固态混菌发酵生产饲用复合酶制剂营养条件和培养条件的研究

黑曲霉变种(A.niger v.Tiegh)CGMCC1182、黑曲霉MA-56(A.niger MA-56)CGMCC2722和黑曲霉XY-1(A.niger XY-1)CGMCC1182分别为α-半乳糖苷酶、β-甘露聚糖酶和木聚糖酶生产菌株。为获得高产α-半乳糖苷酶、β-甘露聚糖酶和木聚糖酶的复合酶制剂,通过单因素实验,研究了黑曲霉三种菌株在固态发酵条件下产复合酶制剂的培养基组成和培养条件。结果表明,黑曲霉混菌发酵生产复合酶的最适培养基组成为:麸皮∶豆粕为7∶3(m/m),在此基础上(以麸皮和豆粕总量为10 g计算)添加玉米芯1.0 g,魔芋粉0.1 g,葡萄糖0.5 g,(NH4)2SO4 0.2 g,NaNO3 0.1 g,MgSO4 0.1 g,KH2PO4 0.2 g,H2O 11 mL。产酶最适培养条件为:培养温度30℃,固形物与加水比1∶1,α-半乳糖苷酶、β-甘露聚糖酶和木聚糖酶接种比例为5∶6∶6,接种混合孢子悬浮液2.5 mL(以一支菌种斜面加30 mL无菌水为标准),300 mL三角瓶中装量8 g培养基,发酵60 h时,复合酶产量达到最优,α-半乳糖苷酶、β-甘露聚糖酶和木聚糖酶三种酶制剂的活力分别可以达到221、894、10188 IU/g。     

团花树α扩展蛋白基因的克隆及表达分析

采用扩增保守区域、基因组步移及3'RACE技术,在团花树中克隆到15个α-expansin基因的cDNA序列,命名为AcEXPA2-16(GenBank注册号JF922686-JF922700),其相对应的基因组序列GenBank注册号为JF922701-JF922715.包括已知的AcEXPA1,这16个EXPA蛋白的大小及序列高度保守,包括N端的信号肽、3个外显子和2个内含子.通过氨基酸序列比对和系统进化树分析,AcEXPA1-16分为4个亚族,分别为A、B、C和D亚族.定量PCR分析显示AcEXPA1-16基因在同一组织中的表达存在冗余现象,而单个基因在不同的组织中又存在特异表达,尤其AcEXPA8在形成层中的表达水平极高.这表明AcEXPA8在木质部发生的过程中可能起到重要的作用.研究结果为在团花树中研究α-expansin基因与木质部的生长和材质的关系提供信息,并最终为林木分子育种提供潜在的候选基因.     

牛α干扰素在毕赤酵母中的高效分泌表达及活性鉴定

为高效分泌表达牛α干扰素(boIFN-α),本研究通过人工合成boIFN-α基因,将目的基因克隆至表达载体pPIC9K中,构建重组质粒pPIC9K-boIFN-α,将其电转化于毕赤酵母菌株GS115,利用抗药选择压力G418筛选重组菌,对重组菌诱导表达,取上清进行SDS-PAGE和western blot检测,并优化重组菌的诱导表达条件.结果显示:筛选获得高效分泌表达boIFN-α的重组菌,其最佳诱导条件为:250 r/min,26℃培养,1％甲醇浓度诱导,诱导72 h.上清中目的蛋白表达量最高可达200 μg/mL,本研究为boIFN-α在生产中的应用奠定了基础.     

Increased Inflammatory Mediators in Horses Naturally Infected with Trypanosoma vivax. A Preliminary Study

The aim of this study was to measure the levels of inflammatory mediators in serum from horses naturally infected with Trypanosoma vivax. Banked serum samples collected during a previously reported T. vivax natural infection were used to analyze proinflammatory cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), and nitrite/nitrate (NO_x) levels. We evaluated 12 serum samples from horses from a farm in southern Brazil, four of which had parasitological and molecular diagnoses for T. vivax and presented with clinical signs of disease. Cytokines were assessed by quantitative sandwich enzyme-linked immunosorbent assay, and NO_x was measured using the modified Griess method. Levels of IFN-γ, TNF-α, IL-1, IL-6, and NO_x were increased in serum of infected animals compared to that in noninfected animals. Therefore, infection with T. vivax caused an increase in proinflammatory cytokines and nitric oxide content.     

Role of estrogen receptor-α (ESR1) and the type 1 insulin-like growth factor receptor (IGFR1) in estradiol-stimulated proliferation of cultured bovine satellite cells

Although the exact mechanism(s) by which estradiol (E₂) enhances muscle growth in a number of species, including humans and cattle, is not known, E₂ treatment has been shown to stimulate proliferation of cultured bovine satellite cells (BSCs). This is particularly significant because satellite cells are the source of nuclei needed to support postnatal muscle fiber hypertrophy and are thus crucial in determining the rate and extent of muscle growth. The objective of this study was to assess the role of estrogen receptor-α (ESR1) and the type 1 insulin-like growth factor receptor (IGFR1) in E₂-stimulated proliferation of cultured BSCs. To accomplish this, we have used small interfering RNA (siRNA) to silence expression of ESR1 or IGFR1 and assessed the effects on E₂-stimulated proliferation in BSC cultures. In BSCs treated with nonspecific siRNA, E₂ significantly (P < 0.05) stimulates proliferation under conditions in which neither IGF-1 nor IGF-2 expression is increased; however, treatment of ESR1- or IGFR1-silenced cells with E₂ does not significantly stimulate proliferation. These results indicate that both ESR1 and IGFR1 are required for E₂ to stimulate proliferation in BSC cultures. The fact that this occurs under culture conditions in which neither IGF-1 nor IGF-2 mRNA expression is increased strongly suggests that E₂ activates IGFR1 via a mechanism that does not involve increased IGF-1 or IGF-2 binding to the receptor.     

Chlamydia infection in cats in New Zealand

Conjunctival swabs collected in 1991-92 from 333 pedigree and non-pedigree cats were tested for the presence of Chlamydia spp. antigen using an ELISA antigen kit. Forty (18.4%) of the 217 samples from cats with conjunctivitis were positive. Seven (6%) of 116 samples from cats which were in contact with cats with conjunctivitis but which showed no clinical signs at the time of sample collection were positive. Positive-testing cats were frequently from multi-cat households. Chlamydia spp. is present and associated with conjunctivitis in cats in New Zealand. Infection may occur concurrently with viral diseases. Feline calicivirus was recovered from 27 (21 with conjunctivitis) of 37 cats tested in five catteries. Four cats (with conjunctivitis) were FIV-positive.     

Pregnancy recognition signaling mechanisms in ruminants and pigs

Maternal recognition of pregnancy refers to the requirement for the conceptus（embryo and its associated extraembryonic membranes） to produce a hormone that acts on the uterus and/or corpus luteum（CL） to ensure maintenance of a functional CL for production of progesterone;the hormone required for pregnancy in most mammals.The pregnancy recognition signal in primates is chorionic gonadotrophin which acts directly on the CL via luteinizing hormone receptors to ensure maintenance of functional CL during pregnancy.In ruminants,interferon tau（IFNT） is the pregnancy recognition signal.IFNT is secreted during the peri-implantation period of pregnancy and acts on uterine epithelia to silence expression of estrogen receptor alpha and oxytocin receptor which abrogates the oxytocin-dependent release of luteolytic pulses of prostaglandin F2-alpha（PGF） by uterine epithelia;therefore,the CL continues to produce progesterone required for pregnancy.Pig conceptuses secrete interferon delta and interferon gamma during the peri-implantation period of pregnancy,but there is no evidence that they are involved in pregnancy recognition signaling.Rather,pig conceptuses secrete abundant amounts of estrogens between Days 11 to 15 of pregnancy required for maternal recognition of pregnancy.Estrogen,likely in concert with prolactin,prevents secretion of PGF into the uterine venous drainage（endocrine secretion）,but maintains secretion of PGF into the uterine lumen（exocrine secretion） where it is metabolized to a form that is not luteolytic.Since PGF is sequestered within the uterine lumen and unavailable to induce luteolysis,functional CL are maintained for production of progesterone.In addition to effects of chorionic gonadotrophin,IFNT and estrogens to signal pregnancy recognition,these hormones act on uterine epithelia to enhance expression of genes critical for growth and development of the conceptus.     

水稻α-淀粉酶基因的表达模式与颖花开放的关系

【目的】淀粉降解与水稻浆片膨大和颖花开放过程密切相关,探究α-淀粉酶基因在颖花开放过程中的作用,为杂交水稻制种效率及产量的提高提供理论依据。【方法】在水稻扬花时,利用稀释碱性品红溶液进行离体穗子吸水试验,观察碱性品红在颖花中残留的组织,通过碘-碘化钾染色法确定11—14期（依据雄蕊发育分期）淀粉粒的分布变化,并通过RT-PCR、RT-qPCR和GUS报告基因检测多个α-淀粉酶基因在此期间的时空表达模式。【结果】水稻颖花开放前,内外稃片通过相互嵌合的钩合槽（marginal tissues of palea,mtp）将浆片和雌雄蕊封闭在内。当颖花开放时,浆片快速膨大,使得内外稃片的钩合点松开。扬花期间,离体穗子在稀释碱性品红溶液中吸水后,碱性品红染料主要残留在内外稃片钩合槽和浆片相连处组织以及花丝中。碘染试验显示,在12期（颖花开放前）,淀粉粒主要分布在雄蕊和内外稃片钩合槽,浆片中也有少量淀粉粒,在13—14期（颖花开放中）,内外稃片钩合槽和浆片中的淀粉粒均降解。RT-PCR分析发现OsRAmy2A和OsRAmy3D的表达量从12期开始上升,至13—14期表达量显著增强,到受精后1 d(...     

大豆α-生育酚的遗传与QTL分析

【目的】 通过对大豆α-生育酚进行遗传和QTL分析,研究其遗传机制,定位其主效QTL,为高α-生育酚含量的大豆品种选育奠定遗传学基础。【方法】 以栽培大豆晋豆23为母本、山西农家品种大豆灰布支黑豆（ZDD02315）为父本杂交衍生的447个RIL作为供试群体构建遗传图谱,试验群体及亲本分别于2011年、2012年和2015年夏季在河南省农业科学院原阳试验基地种植,冬季在海南省三亚南繁基地种植。田间试验采取随机区组设计,2次重复。从6个环境中每个家系选取15.00 g籽粒饱满,大小一致的大豆种子,利用高效液相色谱法定性、定量测定样品中的α-生育酚含量。采用主基因+多基因混合遗传分离分析法和WinQTLCart 2.5复合区间作图法,对大豆α-生育酚含量进行主基因+多基因混合遗传分析和QTL定位。【结果】 基于主基因+多基因混合遗传分离分析法,α-生育酚受4对主基因控制,遗传基因分布在双亲中。4对主基因间加性效应值中3对为正值,表明这些基因来源于母本晋豆23;1对为负值,表明该对基因来源于父本灰布支黑豆;4对主基因之间相互作用的上位性效应表现为正值和负值的各有3对,说明不同基因间上位性效应对α-TOC的影响方向并不完全一致。环境因素引起的变异为0.13%—4.05%。表明α-TOC主要受4对主基因影响,受环境因素影响较小。采用WinQTLCart 2.5复合区间作图（CIM）共检测到17个影响α-生育酚的QTL,分布于第1、2、5、6、8、14、16、17共8条染色体中,单个QTL的贡献率8.35%—35.78%,QTL主要表现为加性效应。qα-D1a-1同时在2011年原阳、2012年原阳和三亚、2015年原阳4个环境下检测到,且均定位在第1染色体Satt320—Satt254标记区间19.79 cM处,解释的表型变异分别为12.55%、12.01%和11.89%、12.61%,加性效应值0.119-0.132,增加α-TOC含量的等位基因来自母本晋豆23;qα-A2-1同时在2011年原阳和三亚、2015年原阳3个环境下检测到,且均定位在第8染色体Sat_129—Satt377标记区间44.53 cM处,解释的表型变异分别为23.18%和22.56%、23.01%,加性效应值-0.195—-0.180,增加α-TOC含量的等位基因来自父本灰布支黑豆。qα-D1a-1和qα-A2-1 2个QTL能够稳定遗传。【结果】 α-生育酚最适遗传模型符合4MG-AI,即4对具有加性上位性效应的主基因遗传模型。其遗传主要受4对主基因影响,受环境因素影响较小。检测到α-生育酚的2个稳定主效QTL,Satt320—Satt254和Sat_129—Satt377是共位标记区间。     

饲粮α-亚麻酸水平对意大利蜜蜂工蜂幼虫生理机能的影响

【目的】探究饲粮中α-亚麻酸的添加水平对意大利蜜蜂（Apis mellifera ligustica）工蜂幼虫抗氧化活性和免疫能力的影响。【方法】移取1日龄意大利蜜蜂工蜂幼虫1 200只,随机分为5组,每组5个重复,每个重复48只;其中1组为对照组,饲喂不添加α-亚麻酸的基础饲粮,4组为处理组,分别饲喂α-亚麻酸添加水平为0.02%、0.04%、0.06%和0.08%的饲粮。按照室内蜜蜂幼虫饲养方法,将1日龄幼虫用移虫针移至温度适宜的加入200 μL饲粮的24孔细胞培养板内,培养板置于恒温培养箱中（温度33℃,相对湿度55%）,试验期间每天更换饲粮。饲养至第6天末或第7天初,幼虫开始有直立或排便现象时,将幼虫转移至提前铺好灭菌纸的24孔细胞培养板内准备化蛹。从饲养第1天开始,每天检查并记录幼虫和蛹的死亡数量,并将死亡个体及时移除,直至未死亡的蛹全部羽化新蜂,记录成功化蛹和羽化新蜂个体数量,统计幼虫化蛹率和羽化率。各组分别取5、6和7日龄幼虫测定抗氧化、免疫、脂质代谢指标及相关基因表达量。【结果】饲粮中α-亚麻酸的添加水平为0.02%和0.04%时,化蛹率和羽化率显著高于与其他处理组（P＜0.05）,而工蜂幼虫血淋巴中甘油三酯（TG）、总胆固醇（TC）和低密度脂蛋白（LDL）含量显著低于对照组,高密度脂蛋白（HDL）的含量却显著高于对照组（P＜0.05）。饲粮中α-亚麻酸的添加水平为0.04%时,工蜂幼虫超氧化物歧化酶（T-SOD）的活性较对照组显著增加,丙二醛（MDA）的含量显著降低（P＜0.05）。饲粮中α-亚麻酸的添加水平为0.02%、0.04%和0.06%时,6日龄工蜂幼虫的溶菌酶（lysozyme）和酚氧化酶（PO）活性显著高于对照组（P＜0.05）。饲粮中α-亚麻酸添加水平为0.04%时,6日龄工蜂幼虫脂肪酸合成酶（FAS）和乙酰辅酶A羧化酶（ACC）活性显著低于对照组（P＜0.05）。饲粮中α-亚麻酸添加水平为0.04%时,5和7日龄工蜂幼虫lysozyme和PO相对表达量显著高于对照组,但饲粮中α-亚麻酸添加水平为0.08%时,lysozyme相对表达量会显著降低（P＜0.05）。【结论】α-亚麻酸对意大利蜜蜂工蜂幼虫抗氧化活性和免疫能力有一定影响,幼虫饲粮中α-亚麻酸适宜添加水平为0.02%—0.04%。