Fusion Expression and Purification of Marek’s disease virus Tegument Protein VP16 and Its Antibody Preparation

马立克氏病病毒(Marek’s disease virus ,MDV)UL48基因编码的蛋白与单纯疱疹病毒1型(Herpes simplex virus ,HSV)衣壳蛋白VP16为同源物，将其克隆入pET-32a载体中，获得pET-VP16, 转化Escherichia coli BL21感受态细胞，经IPTG诱导表达，SDS-PAGE电泳显示:融合蛋白获得可溶性表达，表达蛋白的相对分子量约为67 kD；将表达的融合蛋白过His·Bind柱，获得纯化的蛋白，将该蛋白免疫家兔，制备多克隆抗体。通过间接ELISA和Western blot鉴定制备的多克隆抗体的效价和特异性，结果表明，该抗体具有较高的特异性，间接ELISA效价大于2×10－5。     

侧金盏花CBF转录激活因子基因片段的克隆

[目的]探索CBF转录激活因子基因在侧金盏花中是否存在。[方法]以侧金盏花基因组DNA为模板,根据拟南芥CBF基因序列的保守区设计1对特异性引物,通过PCR扩增获得1个DNA片段,将其克隆到pUCm-T载体中,转化大肠杆菌TG1,筛选阳性克隆并对其进行测序鉴定。[结果]测序结果显示通过PCR扩增获得的基因片段长448 bp。在GenBank中检索,该序列与从拟南芥和其他作物中克隆的CBF同源基因序列同源性较低,说明是一个新的基因片段,初步认定该片段是侧金盏花CBF基因片段。[结论]该研究为进一步克隆侧金盏花CBF转录激活因子基因全长序列、鉴定其生物学意义奠定了基础。     

植物激活蛋白对白术根腐病和斑枯病的诱抗效果及其对产量的影响

植物激活蛋白能显著提高白术对病害的抗性。试验表明700~1300倍液植物激活蛋白喷施白术叶面,隔30 d喷1次,对白术根腐病和斑枯病的诱抗效果分别达53.9%~81.2%和39.7%~63.3%。植物激活蛋白能显著提高白术商品性和产量,白术的单术重量增加35%以上,白术产量提高46%以上。6月初使用700倍激活蛋白,间隔30 d,整个生长期使用5次对白术抗病增产效果最好,对斑枯病诱抗效果达63.3%,增产129.93%。     

禾谷镰刀菌武昌菌株毒素的毒性分析

为提高小麦抗赤霉病体细胞的筛选效果，对禾谷镰刀菌武昌菌株的产毒、毒素提取、纯化、毒素组分及毒素初定量进行了研究。麦粒培养基培养２５天后可检测到两个主要毒性组分（Ａ和Ｂ）。其特征吸收峰值分别为２７０和２７５ｎｍ，Ｒｆ值分别为０．４５和０．５５左右。ＳＰＭ培养基培养只能检测到组分Ａ。Ａ组分与脱氧雪腐镰刀菌烯醇（ＤＯＮ）的结构特征基本相同。经高压灭菌的毒素可渗入到培养基中作抗性愈伤组织的筛选剂。毒素对愈     

UK114基因的克隆和表达载体的构建

本研究采用PCR法扩增得到重组UK114 cDNA序列,将其克隆于T-easy载体并进行了序列测定与分析。结果表明该序列全长1 017 bp,有一个开放的阅读框(40 nt~450 nt),可编码137个氨基酸(14.2 kDa),与UK114的序列比较,同源性为91%,突变的86个核苷酸中都为无义突变,开放阅读框中仅有一个核苷酸突变。此外,本实验构建了pBV114表达载体,选择阳性克隆提取质粒进行酶切,琼脂糖检测获得约1 kb和3.7 kb的两个片段。     

基因活化剂对紫色甘薯块根产量的影响

基因活化剂使用浓度(A)设3个水平375×(A1)、750×(A2)、1 500×(A3)稀释液;使用时间(B)设4个水平浸渍种苗基部1 h(B1)和1次(B2)、2次(B3)、3次(B4)叶面追肥,收获时测定块根鲜重.结果表明A1,A2,A3的块根产量分别比对照提高38.60%,61.70%和42.54%,B1,B2,B3和B4的块根产量分别比对照提高38.74%,64.33%,47.37%和40.06%,与对照差异均达显著水平(P＜0.05).A2B2为最佳水平组合,平均块根产量比对照提高89.77%,且与所有水平组合之间都有显著差异(P＜0.05).用750倍的基因活化剂浸渍种苗基部1 h再追肥1次,可显著提高紫色甘薯的块根产量.     

植物激活蛋白对水稻抗性相关基因转录水平的影响

 在cDNA芯片分析结果的基础上，对从交链孢真菌（Alternaria spp.）中提取的新型植物激活蛋白对水稻抗病相关基因转录水平的影响进行了研究，同时检测了抗病防御相关酶活性和稻瘟菌抗性的变化。结果表明，水稻幼苗经 2 g·ml-1激活蛋白喷雾处理后，NPR1基因从第天时转录水平开始提高，第3天和5天时转录活性继续增强；EIN2基因的转录在第1天、3天时转录水平显著提高，第5天时又有所回复，但仍高于对照；CTR1的转录在第1天时虽无影响，但第3天和第5天转录水平明显降低，而PR4 的转录在各检测时段均未表现明显变化。5 d后叶片的稻瘟菌病情指数和平均每叶病斑数开始显著降低，14 d时对稻瘟菌的诱抗效果最为明显。叶片苯丙氨酸解氨酶（PAL）活性在处理后1 d迅速增加，7 d和9 d时活性增加达到高峰期； -1,3-葡聚糖酶活性在处理后3 d开始增加，5 d时增幅最大，随后增幅降低，14 d时略低于对照水平；几丁质酶活性在处理后前3 d低于对照，5 d后活性平稳增加，14 d时降至略低于对照的水平。     

Study on the Role of AMPK Activator in Cryopreservation of Sheep Semen

This study aimed to investigate the effects of AMPK activators metformin (Met) and acadesine (AICAR) on sheep semen cryopreservation. Firstly, Met and AICAR with different concentrations (0, 100, 200, 300, 400, 500 μmol·L^-1) were added into the frozen diluent. After freezing and thawing, the optimal concentration (400 μmol·L^-1 Met, 200 μmol·L^-1 AICAR) were selected based on sperm motility, motion performance and membrane integrity; Secondly, the collected semen was froze using different frozen diluents (control group:diluent; Met group:diluent + 400 μmol·L^-1 Met; AICAR group:diluent + 200 μmol·L^-1 AICAR). After thawing, the sperm AMPK protein expression, acrosomal enzyme activity, metabolic index, mitochondrial function and antioxidant enzyme activity were examined. The results showed that the addition of 400 μmol·L^-1 Met and 200 μmol·L^-1 AICAR could significantly improve sperm motility, motion performance and membrane integrity(P<0.05) after thawing. In 400 μmol·L^-1 Met group, the total sperm motility, acrosome integrity rate and plasma membrane integrity rate were 43.20%, 91% and 46%, respectively. Compared with the control group, the level of AMPK phosphorylation in the sperm of the Met and AICAR groups was significantly increased after thawing (P<0.05), the acrosome enzyme activity of sperm was significantly increased(P<0.05); the pyruvate level was significantly decreased (P<0.05), the lactate dehydrogenase activity, lactic acid content, and ATP content were significantly increased(P<0.05). Compared with the control group, the dilution of Met group and AICAR group was more conducive to maintain mitochondrial membrane potential (P<0.05), increase ATPase and antioxidant enzyme activity (P<0.05). The addition of appropriate concentrations of Met and AICAR could improve semen quality of cryopreservation in sheep.     

藻蓝蛋白α亚基裂合酶的分子克隆和酶动力学研究

为了比较研究不同藻种中藻蓝蛋白裂合酶CpcE/F的结构与功能的差异,对Anabaena sp.PCC 7120中的CpcE/F进行克隆,并进行大量表达,将表达的裂合酶CpcE/F用于藻蓝胆素(PCB)与Mastigocladus laminosus PCC 7603藻蓝蛋白α-亚基(α-PC)脱辅基蛋白(CpcA)的体外重组,得到天然活性的α-PC,从而表明CpcE/F所编码的蛋白质是α-PC生物合成的裂合酶,并对CpcE/F的酶动力学进行了初步研究。     

Echinacoside promotes bone regeneration by increasing OPG/RANKL ratio in MC3T3-E1 cells

Echinacoside (ECH), isolated from Cistanche tubulosa (Schrenk) R. Wight stems, was subjected to in vitro experiments to investigate its bioactivities on proliferation, differentiation and mineralization of the osteoblastic cell line MC3T3-E1. MTT assay, the alkaline phosphatase (ALP) activity and calcium deposition were determined, and the secretion of collagen I (COL I), osteocalcin (OCN), osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were also assayed by enzyme-linked immunosorbent assay (ELISA). The results showed that ECH caused a significant increase in cell proliferation, ALP activity, COL I contents, OCN levels and an enhancement of mineralization in osteoblasts at the concentration range from 0.01 to 10 nmol·L^− 1 (p < 0.05), suggesting that ECH has a stimulatory effect on osteoblastic bone formation or has potential activity against osteoporosis. In addition, the ratio of OPG/RANKL also could be enhanced by ECH. These findings provide the potent evidence that ECH can promote bone regeneration in cultured osteoblastic MC3T3-E1 cells, which might be done by elevating the OPG/RANKL ratio, and potential evidence for echinacoside to be a promising drug or a lead compound in the development of disease-modifying drug to prevent osteoporosis.