冷应激前后大鼠心脏、肝脏及脾脏中α-烯醇化酶基因mRNA的转录水平

目的研究冷应激前后大鼠心脏、肝脏、脾脏中α-烯醇化酶(α-enolase,ENO1)基因m RNA的转录水平。方法将大鼠随机分为正常对照组(24℃,正常饲喂7 d)和冷应激组(置4℃,应激12 h),提取各组大鼠心脏、肝脏及脾脏细胞总RNA,反转录成c DNA,以其为模板,PCR扩增ENO1基因,应用生物信息学软件DNAstar进行同源性分析,实时荧光定量PCR法检测大鼠心脏、肝脏、脾脏中ENO1基因m RNA的转录水平。结果冷应激前后大鼠心脏、肝脏及脾脏总RNA的1%琼脂糖凝胶电泳可见28S、18S和5S 3条带,A260/A280值为1.8~2.0;ENO1基因经2%琼脂糖凝胶电泳分析可见107 bp的目的基因条带;ENO1基因序列与NCBI上公布序列的同源性为100%;冷应激组大鼠肝脏、脾脏组织中ENO1基因m RNA的转录水平明显高于正常对照组(P0.01),正常对照组大鼠心脏组织中ENO1基因m RNA转录水平明显高于冷应激组(P0.05)。结论冷应激前后大鼠心脏、肝脏及脾脏中ENO1基因m RNA的转录水平均发生显著改变,本实验为动物应激状态评估及应激发生机制的研究提供了实验依据。     

α-烯醇化酶在籽鹅不同级别卵泡壁的定位及定量

目的对籽鹅不同级别卵泡壁中的α-烯醇化酶(α-enolase)进行定位及定量检测。方法分离产蛋期籽鹅等级卵泡(F1、F2、F3、F4、F5)、小黄卵泡(small yellow follicle,SYF)、大白卵泡(large white follicle,LWF)的卵泡壁;应用免疫组化方法检测不同级别卵泡壁的α-烯醇化酶的定位,采用Western blot及实时荧光定量PCR双标准曲线法检测α-烯醇化酶蛋白表达量及mRNA转录水平。结果不同级别卵泡壁均存在α-烯醇化酶表达,颗粒细胞层表达量总体高于膜层,SYF颗粒细胞层α-烯醇化酶表达量较高;F2级卵泡壁α-烯醇化酶mRNA转录水平显著高于LWF、F3及F4级别卵泡,差异有统计学意义(P 0. 01),F1、F5及SYF级卵泡壁α-烯醇化酶m RNA转录水平显著高于其他级别卵泡壁,差异有统计学意义(P 0. 01);SYF级卵泡壁α-烯醇化酶蛋白表达量显著高于其他级别卵泡,差异有统计学意义(P 0. 01)。结论α-烯醇化酶及糖酵解途径可能在卵泡发育中发挥重要功能,为α-烯醇化酶在禽类卵泡选择及生长发育中的功能研究奠定了基础。     

籽鹅卵泡颗粒细胞α-烯醇化酶基因RNA干扰表达质粒的构建及鉴定

目的构建籽鹅卵泡颗粒细胞α-烯醇化酶(α-enolase,ENO1)基因RNA干扰表达质粒,并进行鉴定。方法利用RNAi Designer等网络在线RNA干扰设计软件设计3条可能会干扰籽鹅卵泡颗粒细胞ENO1基因表达的插入DNA序列:shRNA-ENO1-350、shRNA-ENO1-892、shRNA-ENO1-591,将体外合成的3条干扰序列分别与线性化的pGPU6/GFP/Neo载体连接,构建ENO1 RNA干扰表达质粒,在Lipofectamine 2000的介导下转染原代培养的籽鹅卵泡颗粒细胞,转染48 h后,荧光倒置显微镜下观察GFP的表达;实时荧光定量PCR和Western blot法检测转染细胞中ENO1基因mRNA的水平及蛋白的表达水平。结果 pGPU6/GFP/Neo-shDNA重组质粒经单酶切及测序证实构建正确;转染后48 h,转染重组质粒的颗粒细胞在荧光倒置显微镜下可见较强的绿色荧光,转染效率可达60%;与培养液组、转染试剂组和无关序列干扰组(shNC)相比,shRNA-ENO1-350组颗粒细胞中ENO1基因mRNA水平和蛋白表达水平均显著降低(P0.01),其他各组之间差异无统计学意义(P0.05)。结论成功构建了籽鹅卵泡颗粒细胞ENO1基因RNA干扰表达质粒,在原代培养的籽鹅卵泡颗粒细胞中,其表达量显著下降,为后续有关ENO1功能的研究奠定了基础。     

鹅细小病毒VP3基因的克隆及序列分析

目的克隆鹅细小病毒(Goose parvovirus,GPV)黑龙江各分离株和疫苗株的VP3基因,并进行序列分析,为小鹅瘟的诊断与防治奠定理论基础。方法根据GenBank中登录的GPV B株全基因序列设计1对特异性引物,PCR扩增、克隆VP3基因,并进行测序和同源性分析。结果克隆的VP3基因大小699 bp,编码233个氨基酸;7个分离株的VP3基因核苷酸序列同源性为99.4%～100%,各分离株与疫苗株的核苷酸序列同源性为98.7%～99.3%,与标准B株的核苷酸序列同源性为97.6%～97.7%,与MDPV核苷酸序列的同源性为80.8%～81.7%;各分离株之间亲缘关系较近,其中QTH分离株与疫苗株亲缘关系最近,氨基酸序列同源性为97.8%,其他分离株与疫苗株的亲缘关系相对较远,氨基酸序列同源性为96.6%～97.4%;所有分离株和疫苗株与标准B株亲缘关系相对较远,与MDPV的亲缘关系最远。结论黑龙江省各分离株与疫苗株的核苷酸和氨基酸序列存在一定的差异。     

新城疫病毒F48E8株HN基因的克隆和序列分析及与国外NDV HN基因的同源性比较

目的 克隆NDV F48E8株HN基因,与国外NDV HN基因进行比较分析。方法 提取RNA,应用RT－PCR一次性扩增出NDV F48E8株的全长HN基因,将该HN基因插入pKS(-)后进行克隆并测序。结果 NDV F48E8株的HN基因核苷酸长度为1713bp,编码571个氨基酸,有5个糖基化位点,12个极保守的半胱氨酸残基,与国外发表的NDV序列相符。结论 NDV F18E8株HN蛋白基因与国外性遥NDV HN蛋白基因核苷酸同源性在83.3%-89.2%之间,的氨基酸同源性在87.6%-91.1%之间。     

Overexpression of G0/G1 Switch Gene 2 in Adipose Tissue of Transgenic Quail Inhibits Lipolysis Associated with Egg Laying

In avians, yolk synthesis is regulated by incorporation of portomicrons from the diet, transport of lipoproteins from the liver, and release of lipids from adipose tissue; however, the extent to which lipolysis in adipose tissue contributes to yolk synthesis and egg production has yet to be elucidated. G0/G1 switch gene 2 (G0S2) is known to bind and inhibit adipose triglyceride lipase (ATGL), the rate-limiting enzyme in lipolysis. The objective of this study was to determine whether overexpression of the G0S2 gene in adipose tissue could successfully inhibit endogenous ATGL activity associated with egg laying. Two independent lines of transgenic quail overexpressing G0S2 had delayed onset of egg production and reduced number of eggs over a six-week period compared to non-transgenic quail. Although no differences in measured parameters were observed at the pre-laying stage (5 weeks of age), G0S2 transgenic quail had significantly larger interclavicular fat pad weights and adipocyte sizes and lower NEFA concentrations in the serum at early (1 week after laying first egg) and active laying (5 weeks after laying first egg) stages. Overexpression of G0S2 inhibited lipolysis during early and active laying, which drastically shifted the balance towards a net accumulation of triacylglycerols and increased adipose tissue mass. Thereby, egg production was negatively affected as less triacylglycerols were catabolized to produce lipids for the yolk.     

实时荧光定量PCR方法检测人、鼠正常组织及脑癌组织中CD109蛋白的表达

目的分析CD109基因在正常组织及脑癌组织中的分布。方法采用实时荧光定量PCR方法,分析CD109在人、鼠正常组织及脑癌中的表达,以18SrRNA作为内标。结果CD109蛋白的mRNA丰度在睾丸组织中最高,在其它组织中较低,在12份脑癌标本中,检测到9份CD109表达上调。结论CD109基因在多数正常组织中表达很少,有可能是治疗恶性肿瘤的一个潜在的分子靶点。     

植物乳杆菌共轭亚油酸异构酶基因的克隆、序列分析及重组表达

利用MRS培养基培养植物乳杆菌AS1.555,收集菌体后提取总DNA,用PCR技术扩增其亚油酸异构酶基因,克隆到pQE30质粒载体上,并进行测序.测序结果表明,扩增DNA全长1710 bp,编码569个氨基酸,分子量为64.7 ku.经同源性比较,此核酸序列与罗伊氏乳杆菌、短双歧杆菌、疮疱丙酸杆菌亚油酸异构酶基因核苷酸序列差别较大,推测此基因的来源与上述菌种不同.     

Analysis of Gene Expression Changes in Plants Grown in Salty Soil in Response to Inoculation with Halophilic Bacteria

Soil salinity is an increasing problem facing agriculture in many parts of the world. Climate change and irrigation practices have led to decreased yields of some farmland due to increased salt levels in the soil. Plants that have tolerance to salt are thus needed to feed the world’s population. One approach addressing this problem is genetic engineering to introduce genes encoding salinity, but this approach has limitations. Another fairly new approach is the isolation and development of salt-tolerant (halophilic) plant-associated bacteria. These bacteria are used as inoculants to stimulate plant growth. Several reports are now available, demonstrating how the use of halophilic inoculants enhance plant growth in salty soil. However, the mechanisms for this growth stimulation are as yet not clear. Enhanced growth in response to bacterial inoculation is expected to be associated with changes in plant gene expression. In this review, we discuss the current literature and approaches for analyzing altered plant gene expression in response to inoculation with halophilic bacteria. Additionally, challenges and limitations to current approaches are analyzed. A further understanding of the molecular mechanisms involved in enhanced plant growth when inoculated with salt-tolerant bacteria will significantly improve agriculture in areas affected by saline soils.     

Selection and Validation of Reference Genes for RT-qPCR Analysis in Aegilops tauschii (Coss.) under Different Abiotic Stresses

Aegilops tauschii (Coss.) is an aggressive and serious annual grass weed in China. Its DD genome is a rich source of genetic material and performs better under different abiotic stress conditions (salinity, drought, temperature, etc.). Reverse-transcribed quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for reference gene selection and validation. This work aimed to evaluate the stability of reference gene expression in Ae. tauschii under different abiotic stresses (salinity, drought, hot, and cold) and developmental stages (seedling and development). The results show that the ubiquitin-conjugating enzyme E2 36-like (UBC36) and protein microrchidia 2-like (HSP) are the most stable genes under control and salinity conditions, respectively. Under drought stress conditions, UBC36 is more stable as compared with others. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) is the most stable reference gene during heat stress conditions and thioredoxin-like protein (YLS) under cold stress condition. Phosphate2A serine/threonine-protein phosphatase 2A (PP2A) and eukaryotic translation initiation factor 3 (ETIF3) are the most stable genes at seedling and developmental stages. Intracellular transport protein (CAC) is recommended as the most stable gene under different abiotic stresses and at developmental stages. Furthermore, the relative expression levels of NHX1 and DREB under different levels of salinity and drought stress conditions varied with the most (HSP and UBC36) and least (YLS and ACT) stable genes. This study provides reliable reference genes for understanding the tolerance mechanisms in Ae. tauschii under different abiotic stress conditions.     

Gene Expression of Type VI Secretion System Associated with Environmental Survival in Acidovorax avenae subsp. avenae by Principle Component Analysis

Valine glycine repeat G (VgrG) proteins are regarded as one of two effectors of Type VI secretion system (T6SS) which is a complex multi-component secretion system. In this study, potential biological roles of T6SS structural and VgrG genes in a rice bacterial pathogen, Acidovorax avenae subsp. avenae (Aaa) RS-1, were evaluated under seven stress conditions using principle component analysis of gene expression. The results showed that growth of the pathogen was reduced by H₂O₂ and paraquat-induced oxidative stress, high salt, low temperature, and vgrG mutation, compared to the control. However, pathogen growth was unaffected by co-culture with a rice rhizobacterium Burkholderia seminalis R456. In addition, expression of 14 T6SS structural and eight vgrG genes was significantly changed under seven conditions. Among different stress conditions, high salt, and low temperature showed a higher effect on the expression of T6SS gene compared with host infection and other environmental conditions. As a first report, this study revealed an association of T6SS gene expression of the pathogen with the host infection, gene mutation, and some common environmental stresses. The results of this research can increase understanding of the biological function of T6SS in this economically-important pathogen of rice.     

Quantitative Real-Time PCR Analysis of YKL-40 and Its Comparison with Mammalian Chitinase mRNAs in Normal Human Tissues Using a Single Standard DNA

YKL-40 (YKL for the first three N-terminal residues of a 40 kDa protein) belongs to a group of human chitinase-like proteins (CLPs), which are similar to chitinases but lack chitinolytic activity. YKL-40 mRNA and its protein levels have been reported elevated in multiple disorders including asthma, cystic fibrosis, rheumatoid arthritis and malignant tumors. Here, we quantified the YKL-40 mRNA levels and compared them with chitinases and housekeeping genes in normal human tissues. To establish the quantitative real-time PCR (qPCR) system for evaluation of relative YKL-40 mRNA levels, we constructed a human standard DNA molecule by ligating cDNAs of YKL-40, two mammalian chitinases and two housekeeping genes in a one-to-one ratio. We generated cDNAs from various normal human tissues and analyzed the YKL-40 mRNA expression levels using a qPCR system with the standard DNA. We found that YKL-40 mRNA is present widely in human tissues while its expression patterns exhibit clear tissue specificity. Highest YKL-40 mRNA levels were detected in the liver, followed by kidney, trachea and lung. The levels of YKL-40 mRNA in the kidney and liver were more than 100-times higher than those of chitotriosidase mRNA. Our study provides for the first time a comprehensive analysis of the relative expression levels of YKL-40 mRNA versus mammalian chitinases in normal human tissues.