Stability comparison of three candidate internal reference genes in Chinese giant salamander (Andrias davidianus)

The Chinese giant salamander (Andrias davidianus) as food and medicinal product has been an important aquaculture object in China. Study of gene function in the Chinese giant salamander requires accurate normalization though the use of appropriate reference genes. In this study, the expression levels of three candidate reference genes including β‐actin, GAPDH and cytb of different tissues, different developmental stages and different challenges in Chinese giant salamander were evaluated by qPCR. The stabilities of these three reference genes were analysed by geNorm, NormFinder and BestKeeper software. The results showed that the expression of GAPDH was more stable than that of β‐actin and cytb in four tissues and at two developmental stages of Chinese giant salamander. Compared with GAPDH and cytb, β‐actin was the most stable in spleen of Chinese giant salamander treated with LPS or GSIV. Therefore, the result showed that GAPDH was the suitable reference gene in different tissues and at different developmental stages of Chinese giant salamander. The β‐actin could be used as a reference gene in spleen of Chinese giant salamander challenged with LPS and GSIV. This study provides convincing information for the GAPDH and β‐actin as suitable reference gene in Chinese giant salamander of different tissues, different developmental stages and different challenges respectively.     

Effects of dietary mulberry leaf extract on the growth,gastrointestinal, hepatic functions of Chinese giant salamander (Andrias davidianus)

Mulberry leaf extract (MLE), an active substance extracted from mulberry leaves, is known to have a positive effect on several physiological functions. The current study examined the effects of dietary MLE in feed at concentrations 0, 3.0, 6.0, 9.0, 12.0 and 15.0 g/kg on the growth performance and gastrointestinal and hepatic functions of Andrias davidianus for 12 weeks. Results indicated that the final body weight, weight gain rate (WGR), specific growth rate and feed intake (FI) of A. davidianus increased with the increase in the dietary MLE up to 9.0 g/kg and declined thereafter, while the feed conversion ratio (FCR) exhibited an opposite trend. Meanwhile, A. davidianus with a dietary intake of 9.0 g/kg MLE showed higher levels of crude protein in muscles and lower levels of moisture and crude lipid levels in the liver and muscles when compared with the control. In addition, dietary MLE increased the density and length of the villi and decreased the cavity rate in the foregut, enhancing the activities of carbonic anhydrase, H⁺‐K⁺‐ATPase, pepsin, intestinal trypsin, lipase and Na⁺‐K⁺‐ATPase (p < .05) in the stomach. Furthermore, dietary MLE increased the intestinal and hepatic superoxide dismutase activities and total antioxidative capacities but decreased their malondialdehyde contents in A. davidianus. Dietary MLE also significantly increased the immune parameters, and the plasma total protein, albumin and immunoglobulin M contents but significantly decreased the aspartate aminotr‐ansferase, alanine aminotransferase and diamine oxidase activities, and the total bilirubin, direct bilirubin, cholesterol, triglyceride and endotoxin contents (p < .05). In conclusion, a quadratic regression analysis of WGR and FCR indicated that the optimum level of MLE for A. davidianus was between 8.21 and 8.30 g/kg of the diet.     

石首麋鹿国家级自然保护区外来入侵植物种的分析

通过对石首麋鹿国家级自然保护区(以下简称石首麋鹿保护区)外来入侵植物的种类、生活型、原产地、入侵途径等进行野外实地调查和分析,初步确认有外来入侵植物27种,隶属于13科25属,其中以菊科的种类最多,有7种;所有入侵植物均为草本植物,一年生草本占40.7%;除南极洲以外的其他六大洲都有植物入侵该区,而以美洲大陆最多;入侵途径多样,以无意传播途径为主。根据调查和分析的结果,分析了麋鹿对部分入侵植物种的采食利用,从植物入侵特点和对石首麋鹿保护区生物多样性的影响两方面对该区植物入侵现状进行分析,针对上述调查和分析的结果提出对保护区内入侵植物的防治措施。     

中国大鲵病害生态综合防控体系构建

在大鲵(Andrias davidianus)人工养殖过程中,系统构建完善的病害生态综合防控体系是防止和减少大鲵病害发生的技术关键。从大鲵养殖设施的选址,苗种种质,规范的养殖设施建设,分级饲养技术,饵料投喂,水质管理,检验检疫,"四定"、"四勤"、"一隔离"技术措施,信息档案管理,生产管理日志建立等方面进行了系统论述。     

大鲵幼体蛋白质的需求量

为评定大鲵幼体对饲料蛋白质的需求量,以鱼粉为主要蛋白源配制6种蛋白质水平(干样基础)的实验饲料:D1(43.7%)、D2(47.1%)、D3(51.3%)、D4(55.7%)、D5(59.9%)和D6(64.4%),饲喂初始体质量为(20.99±0.15)g的大鲵幼体92 d。结果显示,①饲料蛋白质水平对大鲵增重率有显著影响,在D4组达到最大值,较D1组增加了276.4%,且全鲵蛋白质沉积率和肌肉RNA、RNA/DNA值、胃蛋白酶、H^+-K^+-ATPase、胰蛋白酶、脂肪酶和Na^+-K^+-ATPase、肝脏超氧化物歧化酶(SOD)均在D4组达到最佳,而肝脏和肠道丙二醛(MDA)在该组均达到最低;②随饲料蛋白质水平增加,肌肉粗蛋白线性增加,全鲵脂肪线性下降,全鲵水分和粗灰分在各组间差异不显著,全鲵粗蛋白先增加后趋于稳定,在D4组达到最大;③大鲵皮肤胶原蛋白含量在D4组达到最高,较D1组增加了27.83%。研究表明,以增重率、肌肉RNA/DNA值、蛋白质沉积率和皮肤胶原蛋白含量为评价指标,通过二次回归方程分析得到大鲵幼体饲料的最适蛋白质水平为55.9%~58.3%(干样基础),该饲料蛋白质水平能显著提高大鲵幼体胃的泌酸能力、机体消化吸收和抗氧化能力,增加鲵体营养素的沉积,从而促进生长和饲料的转化;而低蛋白质水平饲料显著抑制大鲵的生长。     

麋鹿产气荚膜梭菌分离鉴定方法的建立与初步应用

探索了产气荚膜梭菌在营养琼脂、甘露醇卵黄琼脂和血琼脂平板上的菌落形态和培养特点,优化了产气荚膜梭菌的分离方法,并对分离菌株进行生化鉴定;在此基础上对北京南海子麋鹿苑和江苏大丰麋鹿国家级自然保护区的麋鹿自然感染情况进行了调查。结果发现,粪样中的产气荚膜梭菌在营养琼脂上生长呈半透明边缘不整的白色菌落,接种甘露醇卵黄琼脂和血琼脂平板,分别出现伴有卵磷脂酶乳光浑浊带的粉红色火山口状菌落和伴有双溶血环的灰绿色勋章样菌落。生化试验结果确认这些分离株均为产气荚膜梭菌。对北京南海子麋鹿苑和江苏大丰麋鹿国家级自然保护区麋鹿粪样检测发现,阳性率分别为13.04%(3/23)和19.51%(8/41)。说明本研究建立的麋鹿产气荚膜梭菌分离鉴定方法简便快速,确实可行;麋鹿产气荚膜梭菌自然感染率较高,应加强麋鹿产气荚膜梭菌病的防控。     

麋鹿致病性大肠杆菌的分离鉴定

从死亡麋鹿的心血、肝脏和肾脏各分离、纯化获得3个分离株,并对分离细菌进行了菌株鉴定及致病性分析。分别应用常规生理生化鉴定、人工感染试验及16S rRNA基因序列分析等手段,确认3个分离株均为致病性大肠杆菌。     

大鲵引进与地下室人工养殖试验研究

2009年11月山东省成功引进大鲵(Andrias davidianus),开晨了大鲵地下室人工养殖试验.其结果表明:放养1 100尾,平均体重5.43 g、体长8.5 cm,经过2年饲养,平均体重1.146 kg、平均体长50.12 cm,成活率98.7％,总重1 244.6 kg,最大个体为2.3 kg.大鲵在海拔108m地区人工养殖的成功,对野生种群修复、保护及开发将起到积极的重要作用.     

大鲵虹彩病毒TaqMan实时荧光定量PCR检测方法的建立

利用PCR技术扩增出大鲵虹彩病毒(giant salamander iridovirus, GSIV)主要衣壳蛋白(MCP)编码区长度为1 392 bp 的片段, 克隆到 pMD19-T载体上, 构建重组质粒 pMD19-T-MCP。经PCR鉴定确认正确后, 以10倍梯度稀释 pMD19-T-MCP重组质粒, 作为标准模板进行 TaqMan实时荧光定量PCR扩增, 制作标准曲线, 建立了大鲵虹彩病毒的 TaqMan实时荧光定量PCR检测方法。制作的标准曲线有极好的线性关系, 且线性范围宽, 相关系数为0.990 19。组内重复试验的CT值标准偏差为0.52%。检测结果显示, 该方法对大鲵虹彩病毒的检测有高度的特异性, 与锦鲤疱疹病毒、弗氏柠檬酸杆菌、嗜水气单胞菌以及鲤上皮瘤细胞基因组DNA之间均无交叉反应, 特异性好, 检测总DNA灵敏度为10个病毒核酸分子拷贝数, 约1.1×10-3 pg/μL病毒核酸, 较之常规PCR的敏感度高出约1 000倍。研究建立的大鲵虹彩病毒TaqMan实时荧光定量PCR方法灵敏度高、特异性强, 对大鲵虹彩病毒病的快速诊断与病毒病原定量检测有重要意义。     

大鲵不同发育阶段及组织中内参基因稳定性评价

【目的】研究18S核糖体RNA(18SrRNA)、延伸因子1-α(EF1-α)、甘油醛-3-磷酸脱氢酶(GAPDH)、β-肌动蛋白(β-actin)和琥珀酸脱氢酶A亚基(SDHA)等5种常见内参基因在大鲵不同组织、不同发育阶段及嗜水气单胞菌感染下的稳定性,为大鲵后基因组学研究提供指导。【方法】采集3龄健康大鲵的肝脏、肠、胃、胰腺、肾脏、肌肉、皮肤、脾脏等组织,3龄、2龄、1龄、9月龄、1周龄健康大鲵的皮肤组织及嗜水气单胞菌感染大鲵后第3,5,7天的皮肤组织,提取其RNA,进行实时荧光定量PCR检测,应用3种内参基因稳定性评价分析工具(geNorm,NormFinder,BestKeeper)分析5种内参基因在大鲵不同组织、不同发育阶段以及不同病理状态下的稳定性。【结果】5种内参基因在大鲵不同组织中表达稳定性较好的是EF1-α和SDHA,而在不同发育阶段及嗜水气单胞菌感染大鲵的皮肤组织中,5种内参基因的表达稳定性不同于在不同组织中的表达,稳定性较好的是EF1-α和GAPDH。【结论】EF1-α表达最为稳定,是大鲵定量PCR首选内参基因,而SDHA、GAPDH以及β-actin表达稳定性受生理状态的影响较大,作为第二内参基因时应视试验目的而定,在不同组织中进行定量PCR比较时应选用SDHA,而在不同生理状态下则应选用GAPDH为第二或备用内参基因。