巴马香猪Toll样受体4基因cDNA的克隆及生物信息学分析

目的研究TLR4在猪自然免疫中的作用及机制,为抗病育种及免疫佐剂的开发提供依据。方法利用NCBI公布的TLR4基因序列设计引物,RT-PCR技术克隆巴马香猪TLR4基因。结果所得基因序列提交GenBank,登录号：GQ304754。经序列分析,发现巴马香猪TLR4基因开放阅读框长2526 bp,编码785个氨基酸,该蛋白等电点为6.58,分子量为96.4×103;与普通猪比对发现巴马香猪TLR4基因有5个碱基发生突变;与小鼠、狗、鸡、牛、羊和人的同源性分别为71.9%、81.5%、54.2%、86.4%、85.5%和81.9%;TLR4膜外区蛋白为背侧多个α螺旋和内侧多个β折叠平行交替排列构成一个弯曲状螺旋结构;N末端存在信号肽,且可能在23～24位氨基酸处存在裂解位点;胞外区有13个明显的LRR,分别位于第53～74、77～100、101～124、149～173、174～192、201～225、372～393、398～429、446～469、470～494、495～518、519～541、543～566位氨基酸区;膜外区含8个N连接的糖基化位点。结论本研究成功克隆巴马香猪TLR4基因,为进一步研究该基因的功能和蛋白质的特性奠定了基础。     

腺病毒介导的Wnt10b对HaCaT细胞迁移的作用及机制

目的:探讨Wnt10b对Ha Ca T表皮细胞迁移的影响及机制。方法:分别用重组腺病毒Ad-GFP、Ad-GFP-Wnt10b感染Ha Ca T表皮细胞,利用细胞免疫荧光技术检测腺病毒Ad-GFP-Wnt10b感染细胞后的过表达情况;采用细胞划痕实验,于不同时间点观察并记录经Wnt10b处理后,Ha Ca T细胞体外迁移功能的改变;进一步采用Westen blot技术检测不同腺病毒处理后,Ha Ca T细胞中Wnt信号关键分子β-catenin、细胞粘附分子E-cadherin表达的改变情况。结果:1Ad-GFP-Wnt10b感染Ha Ca T细胞48 h后,细胞内GFP表达上调,细胞免疫荧光染色显示Wnt10b处理组高表达Wnt10b蛋白;2Ha Ca T细胞经Ad-GFP-Wnt10b处理后,细胞创面愈合速度明显增快;3Wnt10b处理组细胞β-catenin蛋白表达水平显著高于对照组,而E-cadherin蛋白表达水平显著低于对照组。结论:Wnt10b能促进Ha Ca T细胞迁移,且该效应涉及经典Wnt/β-catenin信号的激活及由E-cadherin介导的细胞粘附性的减弱。     

黄瓜CsEXP 10基因的电子克隆及生物信息学分析

以黄瓜CsEXP10 cDNA片段为信息探针,利用电子克隆和序列拼接方法获得了1191bp的cDNA序列。应用生物信息学软件预测了该蛋白的理化性质、卷曲螺旋、疏水性、信号肽、跨膜结构、糖基位点、活性位点、亚细胞定位及二级和高级结构。结果表明：该蛋白是一个疏水性稳定蛋白,定位于细胞壁,含有4个α-螺旋,11个β-折叠,5个跨膜区域,10个糖基化位点,具有催化域和多糖结合域两个结构域。     

猪JARID1C基因的cDNA克隆、生物信息学及组织表达谱分析

JARID1C是高度保守的ARID蛋白家族的成员,该家族的蛋白参与并引起一系列生物学效应,如染色质重塑、细胞增殖与分裂、个体发育以及基因转录调控。JARID1C在人脑中表达丰富,对脑的发育和维持正常功能具有重要作用,突变可引起智力迟钝。本研究采用电子克隆（insilicocloning）的方法并结合5′末端快速扩增技术（RACE）,从猪卵巢中克隆到JARID1C的全长cDNA序列（GenBank登录号：EF139241）。猪JARID1C基因的cDNA全长5,908bp,包括4,551bp的开放阅读框（ORF）、522bp的5′非翻译区（5′UTR）和835bp的3′非翻译区（3′UTR）,polyA加尾信号序列AATAAA位于5,881bp和5,886bp之间。生物信息学分析揭示JARID1C蛋白含有1517个氨基酸残基,定位于细胞核中,该蛋白含有5个保守的结构域：JmjN结构域、ARID结构域、JmjC结构域、C5HC2锌指结构域和PHD锌指结构域。应用ClusterW程序分别对猪、狗、小鼠、大鼠、人和猿的JARID1C核苷酸序列和氨基酸序列进行多重序列比对,发现猪的JARID1C与其他哺乳动物具有很高的相似性。借助Mega3.1软件,采用N-J算法构建JARID1亚家族蛋白的系统进化树,揭示不同物种的进化关系。应用实时荧光定量PCR技术分析该基因在不同组织的表达差异,结果表明该基因在各组织均不同程度地表达,其中在肺和骨骼肌表达水平最低,而在脑和性腺表达水平最高。     

猪Opn4基因cDNA克隆与表达分析

为揭示猪Opn4基因的结构并研究其在猪视网膜中昼夜表达差异与生长各阶段表达规律, 文章克隆了猪Opn4基因全长CDS区序列, 定量检测了Opn4基因在猪视网膜中昼夜的表达量以及其在不同生长阶段的表达量。结果表明, Opn4基因编码区序列为1 437 bp, 编码478个氨基酸, 分子式为C2398H3705N623O651S23; Opn4基因在白昼的表达量极显著高于夜晚(P<0.01); Opn4基因在出生前34 d胎儿视网膜中表达量最低(P<0.01); 1日龄时表达量最高(P<0.01); 1月龄、4月龄、84月龄猪视网膜中Opn4表达量差异不显著(P>0.05)。以上结果提示, Opn4基因可参与调控猪的生物节律, 并且Opn4主要参与出生后猪对外界光照的应答。     

蒙古冰草PLD基因cDNA克隆及生物信息学分析

以蒙古冰草(Agropyron mongolicum Keng)叶片为材料.根据已报道的醉酒毒麦PLD基因片段设计特异引物,通过RT-PCR和RACE技术,获得蒙古冰草PLD基因的全长cDNA(GenBank登录号为EU333811).该基因cDNA全长2 966 bp,包含2 439 bp的完整开放读码框,编码813个氨基酸.BlastP搜索结果显示,该基因推测的氨基酸序列与已克隆的醉酒毒麦、玉米、水稻PLD基因氨基酸序列的一致性为80%～89%.利用生物信息学软件在线分析其序列结构、氨基酸组成及编码氨基酸的性质和结构,结果表明:该基因编码的蛋白为可溶性蛋白,其相对分子量为92 079.2 Da,理论等电点为5.24,无跨膜域、无信号肽;其二级结构主要以无规卷曲为主;三级结构显示紧靠N端处为C2域,在中间和靠近C端处存在PLD的标志序列,即HKD基序.系统进化树显示,蒙古冰草PLD与醉酒毒麦的亲缘关系最近.     

猪CFL2b 基因cDNA克隆初步分析

利用基因表达谱芯片分析法筛选出与大白猪高肌肉产量-肌纤维形成有关的CFL2b基因.参考人和小鼠CFL2b基因序列,采用SMART-RACE技术结合EST序列拼接技术,从猪骨骼肌肌肉中首次克隆了猪CFL2b的全长cDNA序列（GenBank登录号EU561660,EU561661）,Northern杂交检测CFL2b基因 mRNA.结果表明,猪CFL2b基因含有2个转录本,长转录本3　012 bp,短转录本1　466 bp .CFL2b基因在多种真核生物中都有表达,且编码区序列非常保守,开放式读码框501 bp编码了166个氨基酸的蛋白质.氨基酸序列分析表明,猪CFL2基因与人和小鼠氨基酸同源性分别为100%和99.1%.核苷酸序列相似性分别为88.1%和74.9%.     

猪T细胞受体γ链基因的克隆及生物信息学分析

目的:克隆猪T细胞受体γ链(pTCR-γ)基因,用以研究猪T细胞受体分子结构与功能。方法与结果:以GenBank上登载的pTCR-γ基因为参考序列,用RT-PCR法从猪外周血淋巴细胞中克隆pTCR-γ基因。序列分析表明,pTCR-γ基因开放读框为1026bp,编码341个氨基酸残基,含有由23个氨基酸残基构成的信号肽序列;与参考序列相比,在核苷酸和推导氨基酸序列上的同源性分别为95.6%和88.8%;系统进化分析发现,该基因与绵羊、恒河猴、人、马、牛的同源性相对较高,与褐鼠、家犬、家鼠、家猫的同源性次之,而与禽类、鱼类的同源性最低;生物信息学结构预测表明猪T细胞受体γ链含2个结构域,其中1个为IG_LIKE1结构域(IGv结构域),由第14～114位共101个氨基酸残基组成;另一个为IG_LIKE2结构域(IGc结构域),由第143～238位共96个氨基酸残基组成。结论:克隆并应用生物信息学技术分析了猪T细胞受体γ链基因序列及编码蛋白的结构特征,为进一步研究γ链的结构与功能奠定了基础。     

Non-canonical Wnt signaling through Wnt5a/b and a novel Wnt11 gene, Wnt11b, regulates cell migration during avian gastrulation

Knowledge of the molecular mechanisms regulating cell ingression, epithelial–mesenchymal transition and migration movements during amniote gastrulation is steadily improving. In the frog and fish embryo, Wnt5 and Wnt11 ligands are expressed around the blastopore and play an important role in regulating cell movements associated with gastrulation. In the chicken embryo, although Wnt5a and Wnt5b are expressed in the primitive streak, the known Wnt11 gene is expressed in paraxial and intermediate mesoderm, and in differentiated myocardial cells, but not in the streak. Here, we identify a previously uncharacterized chicken Wnt11 gene, Wnt11b, that is orthologous to the frog Wnt11 and zebrafish Wnt11 (silberblick) genes. Chicken Wnt11b is expressed in the primitive streak in a pattern similar to chicken Wnt5a and Wnt5b. When non-canonical Wnt signaling is blocked using a Dishevelled dominant-negative protein, gastrulation movements are inhibited and cells accumulate in the primitive streak. Furthermore, disruption of non-canonical Wnt signaling by overexpression of full-length or dominant-negative Wnt11b or Wnt5a constructions abrogates normal cell migration through the primitive streak. We conclude that non-canonical Wnt signaling, mediated in part by Wnt11b, is important for regulation of gastrulation cell movements in the avian embryo.     

Combinatorial Wnt control of zebrafish midbrain-hindbrain boundary formation

Wnt signaling is known to be required for the normal development of the vertebrate midbrain and hindbrain, but genetic loss of function analyses in the mouse and zebrafish yield differing results regarding the relative importance of specific Wnt loci. In the zebrafish, Wnt1 and Wnt10b functionally overlap in their control of gene expression in the ventral midbrain-hindbrain boundary (MHB), but they are not required for the formation of the MHB constriction. Whether other wnt loci are involved in zebrafish MHB development is unclear, although the expression of at least two wnts, wnt3a and wnt8b, is maintained in wnt1/wnt10b mutants. In order to address the role of wnt3a in zebrafish, we have isolated a full length cDNA and examined its expression and function via knockdown by morpholino antisense oligonucleotide (MO)-mediated knockdown. The expression pattern of wnt3a appears to be evolutionarily conserved between zebrafish and mouse, and MO knockdown shows that Wnt3a, while not uniquely required for MHB development, is required in the absence of Wnt1 and Wnt10b for the formation of the MHB constriction. In zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b, the expression of engrailed orthologs, pax2a and fgf8 is not maintained after mid-somitogenesis. In contrast to acerebellar and no isthmus mutants, in which midbrain and hindbrain cells acquire new fates but cell number is not significantly affected until late in embryogenesis, zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b undergo extensive apoptosis in the midbrain and cerebellum anlagen beginning in mid-somitogenesis, which results in the absence of a significant portion of the midbrain and cerebellum. Thus, the requirement for Wnt signaling in forming the MHB constriction is evolutionarily conserved in vertebrates and it is possible in zebrafish to dissect the relative impact of multiple Wnt loci in midbrain and hindbrain development.     

Effects of Wnt-10b on hair shaft growth in hair follicle cultures

Wnts are deeply involved in the proliferation and differentiation of skin epithelial cells. We previously reported the differentiation of cultured primary skin epithelial cells toward hair shaft and inner root sheath (IRS) of the hair follicle via beta-catenin stabilization caused by Wnt-10b, however, the effects of Wnt-10b on cultured hair follicles have not been reported. In the present study, we examined the effects of Wnt-10b on shaft growth using organ cultures of whisker hair follicles in serum-free conditions. No hair shaft growth was observed in the absence of Wnt-10b, whereas its addition to the culture promoted elongation of the hair shaft, intensive incorporation of BrdU in matrix cells flanking the dermal papilla (DP), and beta-catenin stabilization in DP and IRS cells. These results suggest a promoting effect of Wnt-10b on hair shaft growth that is involved with stimulation of the DP via Wnt-10b/beta-catenin signalling, proliferation of matrix cells next to the DP, and differentiation of IRS cells by Wnt-10b.     

GRB10 binds to LRP6, the Wnt co-receptor and inhibits canonical Wnt signaling pathway

During adipocyte differentiation, the cells experience dramatic alterations in morphology, motility and cell-ECM contact. Focal adhesion kinase (pp125FAK), a widely expressed non-receptor tyrosine kinase in integrin signaling, has been reported to participate in these events in various cells. Utilizing 3T3-L1 cells and primary rat preadipocytes, we explored the role of FAK in adipocyte differentiation. Gradual cleavage of FAK was demonstrated during adipcoyte differentiation, both in vitro and in vivo. This cleavage of FAK was mediated by calpain. Inhibition of calpain activity resulted in the rescue of FAK degradation, accompanied with the disturbance of final maturation of adipocyte. Our study revealed that FAK participated in adipocyte differentiation, and its cleavage by calpain was required to fulfill the final maturation of adipocytes.     

猪CFL2b基因部分序列的克隆及组织表达谱分析

在猪QTL定位基础上,利用比较基因组学原理,参照猪CFL2的cDNA序列,对猪CFL2基因的部分基因组DNA序列进行克隆、测序;利用RT-PCR半定量法检测CFL2基因在不同组织的表达情况.结果显示,所克隆的CFL2基因部分基因组DNA序列长度为2 377bp,包括4个外显子和4个内含子,该基因mRNA序列与已报道的人CFL2b基因序列相似性为89%;猪CFL2基因在多种组织中均有表达,但在骨骼肌和心肌中表达丰度较高.结果表明,本研究成功测序了猪CFL2b基因部分基因组DNA序列,为进一步研究该基因与肌肉发育及生理功能的关系奠定了基础.     

Canonical Wnts,specifically Wnt-10b,show ability to maintain dermal papilla cells

Although Wnts are expressed in hair follicles (HFs) and considered to be crucial for maintaining dermal papilla (DP) cells, the functional differences among them remain largely unknown. In the present study, we investigated the effects of Wnts (Wnt-3a, 5a, 10b, 11) on the proliferation of mouse-derived primary DP cells in vitro as well as their trichogenesis-promoting ability using an in vivo skin reconstitution protocol. Wnt-10b promoted cell proliferation and trichogenesis, while Wnt-3a showed those abilities to a limited extent, and Wnt-5a and 11 had no effects. Furthermore, we investigated the effects of these Wnts on cultured DP cells obtained from versican-GFP transgenic mice and found that Wnt-10b had a potent ability to sustain their GFP-positivity. These results suggest that canonical Wnts, specifically Wnt-10b, play important roles in the maintenance of DP cells and trichogenesis.