%、C 26.16%,长顺绿壳蛋鸡的碱基含量分别是A 27.13%、T 33.58%、G 13.20%、C 26.09%。百宜黑鸡和长顺绿壳蛋鸡的遗传距离是0.036 2,根据分子钟计算,二者大约在181万a前分歧进化。     

牦牛Linc24063的克隆鉴定及其与miRNAs 表达水平的相关性分析

【目的】通过对牦牛长链非编码RNA Linc24063进行克隆鉴定,分析其在乳腺组织中与miRNAs表达量的相关性,为Linc24063通过调控miRNA发挥功能的调控机制研究提供试验依据。【方法】选取4.5岁处于第一泌乳期的健康母牦牛12头,清晨空腹屠宰后,迅速采集大脑、乳腺、肾脏、心脏、肝脏和卵巢组织,提取组织总RNA,利用5′ RACE和3′ RACE方法克隆牦牛Linc24063序列;利用生物信息学对其序列保守性、染色体定位及编码潜能进行分析,然后利用原核表达试验对其编码能力进行验证;利用RT-qPCR检测其在大脑、乳腺、肾脏、心脏、肝脏和卵巢的表达水平。利用普通牛和绵羊的miRNA数据库,结合miRanda和mireap软件预测与Linc24063具有相互作用的物种间保守miRNAs,与前人研究表明在牛不同泌乳时期乳腺组织中具有差异表达的miRNA取交集,然后对其靶基因进行GO富集和KEGG信号通路分析;最后使用皮尔逊相关系数在乳腺组织中进行Linc24063与miRNAs表达量的相关性分析。【结果】Linc24063 5′RACE和3′RACE片段大小分别为476 bp和356 bp,测序分析表明Linc24063大小为758 bp,位于牦牛21号染色体的Dlk1-Dio3印记域,与普通牛的序列保守性最高。生物信息学预测其编码潜能较低,原核表达试验进一步验证Linc24063不能有效的翻译蛋白,表明Linc24063是一个真正的长链非编码lncRNA。组织表达谱分析表明,Linc24063在牦牛乳腺组织中表达量最高,在肝脏和卵巢中表达量较低。生物信息学分析后共筛选到与Linc24063具有相互作用的物种间保守miRNAs 21个,其中有研究报道在乳腺中有差异表达的13个;13个miRNAs靶基因富集到TGF-β、PI3K-Akt、胰岛素等信号通路中,表明Linc24063可能通过这些信号通路参与牦牛乳脂和乳蛋白的生物合成过程。在乳腺组织中,Linc24063表达水平与miR-200a（P=0.001）、miR-141（P=0.02）显著负相关,与miR-27a（P=0.023）显著正相关,与miR-24（P=0.601）不具备相关性。【结论】Linc24063位于Dlk1-Dio3印记域内,在乳腺组织中具有较高表达量,且可能通过与miR-200a、miR-141和miR-27a相互作用从而参与牦牛乳脂和乳蛋白的生物合成过程。为深入探讨牦牛Linc24063在乳脂和乳蛋白合成中的生物学功能提供了基础数据。     

父母代固始鸡十二指肠的发育形态学观察

为了研究固始鸡十二指肠的生长发育规律和组织学特点,应用大体解剖学和组织切片技术,对0~20周龄父母代固始鸡的十二指肠进行了发育形态学研究,并用Logistic方程模型模拟十二指肠质量的生长变化。结果表明,随着周龄的增加,固始鸡十二指肠的长度、质量、周长、绒毛长度、肠腺隐窝深度、肠腺宽度及各肌层厚度均增大;十二指肠相对生长率、十二指肠指数和肠腺密度均呈下降趋势;肠绒毛有分支现象;淋巴组织的发育比较缓慢,到6周龄后才出现有淋巴小结。     

UBE2I stimulates female gonadal differentiation in chicken (Gallus gallus) embryos

Without known analogous sex-determining factors like SRY (sex determining region Y) in mammals, the chicken (Gallus gallus) sex determination mechanism still remains unclear, which highly restricts the biological research on chicken development and poultry single-sex reproduction. Here we not only characterized a new female-biased gene UBE2I and identified the expression pattern by qRT-PCR, but also described the functional role of UBE2I in the gonadal development of chicken embryos. Results showed that UBE2I exhibited a female-biased expression pattern in the early stage of PGCs (primordial germ cells) in embryonic gonads and robust expression in ovaries of newborn chickens. Most importantly, we successfully developed an effective method to interfere or overexpress UBE2I in chicken embryos through the intravascular injection. The qRT-PCR analysis showed that the sex-related genes (FOXL2, CYP19A1 and HINTW) in females were upregulated (P<0.05) under the overexpression of UBE2I and the sex-related genes (SOX9, DMRT1 and WT1) in females were downregulated (P<0.05) after interfering UBE2I. Furthermore, the change of UBE2I expression was associated with the level of estradiol and its receptors (AR and ESR), which suggests that UBE2I is necessary to initiate the female-specific development in chickens. In conclusion, this work demonstrates that UBE2I is a crucial sex differentiation-related gene in the embryonic development of chickens, which provides insights for further understanding the mechanism of sex determination in chickens.     

Influence on Cellular Signal Transduction Pathway in Dairy Cow Mammary Gland Epithelial Cells by Galactopoietic Compound Isolated from Vaccariae segetalis

The galactopoietic mechanism of Vaccaria segetalis is still unknown. Understanding dibutyl phthalate (DBP) separated from Vaccaria segetalis on the expression of lactation signal transduetion genes of mammary gland epithelial cells, including prlr, erα, akt1, socs2, pparγand elf5, will be helpful to reveal the molecular mechanism. Western blot and qRTPCR were used to study the change of prlr, erα, akt, socs2, pparγ, and elf5 expression at mRNA and protein level. Colocalization expression of prolactin receptor (PRLR) and estrogen receptor α (ERα) was observed by immunofluoreseence;the expression changes of miRNAs (21, 125b, 143, and 195) and the secretion of β-casein and lactose were detected byqRT-PCR and RP-HPLC. The results showed that Vaccaria segetalis active compound had similar fuctions as estrogen and/or prolactin (PRL) in dairy cow mammary gland epithelial cells (DCMECs), increased the expressions of prlr, erα, akt1,and elf5 genes, while repressed ppaγ expressions. DBP promoted socs2 mRNA expression, but its protein expressionswere repressed. Furthermore, both DBP and PRL could repress the expressions of miRNA-125b, miRNA-143 and miRNA-195 in DCMECs. DBP could repress the expression of miRNA-21, while the influence of PRL on miRNA-21 was not certain.DBP could promote the lactation ability of DCMECs by regulating the ER and PRLR cellular signal transduction pathway.     

Chinese herbal formulae defend against Mycoplasma gallisepticum infection

Mycoplasma gallisepticum HS strain (MG-HS) is a pathogen that causes chronic respiratory disease (CRD) in chicken, which is characterized by host respiratory inflammatory damage, brings huge economic losses to the poultry industry. Recently, emerging Chinese herbal medicines (CHM) have been used to treat CRD. This study was aimed to investigate the preventive and therapeutic effects and their potential mechanisms of Chinese herbal medicinal formulae (CHMF), which consisted of 10 kinds of Chinese herbal medicine including Scutellaria, Houttuynia cordate and licorice, on MG-induced CRD in chickens. With respect to the preventive effect, the results showed that CHMF could effectively recover the MG-induced decrease on body weight and feed conversion ratio. Histopathological analysis showed that both prevention and treatment of CHMF could significantly alleviate the severe respiratory inflammation induced by MG infection. Moreover, compared with the MG infection group, both the prevention groups and the treatment groups of CHMF could effectively reduce the expression of MG adhesion protein (pMGA1.2) to inhibit the proliferation of MG, and thus effectively inhibit the expression of MG-induced inflammatory factors interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α). In summary, these findings confirm that CHMF can protect chickens from various tissue damage caused by MG infection and has no adverse effects on the performance of chickens in the short term. And its efficacy against MG is equal to or better than that of tiamulin.     

mgr-mir-9 implicates Meloidogyne graminicola infection in rice by targeting the effector MgPDI

MicroRNAs (miRNAs), a class of small non-coding RNAs, are crucial endogenous gene regulators in a range of animals, including plant-parasitic nematodes. Meloidogyne graminicola is an obligate sedentary endoparasite of rice and causes significant yield losses. A number of studies focused on the roles of M. graminicola effectors during the parasitic process; however, how nematode miRNAs regulate its effectors needs elucidating. In this research, we analyzed a cluster of M. graminicola miRNAs obtained at the second-stage juveniles (J2s) stage that are closely linked to the regulation of M. graminicola effectors. There are 49 767 105 total clean reads obtained from three libraries. A total of 233 known miRNAs and 21 novel miRNAs were identified. Among the known miRNAs, mgr-lin-4, mgr-mir-1, mgr-mir-100, mgr-mir-86, mgr-mir-279, mgr-mir-87, mgr-mir-71, mgr-mir-9, mgr-mir-50, mgr-mir-72, and mgr-mir-34 are the most abundant 11 miRNAs families. Moreover, the expression levels of selected miRNAs were validated by real-time quantitative PCR. We hypothesized that these miRNAs might regulate the expression of secreted effectors during the J2s stage to facilitate its infection. Consistent with this, we found that mgr-mir-9 targets MgPDI, an important M. graminicola effector mRNA. In addition to that, J2s treated with mgr-mir-9 mimics showed down-regulation of MgPDI expression and reduced reproductive ability, alluding mgr-mir-9 is involved in nematode infection. These results provide novel insight into the regulatory functions of M. graminicola miRNAs during the infection and identify miRNAs and their effector targets as potential key management targets to limit parasite survival during the early stages of infection.     

青蛤壳色遗传初步研究

本实验对自然种群青蛤的壳色进行了观察和分析;将亲贝分为白壳群体和紫壳群体分别进行人工繁育,对F1壳色分离情况进行了统计和分析;分析了泥质、粉沙质和细沙质不同底质对F1壳色表达的影响。结果表明:(1)F1出现了4种壳色类型,两群体间相同壳色个体百分率差异均显著(P﹤0.01),F1壳色出现相同于亲本的纯化,说明青蛤白、紫壳色能够真实遗传;(2)青蛤紫色壳色的深浅明显受到底质的影响,泥质使壳色变淡并降低了紫壳个体百分率,沙质底则利于紫色壳色的表达,其中细沙质与泥质差异达到显著性水平(P﹤0.05),但底质影响力不超过遗传能力的1/4;(3)紫壳群体F1的紫壳个体百分率达70%以上,接近未分离亲贝18.71%的4倍,使紫壳个体得到显著纯化,为选育纯紫壳品系或群系创造了条件。     

Knockdown of the Meq gene in Marek's disease tumor cell line MSB1 might induce cell apoptosis and inhibit cell proliferation and invasion

Marek's disease (MD), a highly cell-associated and contagious disease of chickens caused by Marek's disease virus (MDV) can result in neural lesions, immunosuppression and neoplasia in chicken. The Meq gene is an important oncogene in the MDV genome, and it is expressed highly in MD tumor tissues and MD T-lymphoblastoid cell lines. An experiment was conducted to elucidate the role of Meq in MD tumor transformation. RNA interference technology was used to block its expression, and then analyzed the biological effects of Meq knockdown on the MD tumor cell line MSB1. A small interfering RNA with an interference efficiency of 70% (P<0.01) was transfected into MSB1 cells to knock down the expression of Meq gene. The cell proliferation, cycle and apoptosis were detected post-Meq knockdown. The results showed that MSB1 cell proliferation was downregulated remarkably at 48 h (P<0.01), 60 h (P<0.05) and 72 h (P<0.01) post-Meq knockdown. The cell cycle was unaffected (P>0.05). B-cell lymphoma 2 gene (BCL2) was anti-apoptotic and caspase-6 was the effector in the apoptosis pathway. The activity of caspase-6 was upregulated (P<0.05) significantly and BCL2 gene expression was downregulated (P<0.05) significantly post-Meq knockdown, suggesting cell apoptosis might be induced. MSB1 cell migration did not exhibit any obvious change (P>0.05) post-Meq knockdown, but the expression of two genes (matrix metalloproteinase 2 (MMP2) and MMP9) that are correlated closely to cell invasion was downregulated (P<0.05) remarkably post-Meq knockdown. The Meq knockdown might affect the main features of tumorous cells, including proliferation, apoptosis, and invasion, suggesting that the Meq gene might play a crucial role in interfering with lymphomatous cell transformation.     

饲粮蛋白质水平对鹌鹑产蛋性能和蛋品质的影响

采用单因子试验设计,将饲粮粗蛋白质(CP)设为6个水平,分别为16％、18％、20％、22％、24％、26％,将鹌鹑分为相应6个组,每组4个重复,每个重复20只鹌鹑,试验分2期进行,前期为6～33周龄,后期为34～41周龄,研究饲粮中蛋白质水平对产蛋鹌鹑产蛋性能和蛋品质的影响.结果表明:第1组鹌鹑后期产蛋量显著低于第2...     

不同剂量党参提取物对青脚麻鸡小肠组织结构影响

为研究饮水中添加不同剂量党参提取物对青脚麻鸡小肠组织结构的影响,将320只14日龄的青脚麻鸡随机分为4组,雌雄各半,分为Ⅰ组(对照,饮用自来水)以及Ⅱ、Ⅲ和Ⅳ组(分别为饮用党参提取物0.5、1和2 g·L-1)。分别于28、35、42、49和56日龄时空腹,各组随机抽样16只,解剖迅速取空肠和回肠标本,Bouin液常温固定24 h后,制作5μm连续组织切片,HE染色,OLYMPNS-CH30型显微摄影系统显微观察并摄影。结果表明,1 g·L-1和2 g·L-1组空肠和回肠绒毛较对照组明显增多,变粗,变长且排列整齐,肠壁增厚,肠腺较发达,回肠段集合淋巴结变大,淋巴细胞聚集明显,其中以2 g·L-1最为显著。说明党参提取物增强了青脚麻鸡小肠吸收功能,对肠道免疫功能亦有一定的促进作用。     

浙江省主要蛋鸭品种蛋品质测定和遗传力估计

测定了浙江省2个地方鸭品种共6个品系各50个蛋的蛋形指数、蛋壳强度、蛋壳厚度、蛋比重、哈氏单位、蛋白高度、蛋黄重和蛋黄比率等8项蛋品质指标,进行了品种和品系间的多重比较和各性状的遗传力估计。结果表明：品种间比较显示,绍兴鸭的蛋壳质量显著高于缙云麻鸭,缙云麻鸭蛋白和蛋黄品质优于绍兴鸭;品系间比较显示,绍兴鸭带圈白翼梢系和绍兴鸭青壳系蛋壳质量最好,优于其他品系,缙云麻鸭3个品系的蛋白和蛋黄品质差异较小;蛋品质各性状的遗传力估计结果显示蛋比重和蛋壳厚度遗传力较高,分别为0.72和0.47,而哈氏单位性状最低（0.06）。结果提示,浙江省2个地方鸭种的蛋比重和蛋壳厚度两项指标可通过遗传选育进行改良,而蛋白高度、蛋黄比率和哈氏单位应通过环境控制来提高。     

miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene

MicroRNAs (miRNAs) have been widely identified in porcine testicular tissues and implicated as crucial regulators of proliferation, apoptosis, and differentiation in porcine spermatogenesis related cells. However, the function roles of most of the miRNAs that have been identified in Sertoli cells are poorly understood. In the present study, six experiments were conducted to study the regulatory role of miR-10b in porcine immature Sertoli cells. In experiment 1, the results showed that the relative mRNA expression level of miR-10b in porcine testicular tissues decreased quadratically (P<0.001) with increasing age, while the relative mRNA expression level of DAZAP1 gene increased (P<0.001). In addition, the mRNA expression of miR-10b was negatively (P<0.01) correlated with DAZAP1 mRNA expression (r=?0.550). In experiment 2, the results from the bioinformatic analysis and a luciferase reporter assay demonstrated that miR-10b directly targeted the DAZAP1 gene in porcine immature Sertoli cells. DAZAP1 mRNA and protein expressions were both regulated (P<0.05) by miR-10b. In experiments 3 to 5, the over-expression of miR-10b or the siRNA-mediated knockdown of the DAZAP1 gene promoted (P<0.05) porcine immature Sertoli cell proliferation, as determined by the Cell Counting Kit-8 (CCK-8) assay and the 5-Ethynyl-2'-deoxyuridine (EdU) assay. However, an annexin V-FITC/PI staining assay and the expression of cell survival-related genes indicated that over-expression of miR-10b or knockdown of DAZAP1 had no effect (P>0.05) on porcine immature Sertoli cell apoptosis. In experiment 6, the co-transfection treatment results showed that miR-10b promoted (P<0.05) porcine immature Sertoli cell proliferation by targeting DAZAP1 gene. Overall, these experiments demonstrated that miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene.