pGH和IGF-Ⅰ双基因共表达载体的构建及转双基因猪的获得与检测

旨在构建可高效表达pGH基因和IGF-Ⅰ基因的双基因共表达载体,制备转双基因(pGH+IGF-Ⅰ)猪,以期探索pGH基因和IGF-Ⅰ基因对猪生长发育的影响,为节粮型高瘦肉率新品种猪的培育奠定理论基础。从长白猪耳样中提取总RNA,经反转录RT-PCR获得pGH基因不含终止密码子的编码序列和IGF-Ⅰ基因完整的编码序列,经酶切连接克隆至pc DNA3.1(+)真核表达载体上,构建pc DNA3.1(+)-pGH-IGF-Ⅰ双基因共表达载体。将其转染PK15细胞,Q-PCR检测2个目的基因在PK15细胞中的表达情况。将构建的双基因共表达载体用纳米材料包裹后转染长白猪精子,采用精子载体法制备转双基因猪。PCR及测序鉴定转双基因阳性个体,Q-PCR检测2个目的基因在转双基因猪体内的表达情况。PCR及测序鉴定追踪检测转双基因猪体内pGH基因和IGF-Ⅰ基因的稳定情况。RT-PCR及测序结果表明,成功克隆了长白猪的pGH基因和IGF-Ⅰ基因的编码序列。酶切和测序分析表明成功构建了双基因真核共表达载体,转染PK15细胞后,Q-PCR检测表明,pGH基因和IGF-Ⅰ基因均在mRNA水平成功表达。母猪妊娠获得13头仔猪,经PCR及测序检测,其中4头仔猪为转双基因阳性,转双基因阳性率为30.76%。Q-PCR检测外源pGH基因与IGF-Ⅰ基因在转双基因猪体内成功表达。1~7月龄均可检测到外源pGH基因与IGF-Ⅰ基因,证明2个外源基因在转双基因猪体内稳定存在,并未随着生长而丢失。在转双基因公猪的精液中均能检测到2个外源基因,证明外源基因存在稳定传代的可能。     

Molecular characterization of Ascaridia galli from Bangladesh and development of a PCR method for distinguishing A. galli from Heterakis spp.

We analyzed the nuclear ribosomal internal transcribed spacer (ITS) 1 and ITS2 sequences for Bangladesh isolates of Ascaridia galli, and we determined that the sequences were unreliable as molecular markers for distinguishing A. galli from other Ascaridia species, because the sequences showed high identity with that of A. columbae. However, the ITS1 sequences were available for designing PCR primers distinguishable between Ascaridia galli and Heterakis spp. Bangladesh isolates of A. galli constituted a monophyletic clade along with other geographical isolates in the cytochrome c oxidase subunit I (COI) phylogenetic tree, however, we could not clarify the phylogenetic relationships between A. galli and other Ascaridia spp., because their available sequences in GenBank were very few. The developed PCR method using DNA from A. galli and Heterakis spp. eggs would enable differential diagnosis of the individual infections in the future.     

绵羊BMP15基因B2突变可视化检测技术的建立

骨形态发生蛋白15（bone morphogenetic protein 15,BMP15）基因突变对绵羊排卵率、产仔数以及繁殖力有很大影响,本研究旨在建立快速可视化检测BMP15基因B2突变的技术。将改良的扩增阻滞突变系统（amplification refractory mutation system,ARMS）与核酸染料SYBR Green Ⅰ结合,针对BMP15基因B2突变设计ARMS特异性引物,使引物下游3'端的最后一个碱基为A,并在下游引物3'端的第3位碱基处设计额外的错配,以提高引物的特异性;经ARMS PCR扩增后,向产物中加入SYBR Green Ⅰ,通过肉眼观察PCR管内的颜色变化来检测BMP15基因的B2突变。经测序发现所采集的样本均为野生型,因此,利用重叠延伸PCR构建BMP15基因B2突变型模板,测序结果显示BMP15基因B2突变型模板构建成功。ARMS PCR结果显示,ARMS特异性引物能够只扩增突变型模板,而不会扩增野生型模板,且扩增效果良好。ARMS PCR扩增后加入SYBR Green Ⅰ发现已知野生型模板与阴性对照一致,呈SYBR Green Ⅰ原始颜色橙黄色,而已知突变型模板呈亮绿色,且色差明显,肉眼可辨。用建立的可视化检测BMP15基因B2突变的技术检测50个小尾寒羊基因组DNA （随机向其中20个样本中加入构建的BMP15基因B2突变型模板）,将可视化检测的突变型样本编号与混合样本时记录的编号对比,结果表明该技术能够准确检测绵羊BMP15基因的B2突变,且准确率高达100%。将构建的BMP15基因B2突变型的模板进行梯度稀释,对本试验可视化检测体系的灵敏度进行检测,发现ARMS可视化检测技术的灵敏度达到0.006 ng/μL。因此,本研究建立的技术能够可视化地检测绵羊BMP15基因B2突变,且准确率高,有望为高繁殖力绵羊的选育提供技术支持。     

绿壳蛋鸡IGF-Ⅰ基因SNPs分析及其与末期产蛋性能的相关性研究

本研究以胰岛素样生长因子Ⅰ(insulin-like growth factors-Ⅰ, IGF-Ⅰ)基因作为候选基因,运用PCR-RFLP法检测基因变异,并分析其变异与鸡末期产蛋性能的相关性。以绿壳蛋鸡(n=113)作为试验材料,对IGF-Ⅰ基因5'调控区进行遗传多样性研究。研究结果表明,该调控区扩增产物经PstⅠ酶切后出现AA、AB、BB共3种基因型,经χ²检验绿壳蛋鸡在IGF-Ⅰ位点上符合Hardy-Weinberg平衡状态(P＞0.05)。相关分析表明,IGF-Ⅰ基因5'调控区PstⅠ位点对于产蛋末期(14和15月龄)的产蛋性能没有显著影响(P＞0.05)。     

Phylogeography and conservation of the populations of Zootoca vivipara carniolica

The lizard Zootoca vivipara has both oviparous and viviparous populations which belong to distinct clades. This study aims to elucidate the geographic distribution, phylogeography and conservation priority of the oviparous subspecies, Zootoca vivipara carniolica, that has recently been identified in Slovenia. We studied seven Slovenian populations, two northeastern Italian populations and five northwestern Italian populations. The seven cytochrome b haplotypes that we identified from the Slovenian and Italian oviparous populations form a monophyletic basal clade. The high homogeneity of the Slovenian and northeastern Italian populations (nucleotide diversity π=0.06%) is a striking contrast with the pattern observed in northwest Italy where each of the five populations studied presents a private haplotype (π=1.03%). The results obtained suggest that all the extant lineages of Z. v. carniolica have originated in an Italian refuge. The reproductive and phylogenetic distinctiveness of Z. v. carniolica clearly justifies conservation of its populations, especially in Italy where the highest genetic diversity and the most ancestral haplotype were observed.     

猪传染性胸膜肺炎重组亚单位疫苗诱导小鼠免疫保护效果的观察

重组表达猪传染性胸膜肺炎放线杆菌6种主要毒力因子基因:apxⅠ、apxⅡ、apxⅢ、apxⅣ、apfa和omp,以重组蛋白rApxⅠ、rApxⅡ、rApxⅢ和rOMP组合免疫小鼠作为试验I组,重组蛋白rApxⅠ、rApxⅡ、rApxⅢr、OMPr、ApxⅣ和rApfa组合免疫小鼠作为试验Ⅱ组,PBS为对照组,分3次免疫小鼠,采用背部皮下多点注射,每次间隔2周,免疫剂量为0.2 mL/只,3免后1周分别以APP1型菌Shope 4074株(5×109cfu)和APP2型菌S1536株(5×1010cfu)进行攻毒试验。通过小鼠保护率与抗体效价的相关性研究、肺部病理变化及肺脏细菌的分布情况等指标进行综合评价。结果显示,试验Ⅰ组4种重组蛋白特异性抗体水平显著高于其他两组(P<0.05),对APP1型菌攻毒的保护率(9/10)明显高于试验Ⅱ组(5/10)和对照组(0/8),小鼠的免疫保护率与抗体效价之间存在显著正相关;且该组对APP2型菌攻毒的保护作用(无肺脏损伤)也明显优于其它两组(典型肺部损伤)。间接免疫荧光试验表明试验Ⅰ组对肺脏细菌的清除效果也明显优于其他两组。本试验揭示试验Ⅰ组对不同血清型APP攻击能够提供很好的交叉保护作用,从而为猪传染性胸膜肺炎新型疫苗的研制提供参考。     

Identification of mutations in the para sodium channel of Bemisia tabaci from Crete, associated with resistance to pyrethroids

We investigated the mechanisms of resistance to α-cypermethrin in a Q biotype, highly resistant Bemisia tabaci strain (GRMAL-RP) isolated from Crete. Cytochrome P450-dependent monoxygenase activity with the substrate ethoxycoumarin, and carboxylesterase activity with the substrates α-naphthyl-acetate, β-naphthyl-acetate, and para-nitrophenol acetate were substantially elevated in the GRMAL-RP, compared to the susceptible SUD-S strain, while glutathione-S-transferase activity with the substrate 1-chloro-2,4-dinitrobenzene was not different. The metabolic inhibitors piperonyl butoxide and S,S,S-tributyl phosphorotrithioate synergised cypermethrin toxicity in the GRMAL-RP strain, however, mortality was still lower than that of the susceptible strain, indicating the presence of an additional resistance mechanism. Analysis of the sequence of the IIS4-IIS6 region of the para sodium channel gene of the GRMAL-RP strain revealed two amino acid replacements compared to that of the SUD-S susceptible strain. One is the leucine to isoleucine substitution at position 925 (L925I) previously implicated in B. tabaci pyrethroid resistance and the other is a novel kdr resistant mutation for B. tabaci, a threonine to valine substitution at position 929 (T929V). Genotype analysis showed that the L925I and T929V were present in all GRMAL-RP males tested, at an approximately 1:1 frequency, but never in combination in the same haplotype.     

Parasitization by Cotesia plutellae enhances detoxifying enzyme activity in Plutella xylostella

Insecticidal tests using diazinon showed that the mortality of Plutella xylostella larvae parasitized by Cotesia plutellae was reduced by 4.6-fold compared to that of the nonparasitized hosts. The use of chemicals with synergistic effect to insecticides in toxicity assay helps to elucidate the kind of enzyme involved in lowering insect mortality. Synergism of diethyl maleate and piperonyl butoxide with diazinon resulted to 2.4- and 1.9-fold increase, respectively, in susceptibility of parasitized larvae compared to those of nonparasitized larvae. These results indicated the possibility that the decrease in susceptibility to diazinon was due to the elevated activities of glutathione-S-transferase (GST) and cytochrome P450 monooxygenase (CYP), respectively. The GST activities in parasitized larvae were significantly higher than those of nonparasitized ones starting from three days post-parasitization until emergence of parasitoid larva. High GST activities during late parasitism could be attributed to both enzyme activities toward diazinon of parasitized P. xylostella larva itself and C. plutellae larva inside larval host. High GST activity one day after parasitization, although statistical significance was not detected, was caused by polydnavirus (PDV) and the venom of C. plutellae not by parasitoid larvae. Artificial injection of PDV plus venom demonstrated that the resulting increase in GST activity is similar to the increase brought by parasitization. High CYP activity after 3 days post-parasitization in parasitized larva was attributed mainly to the activity of parasitoid larva. Carboxylesterase activity in the parasitized host remained at a high level, while that in the nonparasitized host decreased slightly as pupation approaches. On the other hand, acetylcholinesterase activity also remained constant after parasitization until larval emergence, while that of the nonparasitized hosts decreased gradually as the host larvae approach pupation. These results were supported by inhibition tests using diazoxon in vitro.     

Insulin like growth factor-1 protects c17.2 neural stem cells against radiation-induced injury

AIM:To investigate the protective effect of insulin like growth factor-1 (IGF-1) against radiation-induced injury in c17.2 neural stem cells.METHODS:c17.2 neural stem cells were cultured in vitro and identified by nestin immunohistochemistry and X-gal staining.The IGF-1 adenoviral vector was transfected into c17.2 neural stem cells.The expression of IGF-1 was detected by IGF-1 immunohistochemistry.Necrosis and apoptosis of c17.2 neural stem cells were induced by radiation at doses of 4 Gy,6 Gy and were detected by flow cytometry and TUNEL test.The effect of IGF transfection on the degree of necrosis and apoptosis in these cells was also observed.RESULTS:c17.2 cells were identified by nestin and X-gal staining.Immunohistochemistry verified the expression of IGF-1 in the c17.2 neural stem cells transfected with adenoviral vector.At the same doses of radiation,the rates of apoptosis and necrosis in c17.2 neural stem cells transfected with IGF-1 were lower than those in the cells without IGF-1 adenoviral vector transfection.CONCLUSION:The rate of necrosis and apoptosis of c17.2 neural stem cells induced by radiation is decreased by transfection of IGF-1 adenoviral vector.IGF-1 shows a protective effect on c17.2 cells.     

Cytochrome P-based resistance mechanism and pyrethroid resistance in the field Anopheles albimanus resistance management trial

The relative rates of cytochrome P⁴⁵⁰ selection in southern Mexican Anopheles albimanus populations were investigated during a 3 years indoor residual house spraying intervention with a pyrethroid (PYR) or DDT, a mosaic of organophosphate (OP)-PYR, and the annual rotation of OP-PYR-carbamate (CARB). This insecticide resistance mechanism, initially evenly spread in the mosquito population, correlated with PYR resistance during the second treated year, when cytochrome P⁴⁵⁰ contents increased in most villages of the PYR, rotation and mosaic schemes. However, by the third year, mean cytochrome P⁴⁵⁰ contents declined to susceptible levels in mosquitoes of the rotation and one mosaic group but not in the PYR-treated villages. In DDT-treated villages, a continuous decrement of cytochrome P⁴⁵⁰ levels occurred since the first treatment year, and susceptible levels were observed at the end of the intervention. Most correlations of cytochrome P⁴⁵⁰ levels and PYR resistance were lost during the third year, indicating that another mechanism evolved in resistant mosquito populations.