基于CRISPR/Cas9技术建立IGF2R基因修饰PK-15细胞

作为我国特色的地方种质资源,藏猪对高海拔、低氧、强紫外线辐射等恶劣环境具有良好的适应性,前期研究发现藏猪的IGF2R（insulin-like growth factor 2 receptor,IGF2R）基因在其内含子上有1段274 bp的缺失,且该缺失为藏猪独有的变异,关于IGF2R是否参与藏猪对低氧等恶劣环境的适应性调节的相关研究尚未报道。为了建立IGF2R基因274 bp修饰的猪细胞系,研究IGF2R基因对藏猪低氧耐受特性的影响,通过CRISPR/Cas9技术建立基因修饰PK-15细胞系,并通过PCR、实时荧光定量PCR（real time quantitative PCR,RT-qPCR）、蛋白免疫印迹（Western-blot）、免疫荧光（immunofluorescence,IF）等方法鉴定其分子特征,随后分别利用CCK-8和RT-qPCR 、Western-blot对基因修饰细胞进行活力和凋亡基因检测。结果显示,已成功构建IGF2R基因274 bp修饰的猪细胞系,IGF2R基因表达量极显著下调（P < 0.01）。基因修饰细胞活力在72 h时极显著上升（P < 0.01）,并且含半胱氨酸的天冬氨酸蛋白水解酶9 （cysteinyl aspartate specific proteinase,Caspase9）和BCL-2关联 X （BCL2-associated X,BAX） 蛋白的水平显著下调（P < 0.05）,并且B淋巴细胞瘤-2（B-cell lymphoma-2,BCL2）基因的mRNA水平极显著上升（P < 0.01）。成功构建了IGF2R基因精确修饰的PK-15细胞系,并初步进行了功能研究,为研究IGF2R基因功能提供细胞模型,并为后续基因编辑猪的制备奠定基础。     

Identification and quantification of benzimidazole resistance polymorphisms in Haemonchus contortus isolated in Northeastern Brazil

Haemonchus contortus is the most prevalent nematode in Brazil. The objective of this study was to select 6 populations of H. contortus of known or suspected benzimidazole resistance status and characterize these using quantitative real-time polymerase chain reaction (qPCR) for single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A in the β-tubulin isotype 1 gene. qPCR was performed using DNA from a pool of 10 adult male H. contortus from a single animal per farm. Faecal egg count reduction test (FECRT) and egg hatch test (EHT) were used to determine the resistance status. Samples were obtained from 6 farms located in 5 counties in the Ceará State: Tauá, Boa Viagem, Quixadá, Santa Quitéria and Solonópole. The inbred-susceptible-Edinburgh (ISE) isolate was used as reference for comparative purposes in the qPCR. Benzimidazole resistance was detected by FECRT on all farms with efficacy values ranging from 0 to 51%. EC50 values as determined by EHT were all above 1.49 μg/ml. High frequencies of the resistant SNPs F200Y and F167Y alleles were detected but no resistance was detected at SNP E198A. Our results suggest that the SNPs F167Y and F200Y are both important for benzimidazole resistance in the studied populations.     

糖对长白猪精液NAGaseⅡ的抑制机理

以长白猪精液为材料,采用经典的纯化方法获得N-乙酰-β-D-氨基葡萄糖苷酶（EC 3.3.1.52,NAGase）的2种同工酶,命名为：NAGaseⅠ和NAGaseⅡ。其中NAGaseⅡ的比活力为358.21U/mg,纯化倍数为6.37倍。以海藻糖、D-甘露糖、蔗糖、葡萄糖为效应物,研究其对该酶活性的影响。结果表明：在特定浓度范围内,海藻糖对酶活力基本没有影响;D-甘露糖、蔗糖、葡萄糖对该酶均有一定的抑制作用;D-甘露糖对酶的抑制作用表现为非竞争型,抑制常数KI为11.94mmo/L;蔗糖对酶的抑制作用表现为竞争型,抑制常数KI为0.56mmol/L;葡萄糖对酶的抑制作用表现为混合型,抑制常数KI和KIS分别为0.35mol/L和64.29mmol/L。     

Cloning and sequence analysis of nine novel MYB genes in Taxodiaceae plants

Myeloblastosis(MYB) is one of the largest transcribed factor families in plants. To gain an overall picture of the evolution of MYB genes in relict plants, we cloned nine novel MYB genes in Taxodiaceae plants(Taxodium distichum, Taxodium ascendens, Cryptomeria japonica var. Sinensis, Cryptomeria japonica cv. Araucarioides, Cryptomer Japonica, Metasequoia glyptostroboides, Cunninghamia lanceolata, Taiwania cryptomerioides and Glyptostrobus pensilis). The deduced amino acid sequences for MYBs showed that the nine MYB proteins contained two DNA binding domains. The first domain is from amino acid position 29 to 78, wherein three tryptophanes at 33, 53 and 73 were separated by 19 amino acids, respectively. The second domain is from amino acid position 82 to 127, wherein three tryptophanes at 86, 105 and 124 were separated by 18 amino acids, respectively, whereas the first tryptophane at amino acid position 86 is replaced by a phenylalanine. The characterization of these conserved domains at nine MYBs indicated that they all belong to the R2R3-MYB group. The secondary structure analysis showed that α-helix and β-turn are the major motifs of the predicted secondary structure of MYBs. The three dimensional model of each MYB protein showed that the structure is like clip, making it more flexible and mobile. The similarities between the nine MYB proteins in Taxodiaceae were calculated. The highest identical value of 99% is between CjsMYB, CjMYB and CjaMYB, whereas the lowest value of 82% is between TaMYB and ClMYB. According to the phylogenetic tree, the distances between different genera were relatively large whereas those within genera were relatively small. As expected, accessions of the same genus formed a subgroup before being grouped with other genera.     

开花麻竹叶绿素荧光参数测定

叶绿素荧光是研究光合作用的有效探针。为了探讨开花对麻竹光合作用的影响,应用PAM-100分别测定了开花与未开化麻竹的叶绿素荧光参数。结果表明,随着光强的升高[0~2 000μmol/（m^2·s）],在开始阶段[0~200μmol/（m^2·s）]麻竹叶片的NPQ、Y（NPQ）、Y（ND）、ETR（Ⅱ）和ETR（Ⅰ）均迅速升高,Y（Ⅱ）和Y（Ⅰ）却迅速下降,之后变化缓慢并趋于平稳,而Y（NA）和Y（NO）几乎一直维持平稳;其中未开花麻竹的NPQ、Y（Ⅰ）和ETR（Ⅰ）均高于开花麻竹,Y（Ⅱ）、Fv/Fm和ETR（Ⅱ）无明显差异。在本实验培养光照下[230μmol/（m2·s）],未开花麻竹的NPQ、Y（Ⅰ）和ETR（Ⅰ）也均高于开花麻竹。由此表明,开花引起麻竹PSⅠ的Y（Ⅰ）和ETR（Ⅰ）以及PSⅡ的NPQ降低,这意味着开花麻竹的光保护能力下降,使得PSⅠ受体侧电子积累,导致光合效率下降。     

油橄榄叶提取物对海洛因依赖小鼠肺组织结构及IL-1β和TNF-α表达的影响

将100只健康小鼠皮下注射海洛因(10mg/kg)40d,然后分别用不同剂量的(50、100、200mg/kg)油橄榄叶提取物(OLE)进行皮下注射治疗,观察肺脏组织结构的变化,检测肺脏中白介素-1β(interleukin-β,IL-β)和肿瘤坏死因子-α(tumor necrosis factor-A,TNF-α)的表达情况.结果表明:与模型对照组相比,OLE使海洛因依赖小鼠肺泡直径、肺泡隔厚度减小,肺脏中IL-1β和TNF-α的表达明显减弱,且呈较好的量效关系,表明OLE能够对海洛因造成的肺损害起到保护作用,其作用机制可能是OLE抑制了IL-1β和TNF-α的在肺中的表达.     

基于FPGA的Buck变换器新型DPWM技术研究

文章针对Buck型DC-DC变换器中DPWM（数字脉冲宽度调制）技术进行研究,指出传统DPWM技术不足,提出MDPWM（改进的脉冲宽度调制）技术。采用FPGA的QuartusⅡ软件进行仿真,仿真结果表明,MDPWM技术能够减少开关管的开通和关断延迟时间、减小器件的功率损耗,当负载波动时,输出电压瞬态响应快,使输出电压稳定性好。     

Evaluation of β-(1 → 3, 1 → 6)-glucans and High-M alginate used as immunostimulatory dietary supplement during first feeding and weaning of Atlantic cod (Gadus morhua L.)

Stimulation of the non-specific defence enhances the disease resistance and growth, and has good potentials as a measure for increased microbial control in juvenile production of marine fish and shellfish. So far, the most commonly used immunostimulants are β-(1 → 3, 1 → 6)-glucans, and in this study the stimulatory potential of a β-(1 → 3, 1 → 6)-glucan of marine origin, the storage polysaccharide from the marine diatom Chaetoceros mülleri, was examined. The glucan (chrysolaminaran) was extracted from cultures of C. mülleri, and used as a dietary supplement in two first feeding experiments with larvae of Atlantic cod Gadus morhua L. In one experiment the microalgal glucan was compared to the commercial yeast-glucan product MacroGard®, and in the other to an alginate with a high content of mannuronic acid (High-M alginate) isolated from Durvillaea antarctica. The stimulants were given via rotifers, and weaning to formulated feed was initiated at day 17 or 18 after hatching. The survival ± SEM at day 27 after hatching was 24.5 ± 2.0%, 14.8 ± 4.5% and 13.1 ± 1.4% for the groups fed C. mülleri-glucan, yeast glucan and for the control, respectively, in the first experiment. The group fed C. mülleri-glucan group had higher survival compared to the control (P < 0.05) group, whereas the yeast glucan had no positive effect on the survival (p > 0.05). The dry weights of the groups at day 27 were low, with 203.2 ± 52.2, 165.2 ± 43.4 and 198.5 ± 58.1 μg per larva for the C. mülleri-glucan, yeast glucan and control groups, respectively. In the second experiment the survival in the period of feeding formulated feed (days 18-30) were 44.6 ± 4.3%, 44.7 ± 1.3%, and 33.8 ± 4.1% survival for the C. mülleri-glucan, High-M alginate and control group, respectively. The cod larvae fed C. mülleri-glucan reached an average weight of 531.6 ± 17.2 μg at day 30, which was significantly higher (p < 0.05) than the control group that had an average of 473.6 ± 3.5 μg. The larvae fed High-M alginate had an average weight of 470.3 ± 31.6 μg per larva at day 30, and not significantly different from the control (p > 0.05). The early weaning to formulated diet had detrimental effect on the growth of the larvae. In both experiments the C. mülleri-glucan group was the only group showing a positive growth rate in the period of weaning to dry feed. The microbial conditions in larval gut and water were monitored with respect to total colony forming units on Marine agar, and Vibrio- and Pseudomonas-like species on selective agars (TCBS and marine Pseudomonas Agar with CFC-supplement). The larvae were rapidly colonised after hatching, but no or weak effects of the stimulants were observed on the colonisation rates or the composition. The total CFU varied from 10¹ to 10² CFU per μg larva after initiation of the first feeding. The percentages of Pseudomonas-like bacteria increased throughout the period, whereas the levels of Vibrio-like bacteria were low and stable. The chrysolaminaran from the diatom C. mülleri was shown to be a promising candidate for use as an immunostimulatory feed additive, and which should be further explored.     

Evaluation of beta-mannanase and nonstarch polysaccharide-degrading enzyme inclusion separately or intermittently in reduced energy diets fed to male broilers on performance parameters and carcass yield

The experimental design consisted of 5 dietary treatments including a positive control (PC), a negative control (NC; with a reduction of 88 kcal/kg of AME through the starter and grower 1 phase and a reduction of 132 kcal/kg of AME in the grower 2, finisher, and withdrawal phases compared with the PC), and the NC supplemented with either β-mannanase, nonstarch polysaccharide-degrading enzymes (NSPase; cocktail carbohydrase), or β-mannanase and NSPase intermittently fed. The intermittent treatment included β-mannanase from d 1 to 21 (starter and grower 1 phases) and NSPase from d 22 to 47. Each treatment included 9 replicate pens with 35 male broilers placed per replicate (1,575 total chicks placed). The dietary program consisted of 5 dietary phases, including the starter through d 10, grower 1 through d 21, grower 2 through d 32, finisher through d 40, and withdrawal through d 47. Broilers were weighed and feed consumption determined on days of dietary changes. On d 48, following an 8-h feed withdrawal, 6 broilers from each replicate pen were removed and processed for whole bird and fat pad measurements. The reduction in energy in the NC diet reduced BW and increased mortality rate, and the inclusion of β-mannanase and NSPase separately and intermittently in the NC diet improved growth performance and reduced mortality to levels that were comparable to the PC. The NC yielded the highest cumulative mortality-corrected FCR and all enzyme inclusion treatments reduced FCR to levels comparable to the PC for the duration of the trial. The NC diet yielded the lowest processing yields and the inclusion of β-mannanase and NSPase separately and intermittently increased multiple processing parameters to a level similar to the PC. These data confirm the ability of β-mannanase and NSPase inclusion separately and intermittently to improve performance parameters in reduced-energy broiler diets.