马铃薯Y病毒N株系HC-Pro基因在毕赤酵母中分泌表达与鉴定

 以克隆载体pGEMT-PVY-NHC为模板,用PCR方法获得HC-Pro基因,构建表达载体pPIC9K-HC,将重组质粒经Sal I单酶切后电转化Pachia pichia GS115菌株,筛选出对G418有高抗性和在MM培养基上生长缓慢的转化子。经摇瓶发酵和1%甲醇诱导后,SDS-PAGE检测发酵液上清,在66 kD处有1个特异条带表达,目的条带蛋白占上清液总蛋白的40%。Western blot鉴定表明,表达蛋白可以和HC抗体发生结合反应。     

枯萎菌拮抗芽孢杆菌BC98-I抗菌多肽的纯化

 枯萎菌拮抗蜡状芽孢杆菌BC98-I分泌产生的抗真菌物质经粗提、Sephadex G100和DEAE52柱层析分离纯化后,得到了抗菌馏分P₁D₄。该馏分达到了电泳纯,SDS-PAGE电泳检测其分子量约为3.5kD,等电点为4.65。又经MALDI-TOF质谱检测,该馏分为精确分子量小于或等于1531.71D的多肽。氨基酸组成分析结果表明:馏分P₁D₄由Asp、Glu、Ser等12种氨基酸组成,富含酸性和极性氨基酸。结合以往研究结果,目标产物热稳定性好,对胰蛋白酶和蛋白酶K有良好耐受性,初步认为属低分子量的环状多肽。     

聚乙二醇8000对棉铃虫微粒体蛋白沉淀作用的研究

为建立棉铃虫Helicoverpa armigera微粒体P450的分离纯化方案,比较了不同浓度下聚乙二醇8000(PEG8000)对棉铃虫幼虫中肠和脂肪体微粒体蛋白的沉淀作用。结果表明,终浓度为8.0×104 mg/L的PEG8000可以使中肠和脂肪体微粒体蛋白的78%沉淀下来,其中包含的细胞色素P450分别占中肠和脂肪体P450总量的28%和34%。SDS-PAGE显示,中肠微粒体经8.0×104 mg/L的PEG8000沉淀后,被沉淀蛋白的分子质量主要集中在14.1 ~ 40 kD和66 ~97 kD范围内。     

甘薯褪绿斑病毒外壳蛋白的分子变异及其特异抗血清制备

 甘薯褪绿斑病毒(Sweet potato chlorotic fleck virus,SPCFV)是侵染甘薯的主要病毒之一。本研究利用RT-PCR方法克隆了SPCFV中国4个分离物的外壳蛋白(CP)基因。序列分析表明,cp基因全长900 bp,编码299个氨基酸残基。4个分离物cp基因的核苷酸序列一致性为78.3%～89.9%,推导的氨基酸序列一致性为91.3%～95.7%,存在较大的分子变异。不同分离物CP氨基酸序列N末端的第3-32位氨基酸为多变区。将四川分离物的cp基因克隆到原核表达载体pET-28a(+)上,SDS-PAGE分析表明,经IPTG诱导,cp基因在大肠杆菌BL21(DE3)中得到了高效表达。以表达的蛋白为抗原免疫家兔,制备了SPCFV CP的特异性抗血清。ACP-ELISA检测结果表明,制备的抗血清效价达1∶128 000,可用于田间甘薯样品的检测。     

瓜类褪绿黄化病毒外壳蛋白基因在大肠杆菌中的表达及抗血清的制备

 根据已报道的瓜类褪绿黄化病毒(Cucurbit chlorotic yellows virus, CCYV)外壳蛋白(CP)基因的核苷酸序列设计引物,利用RT-PCR方法从感病甜瓜中扩增得到长度为753 bp的CP基因,将其克隆到原核表达载体pGEX-4T-3上,获得重组载体pGexCP,在大肠杆菌BL21菌株中诱导表达,SDS-PAGE分析表明,经IPTG诱导,CP基因在大肠杆菌BL21中得到了高效表达。以表达的蛋白为抗原,免疫家兔,制备了CCYV外壳蛋白的特异性抗血清。ACP-ELISA检测结果表明,当CP蛋白浓度为2.5 μg/mL时,血清效价高达5.12×10⁵。Western blot分析表明制备的抗体检测CCYV侵染的甜瓜叶片得到特异性的条带,表明该抗体的特异性较好。ACP-ELISA检测结果表明,制备的抗血清可用于田间甜瓜病样的检测。本试验为CCYV的快速检测提供了重要的研究材料。     

生防细菌对马铃薯青枯病的防病增产作用研究

将对马铃薯青枯病菌具有抑菌活性的枯草芽孢杆菌 (Bacillus subtilis)菌株0702、GP7-13制成粉状制剂 ,用于马铃薯种薯播前处理。湖北恩施田间小区防病增产试验表明 ,病地上菌株GP7-13对马铃薯青枯病的防效达73.9%～89.7% ,0702菌株达60.9%～88.2%。两生防菌剂在北京郊区南口和河北张北县无病地试验 ,对马铃薯具有促生增产作用 ,菌株GP7-13可增产马铃薯 17.3%～60.3% ;0702增产26.9%～61.2%。     

螺旋毛壳ND35 β-1,3-葡聚糖酶的诱导、性质及其抑菌作用

 以病原菌Rhizoctonia solani的细胞壁为诱导物,模拟毛壳菌自然的重寄生过程,研究了内生真菌螺旋毛壳(Chaetomium spirale) ND35 β-1,3-葡聚糖酶的产酶条件、性质,尤其是不同碳源的调控作用。结果表明,不同种类的真菌细胞壁及几丁质和昆布多糖,均可诱导产生β-1,3-葡聚糖酶,而作为分解代谢产物的葡萄糖则抑制产酶。经硫酸铵沉淀、DEAE-Sepharose阴离子交换层析及Phenyl-Sepharose疏水层析,并通过SDS-PAGE鉴定,纯化了一种分子量约为73 kDa的内切β-1,3-葡聚糖酶GLUC73。其最适反应温度为55℃,在40℃以下较稳定;最适pH值为5.5,在pH 5-9范围内均很稳定;酶活性受Hg²⁺、Fe³⁺、Zn²⁺、Mg²⁺等金属离子不同程度的抑制,Mn²⁺和Co²⁺对酶有激活作用;以昆布多糖为底物时,该酶的米氏常数K_m为0.412 mg·mL^-1,最大反直速度V_max为3.876 U·mL^-1。粗酶液同时具有β-1,3-葡聚糖酶和几丁质酶活性,离体抑菌试验表明,对苹果炭疽病菌(Glomerella cingulata)、杨树腐烂病菌(Valsa sordida)、苹果树腐烂病菌(Valsa mali)的菌丝生长和孢子萌发有明显的抑制作用。通过对β-1,3-葡聚糖进行免疫细胞化学标记和超微结构观察,间接证明了β-1,3-葡聚糖酶在螺旋毛壳重寄生过程中的作用。     

马铃薯X病毒运动蛋白基因的原核表达、抗血清制备及应用

构建马铃薯X病毒运动蛋白(P25)原核表达载体,并在大肠杆菌BL21(DE3)pLysS中诱导表达融合蛋白,所表达的P25蛋白经SDS-PAGE电泳纯化后免疫小鼠,抗血清效价为1∶4000,抗血清与PVX有特异性反应(P/N>2),且具有较强的反应特异性,而与PVY、TMV均不发生反应.该方法制备的抗血清可用于ELISA检测PVX感病植株及Western blot检测转基因植株.     

百合斑驳病毒外壳蛋白基因原核表达、抗血清制备及其应用

为了建立百合斑驳病毒(Lily mottle virus,LMoV)的快速检测方法,采用RT-PCR方法从感染LMoV的百合叶片中克隆该病毒的外壳蛋白(coat protein,CP)基因,然后连接到原核表达载体pET28a(+)上,导入大肠杆菌Escherichia coliBL21(DE3)并诱导表达,以表达的重组蛋白为抗原制备该病毒的抗血清。结果显示:LMoVCP全长为822 bp,编码274个氨基酸;SDS-PAGE及Western blot检测结果表明,经IPTG诱导得到了分子量约为34 kD带有HIS标签的目的蛋白;用该蛋白制备的抗血清经间接ELISA和Western blot检测结果显示,其效价为1∶51 200,具有较高的特异性,可用于感染LMoV百合的检测,其检测结果与RT-PCR检测结果一致。     

UPLC-MS/MS法检测稻米及土壤中扑草净除草剂的残留量

建立检测稻米及土壤中扑草净的UPLC-MS/MS方法。稻米和土壤样品经乙腈和混合液提取,甲醇/二氯甲烷溶解,PSA固相萃取柱净化,氮气吹干后经UPLC-MS/MS测定,外标法定量。建立了水稻及土壤中提取扑草净残留量的液相色谱-质谱/质谱测定方法。扑草净在稻米及土壤中的最低检测质量分数分别为0.01mg/kg;最小检出量5×10~11g;稻米添加回收率为82.7%~105.3%,土壤中添加回收率为79.6%~103.3%;稻米的RSD为2.6%~3.6%,土壤的RSD为3.2%~5.2%。建立方法准确、快速、灵敏度高,能够满足扑草净残留量分析的要求。     

稻瘟菌激发子CSB I专化性及相关性质研究

 以一套水稻抗稻瘟病近等基因系为材料,接种稻瘟菌(Magnaporthe grisea)细胞壁来源的糖蛋白激发子CSB I,其诱导植保素的积累在高度非亲和性互作水稻远高于亲和性互作水稻;研究同时表明,CSB I可专化性诱导完全非亲和性互作和高度非亲和性互作水稻的过敏性坏死反应;表明该激发子具有小种-品种专化性。经热、胰蛋白酶和过碘酸钠处理后的生物活性检测结果表明,CSB I的活性位点为糖基部分。经pH稳定性检测,CSB I在酸性及相对弱碱性条件下较稳定;而在强碱性条件下,激发子活性下降较多,甚至完全丧失。对CSB I诱导活性的有效浓度测定表明,激发子诱导水稻叶片酶活性升高的最低有效浓度为0.07~0.70 nmol/L。     

稻瘟菌GP66激发子诱导的水稻膜脂过氧化及其保护酶活性变化

 以抗稻瘟病水稻近等基因系C101LAC及背景品系CO39为材料,研究稻瘟菌(Magnaporthe grisea)来源的GP66激发子诱导的水稻膜脂过氧化及保护酶活性变化.结果表明,激发子诱导非亲和性互作水稻超氧阴离子(O₂^·)积累和脂氧合酶(LOX)活性在早期明显高于亲和性互作水稻;O₂^·积累和LOX活性的升高进而导致了非亲和性互作水稻的膜脂过氧化,其相对电导率及丙二醛(MDA)含量出现的高峰期和强度也明显要早和高于亲和性互作水稻.超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性均趋于下降,不同亲和性互作水稻间的变化则不明显;非亲和性互作水稻过氧化物酶(POD)活性则在激发子诱导早期明显高于亲和性互作水稻,可能与其参与其它抗性有关.这些结果表明膜脂过氧化的发生是激发子诱导水稻抗性的主要生理机制之一.     

杨盘二孢激发子的分离及稳定性研究

 从杨盘二孢的培养滤液和菌丝中获得2种激发子粗提物,分别测定其糖和蛋白质的含量,发现滤液激发子粗提物(CFE)糖和蛋白质的含量分别为41.07和40mg/mL,菌丝激发子粗提物(CME)糖和蛋白质含量分别为48.07和55mg/mL。滤液激发子粗提物对温度不敏感而对碱性条件敏感;菌丝激发子粗提物对酸碱不敏感而对温度敏感。用Sephadex G-100柱层析的方法初步纯化2种激发子,并且菌丝激发子粗提物过柱后得到2个活性组分J14和J25;滤液激发子粗提物过柱后获得2个活性组分L9和L16。将4个组分进行烟草叶片过敏反应和I-895杨酶活性的诱导,结果表明,菌丝激发子强活性物质集中在J14组分;而滤液激发子强活性物质集中在L9组分。     

棉疫病菌90kD胞外蛋白激发子生物活性与稳定性研究

 对棉疫病菌(Phytophthora boehmeriae Saw.)90 kD胞外蛋白激发子的生物活性与稳定性进行了研究。用不同浓度的激发子处理烟叶测定其诱发过敏反应的有效浓度,结果是所测定的0.5~100 nmol/L各浓度均可诱发过敏反应,但100 pmol/L不能诱发过敏反应,表明该激发子诱导烟草产生过敏反应的最低有效浓度在100 pmol/L~0.5 nmol/L之间。该激发子可诱导不同烟草(Nicotiana tabacum L.)品种产生过敏反应,但不能诱导辣椒(Capsicum annuum L.)和茄子(Solanum melongena L.)等茄科作物及棉花(Gossypium arboreum Linn.)发生过敏反应,初步表明该激发子诱导植物发生过敏反应具有一定的专化性。以10 nmol/L激发子溶液处理烟叶后分别于第0、24、48和72 h接种烟草疫霉(Phytophthora nicotianae),结果证明该激发子可以诱导烟草产生抗性反应,其抗性以处理后立即接种烟草疫霉为最强,接种后48h防效达68%,以后随时间的延长逐渐减弱。激发子分别经p H值2~14的水溶液25℃处理30 min,或分别经4、25、60和100℃处理5 min仍能诱发过敏反应,但是经蛋白酶K处理后不能诱发过敏反应。表明该激发子对酸、碱和温度不敏感,但对蛋白酶K敏感。     

Purification, Elicitor Activity, and Cell Wall Localization of a Glycoprotein from Phytophthora parasitica var. nicotianae, a Fungal Pathogen of Tobacco

ABSTRACT A glycoprotein of 34 kDa (GP 34) was solubilized at acidic pH from the mycelium of Phytophthora parasitica var. nicotianae and was purified by ion exchange and gel permeation chromatography. Whole tobacco plants treated with GP 34 through their roots showed an enhanced lipoxygenase activity as well as hydroxyproline-rich glycoprotein accumulation, indicating that this molecule had elicitor properties. An antiserum raised against the pure glycoprotein allowed localization of GP 34 by immunogold-labeling on the cell surface of the mycelium when the fungus was grown in vitro. In the wall-less zoospores, GP 34 was limited to the flagellum surface. It was then abundantly synthesized at the onset of encystment. During infection of tobacco plants, labeling was very faint at early stages of colonization, particularly in the susceptible host cultivar. It appeared earlier in the resistant host cultivar and was restricted to the living fungus, declining with mycelium cell death.     

Preliminary characterization of race-specific elicitors from<Emphasis Type="Italic">Peronospora parasitica</Emphasis> and their ability to elicit phenolic accumulation in<Emphasis Type="Italic">Arabidopsis</Emphasis>

Intercellular washing fluid (IWF) obtained from the susceptibleArabidopsis accession Ws-eds1 inoculated withPeronospora parasitica isolate Emoy-2, contained an elicitor of necrosis with ecotype specificity towardsArabidopsis accessions with particular resistance genes. This elicitor caused necrosis on the highly resistant accessions La-er, Nd-1 and partly on Col-5, but not on the susceptible accessions Ws-eds1 and Oy-0. In resistant plants, injection of IWF caused hypersensitive reaction (HR)-like cell collapse which was associated with the accumulation of phenolics and lignin-like material in walls of cells undergoing cell death. The elicitor is sensitive to proteinase K and pronase enzymes, heating and autoclaving but insensitive to periodate oxidation, freezing and thawing, and is not dialyzable. Results suggest that the elicitor is a protein. Fractionation experiments using size-exclusion membranes revealed that elicitor activity has a molecular weight in excess of 100 kDa. http://www.phytoparasitica.org posting July 13, 2003.     

Purification of an Elicitor-Inducible Antifungal Chitinase from Suspension-Cultured Rice Cells

Suspension-cultured rice cells showed an appreciable amount of chitinase activity when the cultured cells were treated with an elicitor isolated fromRhizoctonia solani, the rice sheath blight pathogen. A fivefold increase in chitinase activity was observed 24 h after elicitor treatment. The elicitor-inducible chitinase was purified by ammonium sulfate fractionation, chitin affinity chromatography and gel filtration. Its molecular weight is 35 kDa and it has an isoelectric point of 8.3. The 35 kDa basic chitinase inhibited mycelial growth ofR. solani in vitro. Morphological changes appeared within 1 h following exposure of mycelium to the chitinase. The hyphal tip showed marked swelling and subsequently lysis was also observed.     

一种新的90 kD胞外蛋白激发子诱导烟草系统获得抗性研究

 就棉疫病菌(Phytophthora boehmeriae Saw.)培养滤液中提纯获得的90 kD胞外蛋白激发子诱发烟草系统获得抗性进行了研究。以10 nmol/L激发子溶液注射处理Samsun NN烟草叶片24 h后,在处理叶片及其上、下各2片叶片接种TMV,结果是处理叶及其上、下各2片叶片上的枯斑数显著少于对照,诱抗防效达40.9%~53.1%;接种TMV7d后处理叶片的枯斑平均直径为1.23mm,显著小于对照叶片上的枯斑直径2.97mm,但是处理叶片的上、下叶片上的枯斑平均直径与对照没有显著差别。以10 nmol/L激发子溶液注射处理Samsun NN烟草叶片分别立即接种或于1、2、4、7、15 d接种TMV,结果证明该激发子诱导烟草对TMV的抗性以处理后第1d至第4 d接种为较好,防效达30.2%~5 0.4%;处理后立即接种和第7d接种TMV诱抗防效仅4%左右,处理叶片上的枯斑数与对照没有显著差别,表明较强的诱导抗性可持续时间<7d。用不同浓度激发子处理烟叶,所测定的0.5~100 nmol/L各浓度均可显著地诱发烟草对TMV产生获得抗性,诱导抗性效果为34.9%~5 8.2%,诱导抗性效果随浓度的降低呈下降趋势。以10 nmol/L激发子溶液注射处理W38烟草叶片,2 d后分别注射接种烟草野火病菌(Pseudomonas syringae pv.tabaci)菌液或喷雾接种烟草赤星病菌(Alternaria alternata)分生孢子,结果是,注射接种烟草野火病菌5d后处理叶片及其上、下叶片上的野火病病斑均显著小于对照;处理叶片上的赤星病病斑数及病斑面积明显小于对照,表明该激发子可诱导烟草对野火病和赤星病产生抗性。上述结果表明,90kD蛋白激发子诱导烟草产生的获得抗性是一种典型的系统获得抗性,该系统获得抗性对病原菌具广谱抗性。     

A soluble carbohydrate elicitor from Blumeria graminis f. sp. tritici is recognized by a broad range of cereals

Wheat plants rapidly recognize pathogenic and non-pathogenic conidia of the powdery mildew fungusBlumeria (syn. Erysiphe)graminis on their leaf surfaces. This suggests that a chemical signal emanates from conidia at the pre-penetration stage of infection. Conidia of B. graminis f. sp. tritici were found to contain an elicitor that was easily washed off their surface. The elicitor activity is heat stable and could not be removed by phenol extraction. By contrast, elicitor activity is sensitive to periodate oxidation and partial acid hydrolysis suggesting that the elicitor activity resides in a carbohydrate moiety. Analysis of carbohydrates revealed mostly glucose, with smaller amounts of xylose and mannose. The glucosyl residues of the B. graminis elicitor were found to be linked (1 → 2)-, (1 → 4), and (1 → 6)-, with (1 → 4, 1 → 6)- branch point residues, and no 3-linked glucose residues were detected. As treatment with β -mannanase significantly reduced elicitor activity, mixed-linkage (1 → 4), (1 → 6)-mannosyl residues appeared to be important for elicitor activity. The B. graminis elicitor induced the expression of all defence-related genes tested in wheat and also induced resistance to subsequent attack by B. graminis f. sp. tritici. In contrast, a hypersensitive response was not induced by the elicitor in the absence or the presence of a challenging inoculum of B. graminis f. sp. tritici. The elicitor also induced the accumulation of thaumatin-like proteins in barley, oat, rye, rice and maize, but did not induce necrosis in any of these species. This suggests that the B. graminis elicitor represents a host non-specific determinant of non-self recognition in cereals activating general defence responses other than the hypersensitive reaction.     

棉疫病菌90kD胞外蛋白激发子诱导烟草过敏性反应的研究

 就棉疫病菌90 kD胞外蛋白激发子诱导烟草过敏反应(HR)过程中细胞死亡和防卫反应酶系活性变化及病程相关蛋白PR5的诱导进行研究。结果是,以10 nmol/L激发子溶液注射处理W38烟草叶片,HR枯斑周围5 mm宽组织在UV光下呈现蓝色荧光,对处理部位进行Evans blue染色测定结果是至20 h处理部位细胞全部死亡;激发子可诱导烟草防卫反应中苯丙氨酸解氨酶(PAL)的活性提高;可快速诱导PR5基因的转录。上述结果表明90 kD蛋白激发子可诱发烟草的细胞死亡、苯丙烷代谢和PR基因的表达等多条信号途径。