Sex-specific expression of a novel gene Tmem184a during mouse testis differentiation

During mouse embryogenesis, the fate of the bipotential gonads is sealed around 10.5 days post coitum (dpc) when the Y-linked gene Sry specifies the differentiation of testes in males, whereas in females, absence of Sry results in ovary formation. Apart from the pivotal action of Sry, many other genes are known to be involved in sex determination and subsequent differentiation. Much is still unknown regarding the regulatory hierarchy governing these events and many more sex differentiation genes are yet to be discovered. In this study, we investigated the expression of Tmem184a, a novel gene encoding a protein of unknown function, but with predicted kinase activity, during mouse embryogenesis. We show that Tmem184a is expressed at high levels in the developing testis from 11.5 dpc, a time of active proliferation and differentiation. Tmem184a expression is further shown to be expressed exclusively within the Sertoli cells of the developing testis cords, suggesting that it may mediate sex-specific signaling events during Sertoli cell differentiation.     

人乳铁蛋白在原核中的融合表达

设计了特异引物，通过PCR扩增人乳铁蛋白（HLF）基因，将扩增的DNA片段重组到原核表达载体pGEX-4T3中，重组体转化大肠杆菌BL21菌株，经IPTG诱导后，得到了高效融合表达。分析结果表明，获得了特异表达的乳铁蛋白。     

黑曲霉酸性蛋白酶EXPA的克隆表达与酶学性质解析

酸性蛋白酶在食品、酿造、饲料和皮革等行业具有重要的应用价值。然而,现有的酸性蛋白酶在50℃以上或pH 3. 0时不稳定,限制了其应用范围。该研究通过分子克隆技术将黑曲霉CICIM F0510的酸性蛋白酶基因exp A在毕赤酵母中进行了克隆表达,构建获得了重组菌GS115 (p PIC-expA)。摇瓶发酵条件下,重组酶EXPA的酶活为257 380 RFU/h。生物信息学分析的结果显示,该酶属于天冬氨酸蛋白酶A1A家族。酶学性质的研究表明,该重组酶的最适反应温度和pH分别为50℃和3. 0;分别在40～50℃或pH 2. 5～3. 5孵育1 h后,仍能保留80%左右的活力。Zn~(2+)、Ca~(2+)、Fe~(2+)和Mn~(2+)对其活性有一定的促进作用;而Cu~(2+)、Co~(2+)、Fe~(3+)、EDTA和SDS则对其活性有显著的抑制作用。此外,重组酶EXPA对大豆分离蛋白、水溶性玉米蛋白和小麦水解蛋白均具有较好的水解作用。较好的耐热性和pH稳定性为EXPA在食品、饲料等领域的应用奠定了基础。     

Production of inhibin A not B in rams: changes in plasma inhibin A during testis growth,and expression of inhibin/activin subunit mRNA and protein in adult testis

Previous studies have shown that changes in the plasma concentrations of immunoreactive inhibin measured by radioimmunoassay occur in parallel with growth and regression of the testes during a reproductive cycle in adult Soay rams induced by exposure to an artificial lighting regimen of alternating 16 week periods of long days and short days. With the development of new two-site ELISAs for sheep inhibin A and inhibin B, we have re-examined the relationship between FSH and dimeric, biologically active inhibin in the reproductive cycle in adult Soay rams. No signal was generated by sheep testicular extract, ram or ewe plasma, or sheep ovarian follicular fluid in the inhibin B ELISA. In contrast, ram plasma contained significant activity in the inhibin A ELISA, which diluted in parallel to the inhibin A standard, and was abolished by preincubation of ram plasma with monoclonal antibodies specific for the betaA, but not the betaB, subunit. These results indicate that the ram is the first adult male mammalian species identified to date in which the testes produce and secrete dimeric inhibin A and not inhibin B. Northern blot analysis and immunocytochemistry confirmed the presence of alpha, betaA and betaB inhibin/activin subunit mRNA and protein in the testes of adult rams. Changes in plasma inhibin A concentrations occurred in parallel with the growth and regression of the testes during the long day: short day: long day lighting regimen in adult Soay rams, confirming our previous observations with immunoreactive inhibin. During the growth phase of the testes in the first 8 weeks of exposure to short days there was a positive correlation between plasma FSH and inhibin A concentrations, indicating that during this phase the secretion of inhibin A is stimulated by FSH and that inhibin A did not act as a negative feedback hormone on FSH secretion. From week 8.5 to week 16.0 of exposure to short days, there was a negative correlation between FSH and testosterone concentrations, but not inhibin, indicating that when inhibin concentrations are high, testosterone acts as the negative regulator of FSH secretion. Thus, in intact adult rams, when the testes are fully active it appears that inhibin A may sensitize the pituitary to the negative feedback effects of testosterone, at which time they act synergistically to maintain plasma concentrations of FSH.     

Cloning of pig prostaglandin F2alphaFP receptor cDNA and expression of its mRNA in the corpora lutea

Changes in the expression and localization of luteal mRNA for PGF(2alpha) (FP) receptors may be critical in determining the luteolytic action of PGF(2alpha) in pig corpora lutea. In this study, a full-length FP receptor (FPr) cDNA was isolated and cloned from pig corpora lutea. This isolate (GenBank accession no. U91520) contains an open reading frame of 1086 bases coding for a protein of 362 amino acids with seven potential transmembrane domains. The predicted amino acid sequence of this isolate was 83% identical to the FPr amino acid sequence of other species including sheep, cattle and humans. Northern blot analysis showed the presence of an FPr message of about 5 kb in mRNA from pig corpora lutea. Relatively weak FPr mRNA expression was detected on day 4 and day 7 of the oestrous cycle. The expression was greater (P < 0.05) on days 10, 13 and 15 than on days 4 and 7. In situ hybridization analysis revealed that mRNA for FPr was expressed predominantly in the steroidogenic large luteal subtype of cell, although there was some expression in small luteal cells, with histological appearance of steroidogenic small cells. Localization of hybridization signals of FPr was observed in luteal tissue at all stages examined. These data demonstrate that FPr is expressed in pig corpora lutea throughout the oestrous cycle and that upregulation of the FPr mRNA occurs when the corpora lutea becomes sensitive to PGF(2alpha). Direct luteal targets of PGF(2alpha) appear to be primarily large steroidogenic cells in this species.     

微小毛霉凝乳酶基因的克隆与表达

微小毛霉(Mucor pusillus)凝乳酶是微生物凝乳酶的主要来源之一,但与传统的牛凝乳酶比较具有一定的缺陷。为将其采用基因工程的方法进行改造获得理想的凝乳酶,本研究克隆到微小毛霉凝乳酶基因,将其插入原核表达载体pTWlN1中,使之与几丁质结合域(CBD)一内含肽(intein)融合,获得原核表达质粒pTWIN1/M。转化大肠杆菌BL21(DE3),后经IPTG诱导后进行SDS-PAGE电泳分析,获得了重组蛋白。     

Cloning and characterization of a novel phytase from wastewater treatment yeast Hansenula fabianii J640 and expression in Pichia pastoris

Phosphohydrolysis of organic phosphorus compounds by acid phosphatases (EC 3.1.3.1 and EC 3.1.3.2) is an important method for efficient removal of phosphorus from high concentration organic wastewater. Another important method is supplementation of animal feed with phytase (EC 3.1.3.8 and EC 3.1.3.26), which improves the availability of phytate-phosphates (phosphate that are hydrolyzed by phytases), making it possible to add less phosphate to animal feed and resulting in the excretion of less phosphorus by the animals. In the present study, we purified a novel phytase from the wastewater treatment yeast Hansenula fabianii J640 (Hfphytase), cloned the 1456 bp open reading frame (ORF) encoding Hfphytase, and characterized Hfphytase. The molecular weight of Hfphytase after deglycosylation by PNGaseF was 49 kDa. The optimal pH and temperature for enzyme activity were 4.5 and 50 °C, respectively. Hfphytase exhibits 40% identity with Debaryomyces castellii phytase, 37% identity with Aspergillus niger PhyB, and 34% identity with Saccharomyces cerevisiae Pho5p. Recombinant Hfphytase was transformed and expressed in Pichia pastoris. The yield was 23 g/l by jar fermenter cultivation. The marked phosphohydrolysis activity exhibited by Hfphytase on six substrates (pNP-P, sodium phytate, glucose-1 phosphate, glucose-6 phosphate, α-glycerophosphate and β-glycerophosphate) indicated that it is a non-specific acid phosphatase.     

Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis

Germ cell proliferation, migration and survival during all stages of spermatogenesis are affected by stem cell factor signalling through the c-Kit receptor, the expression and function of which are vital for normal male reproductive function. The present study comprehensively describes the c-Kit mRNA and protein cellular expression profiles in germ cells of the postnatal and adult rodent testis, revealing their significant elevation in synthesis at the onset of spermatogenesis. Real-time PCR analysis for both mice and rats matched the cellular mRNA expression profile where examined. Localization studies in normal mouse testes indicated that both c-Kit mRNA and protein are first detectable in differentiating spermatogonia. In addition, all spermatogonia isolated from 8-day-old mice displayed detectable c-Kit mRNA, but 30-50% of these lacked protein expression. The c-Kit mRNA and protein profile in normal rat testes indicated expression in gonocytes, in addition to differentiating spermatogonia. However, in the irradiated adult rat testes, in which undifferentiated spermatogonia are the only germ cell type, mRNA was also detected in the absence of protein. This persisted at 3 days and 1 and 2 weeks following treatment with gonadotrophin-releasing hormone (GnRH) antagonist to stimulate spermatogenesis recovery. By 4 weeks of GnRH antagonist treatment, accompanying the emergence of differentiating spermatogonia, both mRNA and protein were detected. Based on these observations, we propose that c-Kit mRNA and protein synthesis are regulated separately, possibly by influences linked to testis maturation and circulating hormone levels.     

嗜热果糖苷酶的基因克隆及表达优化

从嗜热菌Thermotoga neapolitana DSM4359得到嗜热果糖苷酶基因片段,以p ET-28a(+)为表达载体,在Escherichia.coil BL21(DE3)中高效表达。由SDS-PAGE检测可得构建后嗜热果糖苷酶的分子量约为60 KDa。通过单因素试验和响应面法对该嗜热果糖苷酶的产酶培养基进行优化选择,最终确定最佳产酶培养基为:蔗糖10.11 g/L,硝酸铵12.30 g/L,安琪酵母浸粉FM905 8.24 g/L,MgSO_4·7H_2O 5.61 mmol/L,NaCl 10 g/L。相比优化前,经优化后的培养基所产嗜热果糖苷酶酶活提高了近2.1倍。达到了提高嗜热果糖苷酶表达量的目的。     

大豆CONSTANS同源基因的克隆及表达

CONSTANS（CO）是调控拟南芥由营养生长向生殖生长转换的关键基因。本研究利用RACE法从晚熟（光周期敏感）大豆品种自贡冬豆中克隆得3个大豆CO同源基因,分别为Glyma04g06240、Glyma13g01290和Gly-ma06g06300。电子克隆法检索发现另外5个同源性较高的基因,这些基因均含一个内含子,其编码氨基酸序列与拟南芥AtCO的B-box和CCT功能结构域高度同源。qRT-PCR分析叶片中Glyma04g06240、Glyma06g06300和Glyma13g01290的表达发现,短日下三者表达均呈现昼夜节律模式,表现为白天降低,夜间积累,凌晨达到表达高峰;长日处理仅影响了这些基因在夜间的积累量。此外,Glyma06g06300和Glyma13g01290在大豆叶片、茎尖、花及嫩荚中有大量表达。短日处理时Glyma13g01290的表达在自贡冬豆由营养生长向生殖生长转化的过程中逐渐增加,而长日处理至13d后其表达逐渐被抑制,表明Glyma13g01290可能参与自贡冬豆开花的调控。     

大豆膨胀素GmEXPA4基因的克隆及表达分析

根据植物膨胀素基因保守区设计简并引物,以大豆矮化突变体HK808幼芽的cDNA为模板,克隆到530bp的膨胀素基因保守区片段,并结合RACE技术克隆得到膨胀素基因全长序列,将其命名为GmEXPA4基因。生物信息学分析结果表明,该基因属于膨胀素中的α-expansin亚家族。GmEXPA4基因的实时荧光定量相对表达分析表明,该基因在东农42及其矮化突变体HK808中的各个时期都有表达,且与植物的生长量密切相关。     

牛凝乳酶基因的克隆与表达

为了获得大量高纯度高活性的凝乳酶制剂,采用基因工程方法,从犊牛皱胃黏膜细胞中克隆得到凝乳酶基因,然后将此基因插入原核表达载体pTWIN1中,使之与几丁质结合域(CBD)-内含肽(intein)融合,从而获得原核表达质粒:pTWIN1/EchybF2.经转化大肠杆菌BL21(DE3)后,在IPTG诱导下进行凝乳酶的表达.SDS-PAGE电泳分析和酶活性实验结果显示,CBD-intein-EchybF2融合蛋白在BL21(DE3)中获得高效表达,在低温诱导时主要以可溶性蛋白的形式存在,并具有凝乳活性.     

甘蓝型油菜AnnBn1基因的克隆和表达分析

利用电子克隆和RT-PCR技术从甘蓝型油菜抗旱品系Q2中克隆了膜联蛋白（annexin）基因,命名为AnnBn1（GenBank登录号HM244482）,并对其进行了表达分析。AnnBn1的开放阅读框长度为954bp,编码317个氨基酸。序列分析推测AnnBn1基因编码的氨基酸序列含有4个重复的结构域及钙离子结合位点,与拟南芥、番茄、棉花和玉米等物种的膜联蛋白具有较高的同源性。荧光定量PCR结果发现AnnBn1基因在Q2的根、茎、叶、芽等组织中均有表达,而且表达量基本相同。对3叶期幼苗进行10%PEG（聚乙二醇）溶液模拟干旱时发现,干旱胁迫后30h内,AnnBn1基因在茎、芽中的表达量升高了2～4倍,而在叶、根中显著升高了10倍以上。AnnBn1基因表达峰值也具有时空特点,干旱胁迫后20h茎和芽中表达量高,而在30h之后叶和根中表达量高。     

Effects of ACTH and expression of the melanocortin-2 receptor in the neonatal mouse testis

ACTH has been shown to stimulate androgen production by the fetal/neonatal mouse testis through the melanocortin type 2 receptor (MC2R). This study was designed to localize the expression of MC2R in the neonatal mouse testis and characterize the effects of ACTH on testicular androgen production. Using immunohistochemistry, MC2R was localized to the fetal-type Leydig cell population of the neonatal testis. ACTH caused a time-dependent increase in cyclic AMP (cAMP) and testosterone production by isolated cells with an increase in cAMP apparent in < 3 min. There was no additive effect of maximally stimulating doses of ACTH and human chorionic gonadotropin (hCG). Androgen production in response to ACTH and hCG was reduced by UO126 and dexamethasone, which are the inhibitors of ERK1/2 and phospholipase A2 respectively. Expression of mRNA encoding StAR was increased fourfold by both ACTH and hCG, although expression of mRNA encoding for steroidogenic enzymes was not markedly affected. The potency of N-terminal fragments of ACTH to stimulate androgen production was similar to that seen previously in the adrenal. Data indicate that both LH and ACTH, acting through their respective receptors, stimulate steroidogenesis by fetal-type Leydig cells via arachidonic acid, protein kinase A, and ERK1/2 activation of StAR.