"促育生精方" 对环磷酰胺致大鼠不育模型CatSper1 基因和CatSper2基 因的影响

目的：研究" 促育生精方" 对精子特异性钙通道蛋白CatSper1、CatSper2 表达的影响。方法：采用Real-time PCR 法检测 CatSper1 mRNA、CatSper2 mRNA 在各组大鼠（模型组、低剂量组、中剂量组、高剂量组、空白对照组）精子中的表达;用Western blot 检测各组CatSper1 蛋白,CatSper2 蛋白的表达。结果：成功建立大鼠不育模型。CatSper1 mRNA、CatSper2 mRNA表达量为： 中、高剂量组显著高于模型组（P＜0.05）。CatSper1、CatSper2蛋白表达量：中、高剂量组显著高于模型组（P＜0.05）。结论：促育生精 方能有效提高少弱精症模型大鼠精子特异性钙通道CatSper1、CatSper2 基因及其蛋白的表达。     

Identification of Four SNPs in LHB Gene and Their Associations with Sperm Qualities of Chinese Buffaloes

Luteinizing hormone beta polypeptide (LHB) gene has been considered important for sexual behavior and has associations with sperm quality. In this study, four SNPs (g.276?T>C, g.377A>C, g.401T>C, and g.412A>G) were detected in the LHB gene of 165 water buffaloes by direct sequencing and identification of overlap peaks, each of which was associated with at least one sperm quality trait of ejaculate volume, sperm concentration, post-thaw sperm motilities, and sperm abnormalities by chi-square analysis. Among them, g.276?T>C was associated with ejaculate volume (F?=?2.857, p?C was related to ejaculate volume (F?=?4.178, p?C had a marker effect on sperm abnormalities (F?=?3.332, p?G was associated with sperm concentration (F?=?3.579, p?LHB gene and Chinese water buffalo sperm quality traits. To the best of our knowledge, this is the first report addressing the associations between the SNPs in the LHB gene and the sperm qualities of Chinese buffaloes.     

精子特异性钙通道CatSper1 对精子线粒体呼吸与能量代谢的影响

目的:应用Cat Sper1单克隆抗体抑制Cat Sper1的功能,检测精子线粒体呼吸功能及能量合成能力,观察Cat Sper1对精子线粒体呼吸与能量代谢的影响。方法:健康志愿者20例,检测精液均符合WHO健康标准。手淫法获取精液,经过上游法处理后每份精液分为A、B两组,分别与Earles液以及50μg/m L抗Cat Sper1多克隆抗体共孵育。在1 h,2 h,4 h后分别检测两组精子精液参数、细胞线粒体呼吸控制率RCR及精子细胞ATP含量。结果:与A组比较,B组精子各时间点a+b(%)尤其是a(%)均明显下降,差异具有统计学意义(P0.01);1 h后B组精子a(%)即明显下降,2 h,4 h后精子下降缓慢,与1 h相比无明显差异,无统计学意义(P0.05)。与A组比较,B组精子各时间点态3值、呼吸控制率RCR以及精子细胞ATP含量均明显下降,差异具有统计学意义(P0.01);B组精子中,2 h、4 h节点精子态3值、呼吸控制率RCR以及精子细胞ATP含量与1 h无明显差异,无统计学意义(P0.05)。结论:阻断精子特异性钙通道Cat Sper1,可降低精子细胞线粒体呼吸功能,减少精子生成ATP的能力,从而使精子活力降低。为精索静脉曲张引起精子Cat Sper1表达下降,从而导致不育的机制寻找可能的理论依据。     

Ca2+ Signals Generated by CatSper and Ca2+ Stores Regulate Different Behaviors in Human Sperm

[Ca²⁺]_i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca²⁺ signaling pathway used. Activation of CatSper (by raising pH_i or stimulating with progesterone) caused sustained [Ca²⁺]_i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca²⁺ caused sustained [Ca²⁺]_i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pH_i and mobilized Ca²⁺ stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca²⁺-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca²⁺]_i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca²⁺ at the sperm neck can be mobilized by Ca²⁺-induced Ca²⁺ release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca²⁺ store, which may be regulated by capacitation and NO from the cumulus.     

荷斯坦牛BoLA-DRA基因SNPs筛选及生物信息学分析

为了探讨BoLA-DRA基因多态性,本研究利用DNA混合池扩增产物直接测序的方法对荷斯坦牛BoLA-DRA基因编码区SNPs进行筛选,并利用生物信息学软件预测该基因m RNA二级结构。结果表明:荷斯坦牛BoLA-DRA基因编码区共有4个SNPs (exon2-A82T,exon2-G116A,exon2-C197T,exon2-C233A)。生物信息学预测结果显示,exon2-A82T、exon2-G116A和exon2-C233A增大了m RNA二级结构的稳定性,而exon2-C197T降低了mRNA二级结构的稳定性。本研究结果可为荷斯坦牛抗病和抗逆性能研究积累更多的分子遗传学数据,并为经济性状相关基因筛选提供理论依据。     

秦川牛IGF2基因SNPs检测及其与胴体、肉质性状的相关性

采用PCR-SSCP方法对186头24月龄秦川牛IGF2基因进行了SNPs多态性检测, 并将其与部分胴体和肉质性状进行关联分析。在IGF2基因120碱基处发现C→T 突变, 在279碱基处发现 A→G 突变。方差分析结果表明: BB、DD 两个位点与胴体性状中与宰前活重、胴体重、胴体长、胴体胸深、眼肌面积显著相关(P<0.05), 其中背部皮下脂肪厚达到差异极显著(P<0.01); 与肉质性状大理石花纹、嫩度、pH24 (牛肉排酸24 h后的酸度值)显著相关(P<0.05)。但是在胴体深、系水力指标中差异不显著(P>0.05)。A、D 等位基因是群体中的优势等位基因, AA、DD 基因型是优势基因型, 而含有B、D 等位基因的个体的胴体和肉质性状优于其他个体, 尤其有着极强脂肪沉积能力     

茶树脯氨酸转运蛋白基因鉴定及表达分析

脯氨酸转运蛋白在植物体内脯氨酸的分配及响应多种非生物逆境胁迫过程中发挥着重要作用。为明确茶树体内脯氨酸转运蛋白家族情况,该研究从全基因组水平鉴定获得茶树脯氨酸转运蛋白家族成员,进行了系统进化关系、蛋白结构、基因表达特异性等分析。结果表明:(1)茶树中有6个脯氨酸转运蛋白基因,长度为1 326～1 725 bp之间,编码氨基酸数目在441～574 aa之间,蛋白质分子质量在48.5～63.0 kD之间,等电点为8.51～9.41,大部分为碱性蛋白,其结构中含有大量的α-螺旋和自由卷曲,少量的延长链和β-转角结构。(2)亚细胞定位分析结果显示,茶树CsProT1、CsProT2、CsProT4、CsProT5和CsProT6蛋白定位于细胞膜,CsProT3蛋白则定位于高尔基体。(3)CsProTs蛋白中含9～11个典型的跨膜结构域,其三级结构与保守基序特征均与拟南芥高度相似,具有高度的保守性,不同成员间氨基酸序列相似性达40.14%。(4)基因表达特异性分析显示,CsProT1,CsProT2和CsProT3基因在各个组织部位的表达量均较高,CsProT4、CsProT5和CsProT6表达量均较低,且CsProT1基因的表达量最高;除CsProT5基因外,CsProTs蛋白家族的基因均受到NaCl、干旱及冷胁迫的诱导表达。(5)蛋白相互作用分析结果显示,CsProTs蛋白可与脯氨酸氧化酶ERD5,脯氨酸生物合成限速酶P5CS1、P5CS2和δ-吡咯啉-5-羧酸脱氢酶ALDH12A1等脯氨酸合成,转运及降解有关的蛋白相互作用,共同调控茶树体内脯氨酸的含量。研究认为,茶树6个CsProTs蛋白可共同参与茶树体内脯氨酸的转运平衡及对多种非生物逆境胁迫响应的过程。     

何首乌中Ⅲ型聚酮合酶基因FmPKS2的原核表达及鉴定

目的：对传统中药何首乌中Ⅲ型聚酮合酶基因FmPKS2进行原核表达并鉴定重组蛋白酶活性,为研究该酶基因在何首乌有效成分代谢合成及其调控中的功能奠定基础。方法：根据何首乌FmPKS2（GenBank登录号：GQ984139）基因序列,通过PCR扩增其全长的编码区,克隆至原核表达载体PET-28a,构建重组质粒PET-FmPKS2,将其转化原核表达菌株E.coli BL21（DE3）,并用IPTG诱导表达,SDS-PAGE鉴定融合蛋白可溶性,通过Ni-NTA亲和树脂纯化可溶性蛋白后以丙二酰-COA和香豆酰-COA为底物进行催化反应,TLC鉴定反应产物。结果：经过诱导,重组菌表达分子量为42KD左右的融合蛋白,其中可溶性重组蛋白最优表达条件为IPTG浓度0.5 mmol/L,诱导时间6h,温度25℃。纯化后的可溶性重组蛋白催化丙二酰-COA和香豆酰-COA得到的产物经TLC鉴定为二苯乙烯类化合物白藜芦醇。结论：成功实现FmPKS2的原核表达且融合蛋白以可溶形式存在,催化反应证明FmPKS2融合蛋白具有二苯乙烯合酶的活性。     

人类新基因Bzw2的克隆、抗体制备及表达分析

Bzw2( basic leucine zipper and W2 domains 2)基因是人类心脏发育候选基因,它由bzip结构域和W2结构域构成.为了研究该基因在心脏发育过程中的作用,利用生物信息学信息克隆了人类Bzw2基因全长,将所得的片段插入到原核表达载体pGEXT-4T-1载体中,利用BL21菌株表达该重组...     

猪链球菌2型全基因组DNA芯片的研制及基因表达谱技术平台的建立

目的：研制猪链球菌2型（SS2）全基因组DNA芯片,建立SS2基因表达谱技术平台。方法：利用SS2全基因组序列,挑选出2194条基因,经PCR扩增出2156条基因并将产物纯化,点样制备芯片;将芯片用于表达谱研究,采用实时定量PCR验证表达谱结果,对芯片进行可靠性分析。结果：芯片杂交数据与实时定量PCR验证显示了较高的相关性,二者相关系数r=0.87。结论：研制了一批SS2全基因组DNA芯片,并建立了基于DNA芯片的表达谱技术平台。     

油菜BnMKK2基因的克隆及表达分析

通过实验从陇油6号油菜中克隆得到了一种新的MAPK激酶基因BnMKK2基因的cDNA,全长1 344 bp,其中包括5′非翻译区（5′UTR）111 bp,3′非翻译区（3′UTR）165 bp,开放阅读框（ORF）长1 068 bp,编码355个氨基酸,该基因编码的蛋白质分子量为39.3 kDa,理论等电点为6.8。与拟南芥AtMKK2有很高的同源性,因此命名为BnMKK2(GenBank登录号:HQ848661)。该基因实时荧光定量PCR分析表明,BnMKK2基因的表达受低温胁迫诱导。     

绵羊ESR2基因组织表达及其多态性与产羔数关联分析

为了探究雌激素受体2 (Estrogen receptor 2, ESR2)基因在绵羊各组织的表达及其多态性与产羔数之间的关系,本研究利用半定量PCR和实时荧光定量PCR技术检测ESR2基因在不同繁殖力小尾寒羊群体组织中的相对表达量,同时采用Sequenom MassARRAY誖SNP技术对多羔品种绵羊(小尾寒羊,湖羊,策勒黑羊)和单羔品种绵羊(苏尼特羊,草原型藏羊,滩羊) ESR2基因g.73324006C>T位点进行检测,并与小尾寒羊产羔数进行关联分析。半定量PCR表明,ESR2基因在单、多羔小尾寒羊子宫中高表达,在其它组织中等或低丰度表达;单羔群体、多羔群体间荧光定量PCR表明,ESR2基因在单羔小尾寒羊垂体表达量显著高于多羔小尾寒羊(p<0.05);群体遗传学分析表明,g.73324006C>T在小尾寒羊群体中表现为低度多态(PIC<0.25),在滩羊群体中处于中度多态(0.25T在小尾寒羊群体处于哈代温伯格平衡状态(p>0.05);关联分析表明,g.73324006C>T位点多态性与小尾寒羊第一胎、第二胎、第三胎产羔数及平均产羔数均显著关联(p<0.05),CC型各胎产羔数均高于TC型。与FecB (A746G)基因组合后发现,GG-CC和AG-CC基因型母羊产羔数显著高于AA-TC、AA-CC、AG-TC基因型组合(p<0.05)。综上,ESR2与小尾寒羊产羔数密切相关,g.73324006C>T可作为绵羊产羔性状选育的潜在分子标记。     

重组人FOXL2基因的原核表达和纯化研究

目的通过pET32a（＋）原核表达载体,表达重组人叉头框蛋白L2（human forkhead box12,FOXL21）。并且进行纯化和鉴定。方法从正常人血液中提取基因组DNA,利用PCR扩增FOXL21目的基因片段,构建FOXL21原核表达重组质粒[pET32a（＋）-FOXL21]并转化E．coli的BL21（DE3）菌株,IPTG诱导重组蛋白表达,经HisTrap FF亲和层析柱纯化,再通过SDS—PAGE和Western印迹鉴定。结果成功克隆到大小为1131bp的人源FOXL21基因片段并准确插入表达载体pET32a（＋）,0．1mmol／LIPTG诱导转化菌8h可表达大量的FOXL21蛋白,并可经HisTrap FF柱亲和层析得到高度纯化。结论成功获得纯化的66kD重组人FOXL21蛋白,为后续进行FOXL21蛋白的功能研究奠定了基础。     

Identification of SNPs in Interferon Gamma,Interleukin-22, and Their Receptors and Associations with Health and Production-Related Traits in Canadian Holstein Bulls

Genetic variants in a number of immunoregulatory genes have been previously associated with health and production traits in dairy cattle. Therefore, in the following study, the genes coding interferon gamma (IFNG), IFNG receptor 1 and 2 domains, interleukin-22 (IL22), and IL22 receptor alpha 1, were investigated for single nucleotide polymorphisms (SNPs) in Holstein bulls. These SNPs, along with SNPs previously identified in IL10, IL10 receptor, and transforming growth factor beta 1 (TGFB1) genes, were evaluated for statistical associations to estimated breeding values for milk somatic cell score (SCS), a trait highly correlated to mastitis incidence, and various production-related traits, including milk yield, protein yield, fat yield, and lactation persistency. While no significant associations were found between these SNPs and SCS, SNPs in IL10 receptor beta subunit showed a significant effect on protein yield and lactation persistency. While there is evidence that IL10 plays an important role during lactation, it is also likely that the effects of SNPs in IL10 receptor beta subunit on protein yield and lactation persistency are due to linkage disequilibrium with a neighboring QTL.     

HgCl2对泥鳅精子运动的影响

为了检测不同浓度的HgCl2对泥鳅Misgurnus anguillicaudatus精子运动的影响,以泥鳅精子为实验材料,用含终浓度分别为0(对照)、1、5、10、15和20 μmoL/L的HgCl2待测液分别孵育0、2、4和6 h后激活,激活后立即在显微镜(Olympus IX81)下观察精子运动参数.为进一步探讨HgCl2对泥鳅精子运动影响的机制,用终浓度为20μmol/L的HgCl2保存液孵育泥鳅精子10 min,以含终浓度分别为0、0.1、1、10 mmol/L的2-巯基乙醇和20μmol/LHgCl2混合液为激活液激活.激活后立即在显微镜下观察精子运动,发现2-巯基乙醇町逆转HgCl2对泥鳅精子的抑制作用,为探讨HgCl2对泥鳅精子运动影响的可能机制提供参考.     

HCV E2区基因在原核系统中的表达

用提取的重组表达载体pET-E2转化BL21（DE3）感受态细胞,经IPTG诱导,再进行SDS－PAGE,可得到有一条约34kDa的表达带,与理论推测的蛋白分子量一致,通过Western-blot鉴定,证明此带即为目的蛋白带。该产物有一个六聚组氨酸尾,主要以包涵体形式存在;计算机扫描分析考马斯亮兰染色后的蛋白胶显示：目的蛋白占整个菌体蛋白的36％以上,经Ni-柱纯化的E2蛋白纯度可达95％以上;以纯化的E2蛋白为抗原,用ELISA方法检测了20份抗HCV阳性血清,结果表明15份抗HCV阳必血清中检出5份E2抗体阳性血清,而5份抗HCV阴性血清中没有检测到E2抗体。     

Identification and Functional Expression of HCx31.9, a Novel Gap Junction Gene

By combining in silico and bench molecular biology methods we have identified a novel human gap junction gene that encodes a protein designated HCx31.9. We have determined its human chromosomal location and gene structure, and we have identified a putative mouse ortholog, mCx30.2. We have observed the presence of HCx31.9 in human cerebral cortex, liver, heart, spleen, lung, and kidney and the presence of mCx30.2 in mouse cerebral cortex, liver and lung. Moreover, preliminary data on the electrophysiological properties of HCx31.9 have been obtained by functional expression in paired Xenopus oocytes and in transfected N2A cells.     

基于转录组测序的慈竹笋木质素基因筛选及其表达分析

慈竹（Bambusa emeiensis）纤维素含量丰富,是较好的造纸原料,但竹茎中木质素影响着制浆生产及纸浆质量。目前,对慈竹木质素生物合成机制所知甚少,这限制了遗传调控竹木质素的研究。本文以拟南芥、水稻等植物的已知木质素基因作为查询序列,通过BLASTp和系统进化分析,从10、50、100和150 cm慈竹笋转录组数据中筛选到351个木质素生物合成相关Unigenes,包括51个LAC,37个4CL、26个PAL、34个CCR和25个CAD相关转录子,其数量高于其他已报道的竹类植物。转录丰度和定量基因表达分析发现16个木质素基因,包括2个PAL、5个CCR、3个4CL、2个CADH2和4个LAC,随着笋发育而表达上调,表明其可能与发育性木质素积累相关。     

细粒棘球蚴AgB8/2基因的原核表达及免疫学鉴定

旨在克隆细粒棘球蚴 AgB8/2基因,优化原核表达体系,鉴定纯化蛋白并初步确定其诊断价值.采用 RT-PCR扩增 AgB8/2基因 cDNA,构建原核表达载体pET-AgB8/2并在大肠杆菌 BL21(DE3)中表达,优化表达条件.纯化的AgB8/2融合蛋白经 Western blot鉴定正确后,采用斑点免疫金渗滤法初步验证其血清学诊断价值.结果显示,克隆的 AgB8/2基因包含了 273 bp的完整ORF,序列分析表明与其它已报道的AgB8/2 cDNA序列的同源性在 99%～100%之间.优化的表达条件为,当菌液 OD600值为0.6时,加入终浓度为0.05 mmol/L的 IPTG,37℃,振荡培养5 h.纯化的蛋白经 Western blot鉴定为目的蛋白.克隆的 AgB8/2基因高度保守,原核表达载体pET-AgB8/2构建正确并高效表达可溶性融合蛋白,可作为特异性抗原应用于斑点免疫金渗滤法检测血清抗体.