抗粘液病毒基因（Mx）S631N多态位点与白耳鸡免疫性状和生产性能的关联分析

通过PCR-RFLP方法检测抗粘液病毒基因(Mx)S631N多态位点的等位基因A和G在白耳鸡群体中的分布;通过选配组建三种不同基因型群体(AA,AG,GG),免疫后分别于35日龄、42日龄、49日龄、70日龄和100日龄采用血凝和血凝抑制方法测定不同基因型个体的禽流感(AI)和新城疫(ND)抗体滴度,并测定不同基因型个体的90日龄体重、开产日龄和72周龄产蛋数;分析该多态位点与免疫性状和生长性状的关联.结果表明,在白耳鸡群体中Mx基因S631N多态位点的等位基因频率处于Hardy-Weinberg平衡状态(P>0.05),3种不同基因型频率分别为0.233(AA),0.517(AG)和0.250(GG);在测定的日龄内不同基因型个体的AI抗体滴度和ND抗体滴度水平呈现出类似的增长和消减变化,在多个测定日龄内从基因型个体的AI抗体滴度和ND抗体滴度水平显著高于AG和GG基因型个体(P<0.05);关联分析结果表明,该多态位点与49日龄时的ND抗体滴度峰值和70日龄时的AI抗体滴度峰值间均存在显著关联(P<0.05),但不同基因型个体间的90日龄平均体重、开产日龄和72周龄产蛋数差异均不显著(P>0.05).研究结果证实Mx基因S631N多态位点可以作为提高抗病力的重要候选标记位点.     

鸡卵细胞卵黄生成受体基因(ovr)SNPs检测及其与蛋用性状的关联分析

以6个鸡(Gallus gallus)品种混交群体390个个体为研究材料,采用PCR-SSCP、PCR-RF-SSCP及测序的方法进行鸡卵细胞卵黄生成受体(occyte vitellogenesis receptor,ovr)基因内含子单核苷酸多态性(SNPs)位点筛选,并与鸡群蛋用性状进行关联分析.结果表明,在鸡ovr基因中发现内含子2T3967G,内含子7C8099T和A8296G,内含子9A8839T和G9084A共5个突变位点;其中内含子2HinfⅠ突变位点与开产日龄和蛋壳厚度相关极显著(P＜0.01),与产蛋量、蛋重和蛋黄比例相关显著(P＜0.05);3种基因型中订个体在开产日龄、平均产蛋量、蛋重、蛋黄比例等方面都优于其它基因型个体.研究结果证实了ovr基因对鸡的产蛋性能和蛋品质性状有重要的影响.     

主要组织相容性复合体(MHC)B-LβⅡ基因Hin1Ⅰ位点多态性对鸡免疫功能的影响

鸡主要组织相容性复合体(MHC)基因具有高度多态性和稳定性,,与机体抗病性和免疫应答密切相关,是家禽抗病育种研究中的优选标记基因.本研究采用PCR-RFLP法,分析170只良凤青脚麻鸡(Gallus gallus)MHC B-Lβ(MHC classⅡ)基因第2外显子Hin1 Ⅰ位点的多态性及其对鸡部分免疫性状(淋巴细胞活性,新城疫(ND)抗体浓度,γ干扰素(IFN-γ)、白介素4(IL-4)和白介素12(IL-12)含量)的影响,探讨MHC B-LβⅡ基因第2外显子多态性及其对鸡免疫功能的影响.结果表明:MHC B-LβⅡ基因第2外显子经Hin1 Ⅰ酶切后检测到A、B两种等位基因和AA、AB、BB 3种基因型,其等位基因频率分别为0.553和0.447,基因型频率分别为0.235、0.636和0.129;AA、AB基因型个体淋巴细胞活性、新城疫抗体浓度、IFN-γ和IL-4含量高于BB基因型；IL-12含量在3种基因型间无明显差异.推测MHC B-LβⅡ基因第2外显子Hin1 Ⅰ位点多态性对鸡免疫功能有一定影响,可作为鸡抗病力性状的一个候选基因.     

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.     

郏县红牛跨膜蛋白18基因(TMEM18)SNPs检测及其与生长性状的关联分析

跨膜蛋白18基因(Transmembrane protein 18,TMEM18)在神经系统表达.全基因组关联分析(GWAS)表明,该基因的突变会影响人类肥胖和Body Mass Index(BMI)值变化.本研究采用DNA池测序技术检测了郏县红牛(Bos taurus) TMEM18基因5个外显子和部分内含子区单核苷酸多态(single nucleotide polymorphism,SNP)位点,通过PCR-RFLP技术对所发现的SNP位点进行分型验证,然后分析不同基因型与郏县红牛生长性状的关联性.结果表明,在郏县红牛TMEM18基因上共发现了3处新的SNPs:NW_003099175.1∶g.2303G＞A、g.3835 G＞A和g.3865 A＞G(aa.Gly＞Ser).关联分析结果表明,郏县红牛TMEM18基因g.2303G＞A位点AA型个体体高、体长、胸围和体重显著大于GG型(P＜0.05),郏县红牛TMEM18基因g.3835 G＞A位点AG型个体胸围和体重显著大于AA型(P＜0.05).研究结果提示,这两个位点可作为郏县红牛品种体尺和体重生长性状选择的分子育种候选标记.     

CAST基因5'调控区的多态性与鸡肉质性状的关联性分析

为探讨钙蛋白酶抑制蛋白(Calpastatin,CAST)基因5'调控区的多态性与鸡肉质性状的关系,本研究以中国农业科学院家禽研究所选育的F、D、Y、B和S3等5个优质肉鸡(Gallus gallus)品系为试验素材,利用聚合酶链式反应限制性片段长度多态性(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)技术检测CAST基因的SNPs,并分析其多态性与肌肉pH值、维生素B1、肉色、系水力、肌内脂肪和肌纤维密度之间的关系.结果显示,在距CDS-198位点发现1个突变位点(G→A),G-198A位点检测到3种基因型GG、GA和AA,相关分析显示,AA基因型个体的肌内脂肪和肌纤维密度显著高于GG型和GA型个体(P＜0.05),而其他肉质性状无显著差异(P＞0.05).由此可见,对于肉质性状而言,AA是最有利基因型,本研究结果初步表明CAST基因G-198A多态位点的A等位基因是提高鸡肉质性状的一个潜在的DNA标记.     

沉默鸡B-Fα基因对IL-2、IL-4和IL-6转录水平的影响

主要组织相容性复合体(major histocompatibility complex, MHC)和白介素(interleukin, IL)都是重要的免疫分子,其中MHCⅠ类分子递呈内源性抗原并启动细胞免疫,IL则是重要的免疫调节因子。关于鸡(Gallus gallus) MHCⅠ类分子(又称为B-Fα)与下游调节因子IL基因的关系尚不明确。本研究旨在应用基因沉默法研究下调B-Fα基因表达对IL-2、IL-4、IL-6转录水平的影响。首先构建沉默鸡B-Fα基因的重组慢病毒载体。根据B-Fα基因和短发夹RNA (short hairpin RNA, shRNA)序列的结构要求,筛选3段DNA序列并设计合成shRNA;利用限制性内切酶定向插入慢病毒表达质粒pLL3.7,获得重组质粒。分别将3个重组质粒转染293T细胞,均能表达标签蛋白并包装慢病毒。将3株重组病毒感染HD-11细胞,感染效率达95%以上。利用qRT-PCR进行检测分析显示,重组病毒干扰B-Fα基因表达,分别下调至39%、97%和61%。将其中干扰效果最佳的重组慢病毒(pLL-sh B-Fα-257)感染鸡源巨噬细胞系HD-11细胞,并检测IL-2、IL-4和IL-6的转录水平。结果显示,B-Fα基因表达下调的同时,IL基因转录水平受到不同程度的影响,其中IL-2表达下调(P0.01),IL-4上调(P0.05),IL-6没有明显变化。上述结果提示,应用基因沉默法可抑制B-Fα基因表达,并影响IL基因的转录水平,提示启动细胞免疫的B-Fα基因与下游调节免疫反应的细胞因子之间存在关联。本研究为深入探讨免疫反应的调控机理提供了基础资料。     

肌肉生长抑制素基因(MSTN)外显子1的多态性及其与边鸡生长性状的关联分析

为了对边鸡生长性状的标记辅助选择提供基础资料,本研究采用PCR-SSCP技术检测边鸡(Gallusgallus)及3个对照鸡(G.gallus)群体(京海黄鸡、尤溪麻鸡和Arbor Acre鸡)肌肉生长抑制素(myostatin,MSTN)基因外显子1的单核苷酸多态性,并与边鸡的生长性状进行相关性分析。结果表明,在边鸡、京海黄鸡和尤溪麻鸡中都检测到6种基因型(AA、AB、BB、BC、AC和CC),而在Arbor Acre鸡中只检测到4种基因型(AB、BC、AC和CC)。总共检测到3个多态位点(G2100A、G2109A和C2244G)。在6～8和12周龄时,BB基因型个体的体重显著高于AA基因型个体(P<0.05)。在14周龄时,BB基因型个体的体重显著高于AA和AC基因型个体(P<0.05)。在16～18周龄时,BB基因型个体的体重显著高于其余5种基因型个体(P<0.05)。研究结果提示,MSTN基因可能是影响边鸡生长性状的一个主效基因或是与之存在紧密遗传连锁的一个标记。     

农村散养家禽禽流感和新城疫免疫程序探讨

农村散养家禽虽然在整个家禽饲养行业所占份量很小,但依然普遍存在。饲养散养家禽的多为老年人以及他们在家帮带的学龄前儿童,是散养家禽容易传播的禽流感包括H5亚型、H7N9的易感人群。为切合农村实际,加强散养家禽H5+H7N9以及新城疫免疫工作,对35羽试验鸡进行了禽流感(H5 Re-8+H7N9 Re1)+新城疫同时免疫,抗体水平合格率分别达到97.14%、97.14%和94.29%,证明对农村散养家禽禽流感和新城疫同时免疫效果理想。     

磷脂酶C-zeta基因(PLCz)多态性与中国荷斯坦公牛精液性状相关性研究

睾丸特异性磷脂酶C-zeta(PLCz)在哺乳动物的精卵融合过程及胚胎发育过程中发挥重要作用,主要是激发精卵融合过程中钙波的震荡.本实验选取3个种公牛站的424头中国荷斯坦公牛个体作为实验材料,通过提取种公牛精液中的全基因组DNA,运用DNA测序、PCR-RFLP技术对PLCz基因的全序列进行了单核苷酸多态性分析.实验发现8个新的SNPs位点,其中g.27529 T＞A和g.27597 T＞C位于内含子7,g.47969 G＞A、g.48020 T＞C、g.48079 G＞A、g.48127 G＞T和g.48384 G＞T位于内含子12,而位于外显子8的多态位点g.27768 G＞C发生突变后可以导致第337位的氨基酸丙氨酸改变为脯氨酸,故而此突变为非同义突变.此外,连锁不平衡分析表明g.27529 T＞A、g.27597 T＞C和g.27768 G＞C 3个位点完全连锁,以SNP1 g.27597T＞C代表;g.47969 G＞A、g.48020 T＞C、g.48079 G＞A、g.48127 G＞T和g.48384 G＞T 5个位点完全连锁,以SNP2 g.47969 A＞B代表.通过与公牛精液品质相关性分析表明,SNP2位点与精子畸形率显著相关(P＜0.05),SNP1则与公牛精液品质无显著关联性.然而,突变体SNP1和SNP2所构建的9种单倍型组合与公牛精液品质性状显著相关,单倍型组合TTAB的射精量(P＜0.05)、鲜精密度(P＜0.01)和冻后活力(P＜0.05)显著高于其他单倍型组合,鲜精活力显著高于单倍型组合TTBB(P＜0.05),畸形率则显著低于单倍型组合CTAB(P＜0.05).结果表明,单倍型组合TTAB可以作为中国荷斯坦公牛高精液品质的有效分子标记.     

Identification of oxidation products of (-)-epigallocatechin gallate and (-)-epigallocatechin with H(2)O(2)

(-)-Epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) are two important antioxidants in tea. They also display some antitumor activities, and these activities are believed to be mainly due to their antioxidative effects. However, the specific mechanisms of antioxidant action of tea catechins remain unclear. In this study are isolated and identified two novel reaction products of EGCG and one product of EGC when they were reacted separately with H(2)O(2). These products are formed by the oxidation and decarboxylation of the A ring in the catechin molecule. This study provides unequivocal proof that the A ring of EGCG and EGC may also be an antioxidant site. This study also indicates an additional reaction pathway for the oxidation chemistry of tea catechins.     

Excretion of (14)C-fumonisin B(1), (14)C-hydrolyzed fumonisin B(1), and (14)C-fumonisin B(1)-fructose in rats.

14C-Fumonisin B(1) (FB(1)) was produced by Fusarium proliferatum M-5991 in modified Myro liquid medium and purified to >95% purity with a specific activity of 1.7 mCi/mmol. Nine male and nine female F344/N rats were each dosed by gavage with 0.69 micromol of (14)C-FB(1), (14)C-hydrolyzed FB(1), or (14)C-FB(1)-fructose/kg body weight. Urinary excretion of (14)C-FB(1) and (14)C-FB(1)-fructose was 0.5% and 4.4% of the total dose, respectively, and was similar between male and female rats. Urinary excretion of (14)C-hydrolyzed HFB(1) was significantly greater (P > 0.05) in female rats as compared with male rats (17.3% vs 12.8% of the total dose, respectively). There were no significant (P > 0.05) differences in biliary excretion of the three fumonisin compounds with a mean of 1. 4% of the dose excreted at 4 h after dosing. Lesser amounts continued to be excreted up to 9.25 h after dosing. Although biliary excretion of the (14)C-FB(1), (14)C-hydrolyzed FB(1), and (14)C-FB(1)-fructose was similar, increased urinary excretion of the (14)C-hydrolyzed FB(1) as compared to (14)C-FB(1) and (14)C-FB(1)-fructose indicated a greater absorption of the hydrolyzed form.     

Chemoenzymatic synthesis of homochiral (R)- and (S)-karahanaenol from (R)-limonene

Terpinolene oxide, a monoterpene belonging to the p-menthane group, is easily derived from naturally abundant (R)-limonene. It was isomerized with montmorillonite clay catalyst to karahanaenone (2,2, 5-trimethylcyclohept-4-en-1-one) by ring enlargement. The enantiomers of the corresponding alcohol, karahanaenol (2,2, 5-trimethylcyclohept-4-en-1- ol), known for their individual organoleptic properties, were resolved through Pseudomonas cepacia lipase mediated enantiospecific alcoholysis of its acetate derivative.     

(三唑基-~(14)C-)粉锈宁的标记合成

本文报道了(三唑基-14C)-粉锈宁的制备。由14C-甲酸和重碳酸氨基胍形成(5-14C)-3-氨基-1,2,4-三唑,再经重氮化脱氨得到(5-14C)-1,2,4-三唑,最后再与对氯酚和二氯片呐酮反应得到(三唑基-14C)-粉锈宁。放化收率为26％(从甲酸-14C计),放化纯度大于95％。     

Theoretical speciation of ethylenediamine-N-(o-hydroxyphenylacetic)-N'-(p-hydroxyphenylacetic) acid (o,p-EDDHA) in agronomic conditions

The presence of ethylenediamine-N-(o-hydroxyphenylacetic)-N'-(p-hydroxyphenylacetic) acid (o,p-EDDHA) as the second largest component in commercial EDDHA iron chelates has recently been demonstrated. Here is reported the speciation of o,p-EDDHA by the application of a novel methodology through the determination of the complexing capacity, protonation, and Ca(2+), Mg(2+), Cu(2+), and Fe(3+) stability constants. The pM values and species distribution in solution, hydroponic, and soil conditions were obtained. Due to the para position of one phenol group in o,p-EDDHA, the protonation constants and Ca and Mg stability constants have different values from those of o,o-EDDHA and p,p-EDDHA regioisomers. o,p-EDDHA/Fe(3+) stability constants are higher than those of EDTA/Fe(3+) but lower than those of o,o-EDDHA/Fe(3+). The sequence obtained for pFe is o,o-EDDHA/Fe(3+) >/= o,p-EDDHA/Fe(3+) > EDTA/Fe(3+). o,p-EDDHA/Fe(3+) can be used as an iron chelate in hydroponic conditions. Also, it can be used in soils with limited Cu availability.     

Binding studies of oxovanadium(V) with ovalbumins (OA)

The effect of protein oxovanadium(V) ion concentration and pH on the ratio of diffusion current (id/id₀) was studied in vanadium(V) ovalbumin-S and denatured ovalbumin systems. In both the cases marked decrease in diffusion current was observed at the respective pH values, indicating that binding takes place with cationic groups of the proteins. The binding sites (n) were found to be pH dependent. The uniformity of logK and ΔG ⁰ value at all pH values indicated the involvement of same sites in interaction. Furthermore, the linear scatchard plots in both the systems supported the involvement of single class of independent sites in oxovanadium(V) anion interaction. The difference in binding sites (n) has been attributed to the folded structure of ovalbumin-S while unfolded one of denatured ovalbumin.     

Effect of pH on interaction between As(III) or As(V) and manganese(IV) oxide

The efficiency of As(III) oxidation by MnO₂, and retention of oxidation products varies with system pH. Maximum retention by hydrous Mn(IV) oxide occurs at pH < 5, declining at higher pH to about half total As at pH 10. The adsorption capacities of pyrolusite and cryptomelane at pH ～6.5 for As(V) species were 10 and 25 mmol kg^?1, respectively. HMO surface saturation (～10 mmol kg^?1) was reached with equilibrium As(V) levels of 5 to 8 × 10^?6 M but this was supplemented at higher levels by an absorption process where uptake increased linearly with concentration (e.g., 68 mmol kg^?1 with 2 × 10^?5 M As(V)). Added As(III) was avidly oxidized and most product retained at pH 3. At higher pH increasing amounts of As(III) remained unoxidized due to initial reactions apparently blocking access to internal pores. Added Na⁺ reduced the amount of As retained by the HMO, with the phosphate salt having a significant effect. Extraction studies confirmed that most As could be released by exposure to reducing agents or chelating agents (EDTA). The environmental significance of the results has been considered.