斑点叉尾(鱼回)趋化因子基因SCYA113内含子1的多态性分析

运用变性聚丙烯酰胺凝胶电泳(DPAGE)和PCR产物测序技术检测斑点叉尾(鱼回)(Ictalums punctatus)CC趋化因子基因SCYAll3内含子l的序列长度和多态性.在3个群体的60个个体中,斑点叉尾(鱼回)CC趋化SCYA113内含子1存在15种单元型和8种长度类型,长度类型分别为A、B、C、D、E、F、G和H型.对这8种序列进行比对分析发现,斑点又尾(鱼回)SCYA113内含子1长度为395～443 bp.8种长度类型中A、G、T、c的平均百分比分别为30.01%、15.43%、38.37%和16.19%,而且G+C(31.62%)含量明显小于A+T(68.38%)含量.内含子1与外显子1和2的连接处存在真核基因中mRNA剪接的识别信号GT/AG;引起长度变化的主要原因是短串联重复序列(微卫星序列)TTc和TTA引起的;除了微卫星序列以外,共检测到6个突变位点,其中3个转换位点为118(C→T)、209(G→A)和250(T→c),3个颠换位点为266(T→A)、289(G→T)和387(G→C),核苷酸多样性指数(π)为0.004,平均核苷酸差异(K)为1.576;在变异水平上,3个群体表现出一定的遗传差异,84群体中检测到8种长度类型和14种基因型.97群体中检测到8种长度类型和1O种基因型,04群体中6种长度类型和1O种基因型(缺少A、B等位基因).从3个群体60个个体拥有22种频率较低(O.017～0.150)的基因型可以看出,斑点叉尾(鱼回)的遗传基因资源较为丰富.     

EST-SSR标记对我国斑点叉尾鮰种质资源的研究

采用EST-SSRs对福建和湖北的1984年群体（P1984）、福建与辽宁的1997年群体（P1997）、湖南的2004年群体（P2004）和福建的1984年与1997年群体杂交的F。代群体（P8497）等4个斑点叉尾鮰（Ictalurus punctatus）养殖群体的遗传多样性进行了研究。结果表明,有10对引物可扩增出清晰条带,其中9对引物具有多态性,共获得32个等位基因,4个群体的平均等位基因数（A）为3．00-3．40,平均有效等位基因数（Ac）为1．95～2．30,平均观察杂合度（Ho）为O．4768-0．5982,平均期望杂合度（Ho）为004913-0．5695,平均多态信息含量（PIC）为0．4113-0．4829,群体间的多态性差异不显著,且各群体的遗传多样性处于中等水平。根据群体间遗传相似性系数、遗传距离及UPGMA聚类分析发现,P1997和P2004群体之间的遗传距离最近,P1984和P2004群体之间的遗传距离最远。4个群体有18．75％的位点偏离了Hardy-Weinberg平衡,表明群体各位点的基因频率和基因型频率稳定性较好。F-统计检验发现,P2004和P8497处于不同程度的杂合子过剩状态,P1984和P1997处于杂合子缺失状态。群体间分化程度很弱,遗传变异主要来自群体内个体之间。     

AFLP分析江西省4个斑点叉尾鮰养殖群体遗传多样性

本文运用AFLP分子标记技术,对江西省4个斑点叉尾鮰(Ictalurus punctatus)养殖群体(GZ、XJ、PYA和PYB)进行遗传多样性分析。结果表明,在64对引物组合中,E3/M2、E4/M7、E3/M8和E5/M5引物从4个群体72个体中共扩增出179个位点,其中多态位点为109,占60.89%。其中,PYA、GZ、XJ和PYB群体的多态位点比例(P)分别为56.54%、56.47%、56.32%和56.14%。Shannon多样性指数(I)分别为0.2890、0.3003、0.2896和0.2852,平均杂合度(H)分别为0.1935、0.2027、0.1938和0.1898。4个群体间的遗传分化系数(Gst)为0.1314,说明群体间发生了一定程度的遗传分化,但分子方差分析(AMOVA)显示,群体的遗传变异90.98%来源于群体内的个体间,9.02%来源于群体间。聚类分析也表明,4个群体所产生的遗传分化主要也来源于群体内。本研究为斑点叉尾鮰种质资源的调查和保护提供参考,为斑点叉尾鮰优良品种的选育提供科学依据。     

斑点叉尾鮰铁调素成熟肽在毕赤酵母中的表达及其抑菌活性

铁调素(hepcidin)是一种在动物肝脏中特异表达的碱性小分子抗菌肽,在机体免疫系统中发挥重要作用,被认为是抗生素的理想替代品。本研究根据毕赤酵母(Pichia pastoris)偏爱性密码子人工合成斑点叉尾鮰(Ictalurus punctatus)hepcidin成熟肽基因(mCH)。通过PCR方法在mCH的5’端和3’端分别引入EcoRⅠ和NotⅠ酶切位点,扩增到的目的片段与表达载体p PIC9K连接构建重组表达载体p PIC9K-mCH后,转化至毕赤酵母GS115细胞;以不同浓度梯度的G418筛选高拷贝转化子,1.0%甲醇、30℃、pH6.0诱导表达72 h,获得重组体mCH。结果显示,斑点叉尾鮰hepcidin成熟肽区域含25个残基,8个保守的半胱氨酸残基位于成熟肽的羧基端,表明该区域对hepcidin的抗菌活性具有重要作用。Tricine-SDS-PAGE分析显示,分泌表达的重组体mCH的分子量约为3 800D,通过阳离子交换层析获得纯化的mCH。抑菌实验表明,重组体抗菌肽mCH对金黄色葡萄球菌(Staphylococcus aureus)和大肠杆菌(Escherichia coli)有抑菌活性。本研究首次实现了斑点叉尾鮰hepcidin成熟肽在毕赤酵母中的重组DNA表达,为其产业化制备提供了重要的基础资料。     

EST-SSR标记对我国斑点叉尾(鲴)种质资源的研究

采用EST-SSRs对福建和湖北的1984年群体(P1984)、福建与辽宁的1997年群体(P1997)、湖南的2004年群体(P2004)和福建的1984年与1997年群体杂交的F.代群体(P8497)等4个斑点叉尾鲴(Ictalurus punctatus)养殖群体的遗传多样性进行了研究.结果表明,有10对引物可扩增出清晰条带,其中9对引物具有多态性,共获得32个等位基因,4个群体的平均等位基因数(A)为3.00～3.40,平均有效等位基因数(Ae)为1.95～2.30,平均观察杂合度(Ho)为0.4768～0.5982,平均期望杂合度(He)为0.4913～0.5695,平均多态信息含量(PIC)为0.4113～0.4829,群体间的多态性差异不显著,且各群体的遗传多样性处于中等水平.根据群体间遗传相似性系数、遗传距离及UPGMA聚类分析发现,P1997和P2004群体之间的遗传距离最近,P1984和P2004群体之间的遗传距离最远.4个群体有18.75%的位点偏离了Hardy-Weinberg平衡,表明群体各位点的基因频率和基因型频率稳定性较好.F-统计检验发现,P2004和P8497处于不同程度的杂合子过剩状态,P1984和P1997处于杂合子缺失状态.群体间分化程度很弱,遗传变异主要来自群体内个体之间.     

牛糖基化依赖细胞黏附分子1基因(G1yCAM1)的遗传多态性分析

以中国荷斯坦牛、鲁西黄牛和渤海黑牛为研究材料.利用CRS-PCR、PCR-SSCP及DNA测序技术检测了糖基化依赖细胞黏附分子l基因(GlyCAMl)的遗传多态性.结果表明,在牛ClyCAMl基因外显子3的第2081(A/C)和内含子3的2417(C/T)位点存在突变.两位点在3个牛群中的等位基因频率A/B分别为0.7525/0.2475、O.6112/0.3888和0.3375/0.6625;0.9046/0.0954、O.8383/0.1617和0.7875/0.2125;经х2适合性检验,3个牛群在内含子3的突变达到Hardy-Weinberg平衡状态(P>O.05),在外显子3鲁西黄牛和渤海黑牛达到Hardy-Weinberg平衡状态(P>O.05),中国荷斯坦牛的突变在此位点未达到Hardy-Weinberg平衡状态(P相似文献   

酉州乌羊MC1R基因的克隆鉴定、生物信息学及多态性分析

MC1R是影响皮肤色素沉着中黑皮素系统的重要基因,通过信号分子的相互作用影响黑色素的生成。为探究酉州乌羊 MC1R的生物学特性,本研究对酉州乌羊MC1R进行了分离鉴定、生物信息学分析。结果表明,酉州乌羊皮肤组织MC1R基因的CDS序列为954 bp,共编码317个氨基酸,酉州乌羊与各物种MC1R具有较高同源性。酉州乌羊MC1R编码蛋白的分子式是C₁₆₀₃H₂₅₆₅N₄₁₁O₄₁₀S₂₂,其为疏水蛋白,有7个α-螺旋组成的跨膜结构域和3个保守功能结构域,MC1R氨基酸序列大部分都用于构成α-螺旋。此外,酉州乌羊MC1R基因c.676A>G突变,其编码的氨基酸由赖氨酸转变为谷氨酸,位于受光刺激和G蛋白配体结合的保守结构域上,氨基酸的异义替换还造成了酉州乌羊MC1R空间构象的差异。而对重庆市山羊群体多态性检测发现,山羊MC1R基因c.676A>G变异位点在重庆合川白山羊、巫溪白山羊、本地白山羊、酉州乌羊中共享,且c.676A>G位点的各基因型在皮肤白色和乌色山羊群体中的分布差异不显著(P>0.05)。本研究初步揭示酉州乌羊MC1R的序列特征和蛋白结构,为酉州乌羊MC1R影响皮肤色素沉积的分子机制研究提供了理论数据。     

草鱼羧肽酶A1基因(CPA1)部分片段的单核苷酸多态性(SNP)多态性及其与生长性状的关联分析

羧肽酶A (EC 3.4.16)是一类水解蛋白和多肽底物C端芳香族氨基酸或脂肪族氨基酸残基的消化酶.在草鱼生长相关的功能基因上研究单核苷酸多态性(SNP)与生长的相关性可为草鱼的分子辅助育种提供依据.本研究根据草鱼(Ctenopharyngodon idella)EST-SNP库的羧肽酶A1基因(CPA1)重叠群的2个Contig扩增该基因的序列片段,采用直接测序法,经过序列比对,共筛到2个颠换SNP位点:C+412A和A36C,分别位于CPA 1基因外显子5的34bp和内含子3的36 bp处,前者为错义突变.然后用一个群体的296尾草鱼对这2个位点用SnaPshot的方法进行检测和分型,统计基因型频率:A36C位点的AA基因型占26.7％,AC基因型占52.0％,CC基因型占21.3％.C+412A位点的AA基因型占15.5％,AC基因型占40.5％,CC基因型占43.9％.利用一般线性模型分析2个SNP位点与草鱼体质量、体长等重要生长性状的关系.关联分析结果显示,C+36A位点不同基因型在体质量、眼间距均值上存在显著差异(P＜0.05).并且AA基因型和CC基因型在体质量、体长、体宽和眼间距上存在显著差异(P＜0.05).AA基因型各项指标均值显著高于CC基因型.C+412A位点在体质量等生长性状上差异不显著(P＞0.05),该位点和生长不相关,但是CC基因型和AC基因型在体重和肛前距上差异显著(P＜0.05),该位点CC基因型的6个生长性状均值都高于AA基因型.由两个位点组成的5种双倍型在体质量上存在显著差异(P＜0.05).双倍型D3和D5在体质量上存在差异显著(P＜0.05).双倍型D3和D8在体质量、体宽、体长、体长/头长等生长性状上都存在显著差异(P＜0.05).D3的各项指标均值最高,D8的各项指标均值最低.本研究结果显示,可以考虑将草鱼CPA1基因作为候选基因,用于草鱼的分子辅助育种.     

7个牛品种PON1基因的EcoRⅤ和AluⅠ酶切多态性分析

对氧磷酶是一种与高密度脂肪酸高度相关的脂酶,且被认为在脂肪代谢和心血管病发生方面起着关键的作用。本研究旨在对对氧磷酶１基因的酶切多态性与牛的生长和胴体性状进行关联分析。基于这一目标,应用RFLP技术对18头安格斯,23头晋南牛,20头利木赞,28头鲁西牛,26头秦川牛,29头西门塔尔牛和29头夏洛莱牛进行了基因型分型,SAS 9.1的GLM过程中的Duncan’s 检验对不同基因型之间进行了多重比较分析,结果表明,EcoRⅤ位点与体重、日增重、眼肌面积和嫩度之间存在显著生物学相关,并且AG基因型个体与其它基因型相比在日增重和嫩度上存在显著差异（p< 0.05）,而AA基因型个体相对于其它基因型具有较大的眼肌面积（p< 0.05）。而AluⅠ位点则与体重、净肉重和嫩度之间具有显著的生物学意义（p< 0.05）.且AA型个体相对来说均优于其它基因型个体（p< 0.05）。品种间的比较表明,除实验牛始重相对趋于一致外,国外肉牛专门化品种在其它方面均优于国内品种。因此,本实验所研究的两个位点可作为牛肉多汁性（嫩度）的标记,为肉牛的选种提供生物学基础。     

B细胞异位基因2（Btg2）多态性与河北小尾寒羊多脊椎性状的关联分析

利用直接测序、PCR-RFLP及dCAPS技术研究了河北小尾寒羊(Ouis aries)B细胞异位基因2的外显子3及3'-uTR上的多态性与河北小尾寒羊多脊椎性状的相关性.经测序结果分析,发现了A150G、C243T和A607G 3个单核苷酸多态性,3个位点的PCR-RFLP分析表明,150位点产生AA、AG和GG基因型;243位点产生CC、CT和TT基因型;607位点产生AA、AG和GG基因型.独立性卡方检测结果表明,150位点的不同表型个体基因型频率差异不显著(P>0.05),243位点的差异极显著(P＜0.01),607位点的差异呈显著水平(P<0.05);进一步对这3个位点进行单倍型分析,其独立性卡方检测表明单倍型在不同表型中差异显著(P＜0.05).其结果表明243位点、607位点以及单倍型GTA可能与小尾寒羊多脊椎性状有关.     

Residue depletion of nitrofuran drugs and their tissue-bound metabolites in channel catfish (Ictalurus punctatus) after oral dosing

The depletion of the nitrofuran drugs furazolidone, nitrofurazone, furaltadone, and nitrofurantoin and their tissue-bound metabolites [3-amino-2-oxazolidinone (AOZ), semicarbazide (SC), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AH), respectively] were examined in the muscle of channel catfish following oral dosing (1 mg/kg body weight). Parent drugs were measurable in muscle within 2 h. Peak levels were found at 4 h for furazolidone (30.4 ng/g) and at 12 h for nitrofurazone, furaltadone, and nitrofurantoin (104, 35.2, and 9.8 ng/g respectively). Parent drugs were rapidly eliminated from muscle, and tissue concentrations fell below the limit of detection (1 ng/g) at 96 h. Peak levels of tissue-bound AMOZ and AOZ (46.8 and 33.7 ng/g respectively) were measured at 12 h, and of SC and AH (31.1 and 9.1 ng/g, respectively) at 24 h. Tissue-bound metabolites were measurable for up to 56 days postdose. These results support the use of tissue-bound metabolites as target analytes for monitoring nitrofuran drugs in channel catfish.     

Determination of niclosamide residues in rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) fillet tissue by high-performance liquid chromatography

Bayluscide [the ethanolamine salt of niclosamide (NIC)] is a registered piscicide used in combination with 3-(trifluoromethyl)-4-nitrophenol (TFM) to control sea lamprey populations in streams tributary to the Great Lakes. A high-performance liquid chromatography (HPLC) method was developed for the determination of NIC residues in muscle fillet tissues of fish exposed to NIC and TFM during sea lamprey control treatments. NIC was extracted from fortified channel catfish and rainbow trout fillet tissue with a series of acetone extractions and cleaned up on C(18) solid-phase extraction cartridges. NIC concentrations were determined by HPLC with detection at 360 and 335 nm for rainbow trout and catfish, respectively. Recovery of NIC from rainbow trout (n = 7) fortified at 0.04 microg/g was 77 +/- 6.5% and from channel catfish (n = 7) fortified at 0.02 microg/g was 113 +/- 11%. NIC detection limit was 0.0107 microg/g for rainbow trout and 0.0063 microg/g for catfish. Percent recovery of incurred radioactive residues by this method from catfish exposed to [(14)C]NIC was 89.3 +/- 4.1%. Percent recoveries of NIC from fortified storage stability tissue samples for rainbow trout (n = 3) analyzed at 5 and 7.5 month periods were 78 +/- 5.1 and 68 +/- 2.4%, respectively. Percent recoveries of NIC from fortified storage stability tissue samples for channel catfish (n = 3) analyzed at 5 and 7.5 month periods were 88 +/- 13 and 76 +/- 21%, respectively.     

Matrix solid phase dispersion isolation and liquid chromatographic determination of oxytetracycline in catfish (Ictalurus punctatus) muscle tissue

A method for isolation and liquid chromatographic determination of oxytetracycline in catfish (Ictalurus punctatus) muscle tissue is presented. Blank control and oxytetracycline-fortified fish muscle tissue samples (0.5 g) were blended with octadecylsllyl (C18, 40 microns, 18% load, endcapped) derivatized silica packing material (2 g) containing 0.05 g each of oxalic acid and disodium ethylenediaminetetraacetate. A column made from the C18/fish tissue matrix was first washed with hexane (8 mL), following which the oxytetracycline was eluted with acetonitrile-methanol (1 + 1, v/v) containing 0.06% w/v each of butylated hydroxyanisole and butylated hydroxytoluene. The eluate contained oxytetracycline analyte that was free from interfering compounds when analyzed by liquid chromatography with UV detection (photodiode array set at 365 nm). Standard curves for oxytetracycline isolated from fortified samples were linear (0.998 +/- 0.002) with an average absolute percentage recovery of 80.9 +/- 6.6% for the concentration range (50, 100, 200, 400, 800, 1600, and 3200 ng/g) examined. The interassay variability was 11.3 +/- 5.2% with an intra-assay variability of 1.1%.     

Matrix solid phase dispersion isolation and liquid chromatographic determination of sulfadimethoxine in catfish (Ictalurus punctatus) muscle tissue

A method for the isolation and liquid chromatographic determination of sulfadimethoxine in catfish (Ictalurus punctatus) muscle tissue is presented. Blank control and sulfadimethoxine-fortified fish muscle tissue samples (0.5 g) were blended with octadecyisilyl (C18, 40 micrograms, 18% load, endcapped) derivatized silica packing material. A column made from the C18/fish tissue blend was first washed with hexane (8 mL), following which the sulfadimethoxine was eluted with dichloromethane (8 mL). The eluant contained sulfadimethoxine analyte that was free from interfering compounds when analyzed by liquid chromatography with UV detection (photodiode array, 270 nm). Standard curves for sulfadimethoxine isolated from fortified samples were linear (0.999 +/- 0.001) with an average relative percentage recovery of 101.1 +/- 4.2% for the concentration range (50, 100, 200, 400, 800, and 1600 ng/g) examined using sulfamethoxazole as the internal standard. The interassay variability was 10.7 +/- 8.2% with an intra-assay variability of 2.2%.     

Multiresidue matrix solid phase dispersion (MSPD) extraction and gas chromatographic screening of nine chlorinated pesticides in catfish (Ictalurus punctatus) muscle tissue

A multiresidue technique for extraction and gas chromatographic screening of 9 insecticide (lindane, heptachlor, aldrin, heptachlor epoxide, p,p'-DDE, dieldrin, endrin, p,p'-TDE, and p,p'-DDT) residues in catfish (Ictalurus punctatus) muscle tissue is presented. The 9 insecticides, plus dibutyl chlorendate internal standard, were fortified into catfish muscle tissue (0.5 g) and blended with 2 g C18 (octadecylsilyl derivatized silica reverse-phase material). The C18/muscle tissue matrix blend was fashioned into a column by adding the blend to a 10 mL syringe barrel containing 2 g activated Florisil. The insecticides were then eluted from the column with acetonitrile (8 mL), and a portion (2 microL) of the acetonitrile eluate was then directly analyzed by gas chromatography with electron capture detection. Unfortified blank controls were treated similarly. The resultant extracts contained pesticide analytes (31.25-500 ng/g) free of interfering compounds when analyzed. Correlation coefficients for the 9 extracted pesticide standard curves (linear regression analysis, n = 5) ranged from 0.9967 (+/- 0.0018) to 0.9999 (+/- 0.0001). Average percentage recoveries (82 +/- 4.8% to 97 +/- 3.6%, n = 25 for each insecticide), interassay (5.0 +/- 2.7% to 16.9 +/- 6.5%, n = 25 for each insecticide) and intraassay (1.8 to 4.7%, n = 5 for each insecticide) variabilities were indicative of an acceptable methodology for the analysis and screening of these residues in catfish muscle tissue.     

鸭脂肪型脂肪酸结合蛋白基因（A-FABP）内含子1序列多态性分析

根据鸭A-FABPmRNA序列和鸡A-FABP基因组序列设计1对引物扩增出鸭脂肪型脂肪酸结合蛋白基因(A-FABP)内含子1序列(GenBank登录号:FJ536257),并根据扩增产物设计4对引物,利用PCR-SSCP对鸭A-FABP基因部分序列进行SNP检测,且对不同鸭群体进行群体遗传学分析.结果表明:克隆序列包含鸭A-FABP基因外显子1、2部分序列和完整的内含子1序列,与鸡A-FABP基因内含子1同源性为75.1%;经SSCP检测,发现引物P1扩增片段有3处碱基突变,引物P3扩增片段有6处碱基突变,这些突变分别产生了3种和8种基因型.在P1位点樱桃谷鸭和苏牧麻鸭偏离了Hardy-Weinberg平衡(P0.05),在P3位点3个群体均符合Hardy-Weinberg平衡(P＞0.05).同时,这些基因型在不同鸭群体中分布存在显著或极显著差异(P<0.05或P<0.01).群体遗传学分析结果显示A-FABP基因在不同鸭群体中具有丰富的单核苷酸多态性,可以进一步作为候选基因来分析其与脂肪性状相关性.     

应用人工神经网络预测马尾松毛虫的有虫面积

运用人工神经网络的原理和方法，选取与马尾松毛虫发生量相关关系密切的8个气象因子作为样本的输入特征，建立马尾松毛虫有虫面积与气象因子的BP网络模型，结果表明：所建立的BP模型，具有令人满意的拟合精度和预测精度。当隐含层神经元个数为15个，输入因子数为8个时，18组有虫面积的平均拟合精度为100％，相关系数为1．0000，2组预留有虫面积的平均预测精度为96．85％，预测准确率为100％。     

牦牛瘦素受体基因(LEPR)多态性分析

瘦素受体(LEPR)是瘦素(leptin)发挥作用的重要中介物.LEPR基因突变与人的肥胖,以及普通牛、猪等动物的脂肪沉积相关.本研究根据GenBank中普通牛(Bos tauras)LEPR基因序列设计3对引物,采用PCR-SSCP方法检测天祝白牦牛、甘南牦牛和青海牦牛(Bos grunniens)LEPR基因第4、5及8外显子及其相邻区域多态性.结果表明,牦牛LEPR基因第4外显子和第8外显子及其相邻区域分别存在多态性.对应普通牛LEPR基因序列,3类群牦牛该基因第4外显子及相邻区域检测到5处单碱基突变并形成2种等位基因A和B,第4外显子存在2处错义突变;该区域A为优势等位基因,PIC＜0.25为低度多态.3类群牦牛LEPR基因第8外显子及其相邻区域发现5处SNPs,其中第8外显子存在3处错义突变;等位基因在牦牛群体间分布不均衡,除天祝白牦牛发现6种等位基因A-F外,其余两类群牦牛仅发现3种等位基因A-C;3类群牦牛中A均为优势等位基因,该位点PIC>O.25,为中、高度多态且显著偏离Hardy-Weinberg平衡状态(P＜0.05).3类群牦牛LEPR基因第5外显子及相邻区域未发现多态性,但相对于普通牛该基因序列,在第4、5内含子区存在3处SNPs.LEPR基因第8外显子编码识别和结合相应配体的C2区,牦牛LEPR基因存在较丰富的多态性,可作为其调控性状的潜在分子标记位点.