伪狂犬病病毒在NF-κB家族p65基因敲除细胞系的复制规律研究

试验旨在研究伪狂犬病病毒(PRV)在NF-κB家族p65基因敲除细胞系中的复制规律。利用慢病毒介导的CRISPR/Cas9基因定点修饰技术构建猪肺泡巨噬细胞(3D4/21)p65基因稳定敲除细胞系。通过构建p65-sgRNA重组质粒,转染至HEK293T/17细胞,收取慢病毒,感染3D4/21细胞后利用嘌呤霉素筛选获得多克隆细胞系,T7核酸酶检测敲除效率,再通过有限稀释法获得3D4/21-p65^-/-的稳定细胞系。CCK-8试剂盒检测3D4/21细胞中敲除p65基因后对细胞增殖的影响;流式细胞术检测PRV-GFP感染3D4/21及3D4/21-p65^-/-细胞后病毒增殖的差异;实时定量PCR检测PRV感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、TK基因mRNA表达水平及PRV感染细胞诱导的IL-1β和IL-6基因mRNA水平表达的变化;Western blotting检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、gE蛋白的表达;滴度测定检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后子代病毒滴度。结果表明,sgRNA2和sgRNA3的基因编辑效率较高,对其进行克隆化培养进而获得敲除p65基因的稳定表达细胞系;CCK-8试剂盒检测细胞活力表明,p65基因敲除对细胞活力无影响;流式细胞仪检测表明,同一时间点PRV-GFP在3D4/21-p65^-/-中的增殖显著高于对照细胞;实时荧光定量PCR表明在3D4/21细胞中敲除p65基因促进了PRV gB、TK基因的mRNA表达水平,而抑制了IL-1β、IL-6基因的mRNA表达;Western blotting结果表明,在3D4/21细胞中敲除p65基因促进了PRV gB、gE蛋白的表达;滴度测定结果表明,同一时间点PRV-QXX在3D4/21-p65^-/-细胞中子代病毒的复制显著高于对照细胞。以上结果均表明,p65基因敲除可促进PRV在3D4/21细胞中复制。     

伪狂犬病病毒在NF-κB家族p65基因敲除细胞系的复制规律研究

试验旨在研究伪狂犬病病毒（PRV）在NF-κB家族p65基因敲除细胞系中的复制规律。利用慢病毒介导的CRISPR/Cas9基因定点修饰技术构建猪肺泡巨噬细胞（3D4/21）p65基因稳定敲除细胞系。通过构建p65-sgRNA重组质粒,转染至HEK293T/17细胞,收取慢病毒,感染3D4/21细胞后利用嘌呤霉素筛选获得多克隆细胞系,T7核酸酶检测敲除效率,再通过有限稀释法获得3D4/21-p65^-/-的稳定细胞系。CCK-8试剂盒检测3D4/21细胞中敲除p65基因后对细胞增殖的影响;流式细胞术检测PRV-GFP感染3D4/21及3D4/21-p65^-/-细胞后病毒增殖的差异;实时定量PCR检测PRV感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、TK基因mRNA表达水平及PRV感染细胞诱导的IL-1β和IL-6基因mRNA水平表达的变化;Western blotting检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、gE蛋白的表达;滴度测定检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后子代病毒滴度。结果表明,sgRNA2和sgRNA3的基因编辑效率较高,对其进行克隆化培养进而获得敲除p65基因的稳定表达细胞系;CCK-8试剂盒检测细胞活力表明,p65基因敲除对细胞活力无影响;流式细胞仪检测表明,同一时间点PRV-GFP在3D4/21-p65^-/-中的增殖显著高于对照细胞;实时荧光定量PCR表明在3D4/21细胞中敲除p65基因促进了PRV gB、TK基因的mRNA表达水平,而抑制了IL-1β、IL-6基因的mRNA表达;Western blotting结果表明,在3D4/21细胞中敲除p65基因促进了PRV gB、gE蛋白的表达;滴度测定结果表明,同一时间点PRV-QXX在3D4/21-p65^-/-细胞中子代病毒的复制显著高于对照细胞。以上结果均表明,p65基因敲除可促进PRV在3D4/21细胞中复制。     

外源肌苷酸对种鸡产蛋性能、蛋品质及种蛋孵化性能的影响

本试验通过在种鸡的基础饲粮中加入肌苷酸(IMP)来研究其对种鸡产蛋性能、蛋品质和种蛋孵化性能的影响。选择遗传背景相同、体重相近,产蛋率达到5%的20周龄健康AA种鸡864羽,随机分为2组(对照组饲喂基础饲粮;试验组饲喂基础饲粮+0.5%肌苷酸),每组6个重复,每个重复72羽,试验期30 d。测定试验期间所有种蛋的平均蛋重、产蛋率和合格蛋率;每组随机选取部分种蛋进行蛋品质测定;于孵化开始第17天,通过照蛋处理统计种蛋受精率,第21天出雏时,计算受精蛋孵化率、入孵蛋孵化率和健雏率。试验结果表明:与对照组相比,试验组种鸡的平均蛋重、产蛋率和合格蛋率均有升高的趋势,但差异不显著(P>0.05);蛋形指数显著升高(P<0.05),蛋壳强度、蛋壳厚度和蛋黄颜色显著降低(P<0.05);受精率、受精蛋孵化率和入孵蛋孵化率显著升高(P<0.05),健雏率和雏鸡体重有升高的趋势,但差异不显著(P>0.05)。综上,0.5%外源肌苷酸对种鸡的产蛋性能和种蛋孵化性能均有促进作用。     

Influence of four soil maintenance practices on Collembola communities in a Mediterranean vineyard

We studied the influence of four soil maintenance practices on Collembola communities in the soil of a Mediterranean vineyard: (a) postemergence herbicide with glyphosate; (b) postemergence and pre-emergence herbicides with glyphosate, terbuthylazine, diuron and oryzalin; (c) natural flora and (d) tillage to a depth of 10–15 cm. Total Collembola abundance, species diversity and species richness significantly varied between the four practices. Notably, the practice using postemergence and pre-emergence herbicides had significantly lower values. Identification of Collembola at species level allowed an interspecies comparison and revealed significant differences for the most common species between the four practices, with each practice being characterized by a different set of species. None of the species were found to be significantly more abundant in the plots treated with postemergence and pre-emergence herbicides.     

内蒙古红豆草黄萎病菌的分离鉴定及其生物学特性

依照柯赫氏法则,利用形态学结合分子生物学的方法首次对内蒙古自治区呼和浩特市中国农科院草原研究所沙尔沁基地红豆草疑似黄萎病的病样进行分离和鉴定,利用蘸根接种法对病原菌的致病性进行测定。同时,首次在室内条件下对病菌的生物学特性,如生长最适温度、酸碱度以及碳源、氮源对病原菌生长速率和产孢量的影响进行了研究。结果表明,引起沙尔沁基地红豆草黄萎病的病原菌为大丽轮枝孢菌,菌株具有较宽的温度和酸碱度适应范围,最适于菌株生长和产孢的温度均为25℃,最适于菌株生长和产孢的pH值范围分别为7~10和5~6。在碳源选择上,大丽轮枝孢菌在可溶性淀粉上的生长速度最快,而蔗糖最有利于其产孢。在氮源选择上,大丽轮枝孢菌在牛肉膏和L-脯氨酸上的生长速度较快,且L-脯氨酸最有利于其产孢。     

Production and Identification of Clone Sheep Using Bone Marrow Mesenchymal Stem Cells as Donor Cell

In order to obtain the cloned sheep using bone marrow mesenchymal stem cells（BMSCs）as the donor cells,the BMSCs of sheep were chosen and reconstructed embryos were built to transfer.10 published microsatellite markers were chosen,and the DNA samples from clone sheep,donor cells and surrogate ewes were amplified,and the relationship of father-son（RCP）was analyzed using the Quantity One for genotyping.The results showed that the reconstructed embryos were successfully built for electric fusion using sheep BMSCs as nuclear donor,and making nuclear transplantation into enucleated mature oocytes of which the fusion rate was 80.62%.20 surrogate ewe were chosen to be implanted with the reconstructed embryos at morula stage by implant surgery,and 5 lambs were born and only 3 were survived.The genotype of cloned sheep was in line with the dornor cell and the RCP were more than 99.999%.In conclusion,the first clone sheep were obtained successfully by using BMSCs as a nuclear donor in this experiment.     

Water Pricing and Sustainability of Self-Governing Irrigation Schemes

The sustainability of self-governingirrigation schemes is currently underpressure in many countries as publicfinancial support is decreasing.Furthermore, growing global concernregarding water scarcity means thatefficient water use is essential.Theoretically the choice and implementationof a water-pricing system should play acentral role in achieving this objective,both by recovering water costs and byencouraging farmers to adopt more efficientbehaviour. In the Senegal River Delta newlyestablished water users associations (WUAs)have chosen low water charges, which areaffordable for the majority of farmers butwhich underestimate long-term maintenancecosts. Combined with the difficulty ofcollecting and managing farmers' fees, thischoice has drawn them into a vicious circleleading to scheme deterioration and poorwater service. New alternatives have beendiscussed with them using a simulation toolthat takes into account both the watercosts and the farmers' incomes. Comparedwith the present fixed water charge basedonly on cultivated land, a two-part optionbased both on equipped and irrigated areasensures the recovery of fixed expenses suchas maintenance, while encouraging farmersto intensify their farming systems byadopting double cropping. Neverthelesschoosing a relevant water charge does notensure by itself the sustainability of anirrigation scheme. Combined supportproviding WUAS and farmers with efficientadvice regarding management andorganisational skills should alsocontribute significantly to achieve thisprime objective.     

用于车辆紧急制动仿真的动态轮胎模型

提出一种可用于车辆紧急制动仿真的动态轮胎模型——LuGre轮胎模型。该动态模型不仅具有与经典稳态模型（如魔术公式）相似的稳态特性,可以方便地通过试验数据进行参数拟合和在线整定,而且能够精确捕捉汽车紧急制动过程中的瞬态特性,又由于采用微分方程形式来描述,更有利于开发高性能的电子制动控制系统来提高汽车的行驶安全性。得到了LuGre轮胎模型的基本特性,并分析了其在车辆紧急制动仿真中的稳态特性和动态特性。     

“上膜下秸”措施对温室土壤水热盐调控试验研究

为了明确温室生产中不同耕作措施的水热盐调控效果,采用温室小区试验方法,以翻耕(CK)、翻耕结合地表覆盖地膜(M)、翻耕结合秸秆深埋(J)为对照,研究了翻耕结合地表覆盖地膜结合秸秆深埋(J+M)措施对温室土壤水热盐动态变化的影响。结果表明,与M、J及CK处理相比,J+M处理全生育期平均土壤含水率可分别提高8.96%、5.45%、14.32%,土壤温度分别提高3.35%、5.21%及8.32%,土壤全盐量分别降低24.44%、19.65%、26.64%,这一结果可为宁夏温室种植实施地膜覆盖与秸秆深埋改良措施提供参考。