牦牛StAR基因克隆及其生物信息学与组织表达特性的研究

旨在克隆获得牦牛StAR基因编码序列（CDS）并进行生物信息学分析,探究其mRNA组织表达特性。本研究以屠宰场采集的成年母牦牛心、肝、脾、肺、肾、卵巢、输卵管、子宫组织（n=5）,不同年龄（胎牛、1岁、2岁）牦牛的卵巢（n=3）,不同发情周期（卵泡期、黄体期）的牦牛卵巢（n=3）,黄体期黄牛的卵巢（n=3）及实验室冻存的牦牛颗粒细胞为研究材料。以牦牛黄体期卵巢cDNA为模板,用逆转录PCR克隆StAR基因,并使用MEGA7.0和ExPASy-ProtParam等软件分析其生物信息学特性;采用实时荧光定量PCR技术分析牦牛StAR基因组织表达特性。结果发现,StAR基因CDS区长858 bp,编码285个氨基酸,StAR蛋白总体带正电荷,属于碱性亲水稳定蛋白,无跨膜结构及信号肽,主要存在于细胞质和线粒体; StAR基因具有较高的保守性,符合物种进化规律。牦牛StAR基因在卵巢表达水平最高（P<0.01）,且2岁时卵巢表达水平极显著高于胎牛和1岁龄（P<0.01）,黄体期卵巢表达水平极显著高于卵泡期（P<0.01）;黄体期黄牛卵巢中StAR基因的表达量极显著高于牦牛（P<0.01）;在颗粒细胞的体外培养过程中StAR基因表达量逐渐上升,在培养24 h时达到高峰（P<0.01）,随后显著降低。综上所述,StAR基因序列较为保守,在牦牛卵巢组织中表达最高,且表达水平随年龄与卵巢周期而变化,提示StAR基因可能参与牦牛卵巢及黄体功能相关的繁殖调控。     

Effects of dietary Bacillus coagulans and yeast hydrolysate supplementation on growth performance,immune response and intestinal barrier function in weaned piglets

The present study investigated the effects of Bacillus coagulans and yeast hydrolysate supplementation on growth performance, immune response and intestinal barrier function of weaned piglets. Twenty-four weaned piglets with an average body weight (BW) of 6.89 ± 0.15 kg were divided into four diets for 28 days. The treatments were basal diet (control), basal diet supplemented with antibiotic (20 mg/kg colistin sulphate and 40 mg/kg bacitracin zinc, AT), probiotics (400 mg/kg Bacillus coagulans ≥5 × 109 CFU/g, BC) or yeast hydrolysate (5000 mg/kg yeast hydrolysate, YH). Average daily gain (ADG) and average daily feed intake (ADFI) were improved by AT and YH diets (p < 0.05), while BC diet only increased ADG (p < 0.05). The complement 3 (C3), lysozyme (LZM) and tumour necrosis factor-α (TNF-α) concentrations in serum were increased in BC diet (p < 0.05). Feeding AT and YH caused the increase of jejunal villus height (p < 0.05), and a higher ratio of villus height/crypt depth was observed in AT, BC and YH groups (p < 0.05). The mRNA expression of zonula occludens-1 (ZO-1) in jejunal mucosa was up-regulated by AT, BC and YH diets (p < 0.05). Dietary AT, BC or YH inclusion decreased the interleukin-1β (IL-1β) concentration and TNF-α mRNA expression (p < 0.05), and YH supplementation even down-regulated toll-like receptor 4 (TLR4) and CD14 expressions (p < 0.05). In summary, the dietary administration of BC or YH both improves growth performance through promoting the intestinal barrier function, indicating both of them can serve as potential alternatives to antibiotics growth promoters for the piglet production.     

新时代饲料跨境电商面临的问题及应对措施研究

随着饲料行业国际市场竞争的日趋加剧,我国饲料企业跨境电商业务受到了严重冲击。近几年,由于农业、畜牧业及肉、蛋、奶等产品的市场价格波动,放大了跨境电商活动中原本就存在的跨境物流、金融支付、网络安全、国际法律公约缺失等问题。为在新的竞争环境下有效降低产品市场价格波动对跨境电商造成的不利影响,应从强化国际间贸易协作,拉动国内市场需求,提升国际贸易一站式服务平台功能,增加互联网软硬件技术投入及促进新的国贸贸易法律体系建立等方面做出努力。     

口蹄疫病毒VP1基因密码子偏好性分析

VP1蛋白是口蹄疫病毒(FMDV)的主要结构蛋白,可诱导机体产生中和抗体以及激发保护性免疫应答。为探究VP1基因密码子偏好性,筛选出其最佳体外表达系统,使用Visual Gene Developer、CodonW、GraphPad Prism等软件,对VP1基因进行密码子偏好性分析,同时将VP1基因密码子使用频率与大肠杆菌、毕赤酵母、昆虫杆状病毒和哺乳动物细胞表达系统作了比较。结果显示,VP1基因的CAI为0.21,ENC为55.43,GC3S为65.26%,表明该基因密码子使用偏好较弱并且密码子偏好以G/C碱基结尾。结合VP1基因与各表达系统密码子使用频率比值,发现其与昆虫杆状病毒表达系统频率比值差异最小且CAI最大,因此推断昆虫杆状病毒表达系统是FMDV VP1基因最适体外表达系统。本研究为FMDV亚单位疫苗研制等提供了技术基础。     

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。     

Betaine Alleviates Heat Stress-Induced Hepatic and Mitochondrial Oxidative Damage in Broilers

The aim of this study was to evaluate the effects of dietary betaine (BET) on growth performance, redox state, and related gene expression in broilers under heat stress (HS). A total of 144 21-day-old male broiler chickens with similar body weights were assigned randomly to three treatments with six replicates (eight chickens per replicate cage). Broilers in the control (CON) group were kept at thermoneutral (TN, 22±1°C) conditions and fed a basal diet until they were 42 days of age. Broilers in the other two groups (defined as HS and HS + BET) were exposed to HS (34±1°C, 8 h/day) and fed the basal diet without or with 1000 mg/kg BET, respectively. Rectal and cockscomb temperature of broilers was increased (P<0.05) in HS and HS + BET groups compared with the CON group, whereas there was no difference between HS and HS + BET groups. Dietary BET supplementation restored (P<0.05) average daily gain (ADG) and average daily feed intake (ADFI) of broilers and reversed (P<0.05) the increase in serum alanine transaminase (ALT) activity and malondialdehyde (MDA) content in the liver tissue of broilers under HS. The HS + BET group had higher (P<0.05) activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) in the liver tissue and mitochondria than the HS group, and the same pattern was observed for glutathione (GSH) and GSH/glutathione disulphide (GSSG) in the liver tissue. The decreased mRNA levels of GPX1 and uncoupling protein (UCP) in the liver induced by HS were restored by BET supplementation. In conclusion, dietary BET supplementation can alleviate HS-induced hepatic and mitochondrial oxidative damage of broilers by regulating mRNA expressions of GPX1 and UCP.     

基于SPCE061A的车载灯光语音识别系统设计

在介绍车载灯光非特定人语音识别系统的组成和原理的基础上,重点分析了基于隐马尔可夫模型(HMM)的模板训练和语音识别算法。选用凌阳SPCE061A的16位单片机进行了系统的硬件和软件设计。制定了本语音识别系统的实验方案,实验结果基本满足灯光控制的要求。     

4LMZ160型履带式苎麻联合收割机的研究

分析了我国苎麻联合收割机现状及存在的问题,设计了适合我国苎麻主产区收获作业的履带式苎麻联合收割机,并对样机的关键部件进行了详细说明。该机一次可完成割幅160mm收割,整机转弯半径小,操作方便,对麻地高垄有较好适应性。其整机结构配置新颖独特,使用半喂入稻麦联合收获机底盘,采用全液压传动技术直接驱动苎麻收割机行走和机具作业,可一次性完成切割、输送、收集功能。田间性能测试结果表明:该机漏割率、割茬高度、作业小时生产率等各项性能指标均到达设计要求。