鸡白痢沙门氏菌鸡胚感染模型建立方法的筛选

为筛选出理想的鸡白痢沙门氏菌宿主感染模型的建立方法,本研究通过选择不同接种方式配合不同菌液浓度接种不同胚龄鸡胚建立感染模型,将90枚SPF鸡胚随机分成9组,每组10枚,分别为A、B、C、D、E、F、G、H、I组,A组为空白对照组,其余各组为模型组,模型组分别以气室接种、蛋壳接触接种的方式对不同胚龄（11和14胚龄）SPF鸡胚接种不同浓度（1×10³、3×10³、9×10³、3×10⁵、9×10⁵ CFU/mL）的鸡白痢沙门氏菌C79-3菌株。每天观察情况直至出雏,待雏鸡孵出后按组分笼饲养,每隔12 h观察1次,连续观察7 d,观察各组雏鸡的临床症状、死亡情况,观察期结束对死亡和存活雏鸡进行剖检、组织病理学检查及鸡白痢沙门氏菌的分离鉴定。结果显示,各模型组均有雏鸡出现明显的鸡白痢病症状并死亡,相较于其他模型组,以11胚龄蛋壳接触感染方式接种9×10⁵ CFU/mL的C79-3菌液的F组和14胚龄气室感染方式接种3×10³ CFU/mL C79-3菌液0.1 mL的H组,鸡胚出壳率较高,可分别达到80%和90%,出壳雏鸡呈现明显鸡白痢病特征,剖检可见肝脏出血、盲肠膨大、卵黄囊吸收不良;组织病理切片可观察到肝脏、盲肠、心脏各有不同程度的损伤,发病率分别100%和88.8%,并且鸡白痢沙门氏菌阳性检出率分别达70%和90%。本研究结果表明,以11胚龄蛋壳接触感染方式接种0.1 mL 9×10⁵ CFU/mL的C79-3菌液和14胚龄以气室感染方式接种0.1 mL 3×10³ CFU/mL的C79-3菌液的两种方法均可用于建立稳定的鸡白痢沙门氏菌宿主感染模型。     

Current knowledge about the processes of luteolysis and maternal recognition of pregnancy in camelids

Camelids have many unique reproductive features that considerably differ from those of other domestic species. Females are induced ovulators with subsequent development of a corpus luteum (CL) with a short lifespan. Plasma progesterone concentration starts to increase on day 4, peaks on day 8–9 and, in non-pregnant animals, basal concentration is reached around day 10–11 post-induction of ovulation. Luteolytic pulses of prostaglandin F_2α (PGF_2α) are firstly detected on day 7 or 8 (approximately on day 5–6 after ovulation), with maximal luteolytic peaks observed between days 9 and 11 post-mating, in coincidence with a high endometrial expression of cyclooxygenase 2, a limiting enzyme in prostaglandins synthesis. Unlike other species, oxytocin seems not to be involved in the luteolytic process in these species. The CL is the main source of progesterone secretion, and its function is required to support pregnancy. Despite constant research efforts, aspects of reproduction and maternal recognition of pregnancy in camelids remain not fully understood. A transient decrease and subsequent recovery in plasma progesterone concentration are observed after day 9 post-mating in pregnant animals in association with a pulsatile release of PGF_2α and a transitory decrease in CL vascularization. Thus, embryo recognition should occur between days 8 and 12 post-mating. In camels, conceptus tissues exhibit aromatizing activity with the capacity to synthesize large amounts of oestradiol. Similarly, llama blastocysts secrete oestradiol-17β during the preimplantation stage, with a higher production during the elongation period. An increase in the endometrial expression of oestrogen receptor α is also observed on day 12 post-mating. All these evidences suggest that oestrogen could be the signal released by the embryo at the time of its recognition in camelids. Besides, nearly 98% of pregnancies are carried out in the left horn. A decrease in the endometrial expression of mucin 1 and 16 genes has been reported, suggesting that these changes are crucial for successful embryo implantation; however, no differences have been observed between horns. Thus, maternal recognition of pregnancy in camelids is a particularly complex process that must occur in a concise time to allow the rescue of the CL and embryo survival.     

小麦品种无性系变异改良技术研究

针对晋阳345和晋麦73两个定型品种存在抗寒性较差的缺点,对其幼胚愈伤组织进行了4种处理:1、愈伤组织在MD培养基上继代2～3次;2、愈伤组织在紫外灯下约10cm照射2h;3、愈伤组织在低温2℃和常温25℃下交替培养30d;4、愈伤组织在紫外灯下照射2h后,再在低温2℃和常温25℃下交替培养30d。结果表明,4个处理后代R2主要农艺性状的变异率差异显著,且紫外灯照射幼胚愈伤组织的变异率比低温2℃和常温25℃交替处理幼胚愈伤组织高,其后代各种农艺性状的变异范围较大,变异率较高。但后者有利于改良抗寒性较差的定型小麦品种。     

基于多信息融合的疫苗制备中鸡蛋胚体分拣系统

采用多信息融合方法研究疫苗制备中鸡蛋胚体状态的识别与分拣技术。在研究不同状态鸡蛋胚体图像特征、温度衰减和透光度变化情况的基础上,得出活胚图像中血管多、粗且呈放射状,弱胚图像中血管少、细且断裂,死胚图像内部均匀无血管,污染胚图像内部有明显黑块特征;鸡蛋胚体从37.8℃的孵化箱中取出置于25℃室温环境,活胚、弱胚、污染胚、死胚的温度衰减速度依次增大;活胚的透光度随孵化时间增加而逐渐降低,其他胚体透光度变化相对较小。将图像、温度、透光度信息特征融合,建立BP神经网络信息融合模型对鸡蛋胚体状态进行识别。最后,从37.8℃孵化箱中抽取80枚孵化6 d的鸡蛋胚体放置于室温10 min后,采集图像、温度和透光度信息,进行试验验证。结果表明多信息融合系统的识别准确率为96.25%,比单用图像、温度和透光度传感器进行识别的准确率分别提高了6.25%、13.75%和8.75%。     

陆地棉×亚洲棉胚胎在培养条件下的发育研究

离体培养陆地棉与亚洲棉杂种胚珠,并以陆地棉自交为对照。观察结果表明,杂种胚胎表现出明显的生长优势,能顺利地通过胚胎发育的各个时期而达成熟胚。其原因是,胚乳游离核迅速解体,珠心组织也随之解体,而不形成胚乳细胞并使胚囊腔迅速扩展,使杂种胚有足够的营养源,并提供了足够的生长空间。自交胚胎的生育速度远不及杂种胚,发育受抑,尤其是球形胚到心形胚的时期较长。     

农杆菌侵染小麦的优化方案

本实验选用优质高产小麦品种洛阳8716和绵阳19幼胚愈伤作为受体材料,以pCAMBIA3301为载体,GV3101,EHA105,LBA4404农杆菌为供体材料进行侵染过程的优化研究.采用3种菌株,4个浓度梯度:OD600=0.2,0.5,0.7,1.0;3个时间参数:10 min,30 min,60 min;一共36个处理,每个处理4个重复.结果显示,农杆菌GV3101和EHA105,OD600 1.0×10 min和OD600 0.5×30 min两个侵染处理结果比较好,瞬时表达率达到50%-70%.以小麦幼胚愈伤组织为受体,预培养3～5 d,在24±1℃下,共培养3～4 d和350 mg/L羧苄青霉素除菌及5 mg/L PPT筛选处理后,转化效果较好,平均转化率为0.45%.     

利用shRNA真核表达载体干涉雌性鸡胚性腺中芳香化酶基因(CYP19A1)的表达

本研究通过把构建的短发夹结构RNA(shRNA)真核表达载体导入鸡胚,检测鸡胚性腺分化期质粒载体在鸡胚体内代谢情况及雌性鸡胚性腺中芳香化酶基因(CYP19A1)mRNA表达效率,进而探讨利用该方法在鸡胚体内进行特定基因干涉的可行性.实验针对CYP19A1基因构建了4条特异性表达载体,一条非特异性表达载体.每个实验组选取45个新鲜种蛋作为实验材料进行胚盘下腔注射,并设立空白对照组.鸡胚发育12d时,检测各处理组鸡胚肝脏组织中质粒存在情况,并取其左侧性腺组织进行目标基因的荧光定量分析.研究结果表明,导入组在12日胚龄时所有鸡胚基因组中均可检测到绿色荧光蛋白基因(EGFP);荧光定量结果显示,导入特异性表达载体cyp-580、cyp-1083和cyp-1295后,对应雌性鸡胚性腺CYP19A1 mRNA表达效率显著低于空白对照组,干涉效率分别为:73％、52％和85％;特异性表达载体cyp-1403组CYP19A1mRNA表达效率与空白对照组相比有所降低但无显著性差异.本实验为诱导鸡胚性反转提供了新方法并建立了鸡胚发育期特定基因体内干涉新平台.     

siRNA干涉禽流感病毒PA基因在鸡胚成纤维细胞中的表达

用阳离子脂体质转染试剂将人工合成的小干扰RNA(Small interference RNA,siRNA)转染鸡胚成纤维细胞(CEF).结果表明,针对禽流感病毒(AIV)H5N1亚型核蛋白(NP)基因的siRNA能在AIV感染后1,2,3 h显著抑制NP基因的表达,表达水平分别降低3,1.4和2.5倍.