水分胁迫对抗旱性不同冬小麦品种可溶性蛋白质的影响

利用SDS－PAGE研究了8个抗旱性不同的冬小麦品种在大田自然水分胁迫和供水处理下小麦旗叶和籽粒可溶性蛋白质的动态变化情况及其与品种抗旱性的关系，结果表明：（1）在干旱诱导条件下，同一品种冬小麦可溶性蛋白种类在不同生育时期有较大变化，分子量呈现以76．5kD蛋白带为中心先升后降的趋势，品种间的变化类似，各品种不同时期可溶性蛋白在70－80kD附近波动，籽粒中可溶性蛋白变化不同，开花后14d时为60－70kD，开花后21d 8个品种处理与对照各差异蛋白质带消失，（2）在拔节期抗旱品种和抗旱高产品种中76．5kD和68．7kD2条带显著增强，不抗旱品种只有68．7kD1条带显著增强。     

一种促进植物根系生长的极细链格孢菌蛋白质分离、纯化和生物功能

通过硫酸铵沉淀、有机溶剂沉淀、离子交换和分子筛连用的方法，从极细链格孢菌JH505菌株中分离纯化到分子量约为35kD的植物激发子。利用固相梯度凝胶双向电泳方法测定了该蛋白的等电点为4．22。将该纯化蛋白稀释液浸泡小麦种子8h，7d后小麦根系琥珀酸脱氢酶活性提高65．27％，经蛋白处理的小麦根长比对照提高13．1％。     

枯草芽胞杆菌胞外抗菌蛋白的初步分离及性质的研究

土壤中分离的枯草芽胞杆菌,其发酵液对苹果炭疽病菌具有很强的抑制作用。为了确定其抑菌物质的主要成分,本试验采用硫酸铵分级沉淀获得无菌滤液中的抗菌蛋白粗提物,超滤后经过聚丙烯酰胺凝胶电泳,胶带回收进行分离鉴定,得到1种具有抑菌活性的蛋白条带。该蛋白对供试的6种植物病原真菌具有抑制作用,该蛋白的活性表现在80℃,30min仍有抑菌作用,对pH(4~7)具有一定的适应范围、另外对紫外照射(0~12h)、抑制剂、有机溶剂均不敏感。质谱鉴定该条带含有两种蛋白,分别为假定蛋白BSU35980gi|16080651,相对分子量11 079Da,等电点4.78,匹配率为68%;丝氨酸羟甲基转移酶gi|16080743,相对分子量为45 575Da,等电点为5.56,匹配率为67%。显微观察发现抑菌蛋白可以使菌丝发生断裂、扭曲、畸形、肿大。     

八字地老虎β-N-乙酰葡萄糖胺糖苷酶基因cDNA序列的克隆及原核表达研究

［目的］ 克隆八字地老虎β-N-乙酰葡萄糖胺糖苷酶基因的cDNA序列并进行原核表达。［方法］ 以八字地老虎预蛹期幼虫为材料提取总RNA,利用RT-PCR和RACE技术克隆基因cDNA序列,将cDNA序列编码区连接到原核表达载体pET21b并转化BL21进行原核表达,并使用His tag纯化试剂盒将目的蛋白进行纯化。［结果］ 得到cDNA全长序列,含有2 488个碱基,包括一个1 785个碱基的开放阅读框,编码一个含594个氨基酸的蛋白,分子量为67.8 ku,等电点5.38,该序列登录GenBank并获得登录号FJ848784。诱导表达蛋白经SDS PAGE电泳得到目的蛋白带,纯化结果也获得了目的蛋白带。［结论］获得了一条新的八字地老虎β-N-乙酰葡萄糖胺糖苷酶基因的cDNA序列并对其在原核表达系统中进行了成功表达和纯化。     

稻白叶枯病菌外泌蛋白激发子的活性测定及其纯化

采用(NH4)2SO4分步沉淀法,从水稻白叶枯病菌的培养液中分离到具有激发子活性的物质。该蛋白处理水稻,可使水稻体内与抗病性相关的苯丙氨酸解氨酶和过氧化物酶活性显著提高;在田间喷洒水稻叶片,可以显著提高水稻对白叶枯病菌的抗性,病斑长度明显降低。经滚动式等电聚焦电泳和离子交换层析分离,生测结果表明第II吸收峰具有生物活性;再经SDS PAGE电泳和考马斯亮蓝染色,第II吸收峰收集物有明显的一条带,相对分子质量为47 9kD。     

壳二孢叶枯病病原菌Ascochyta rabiei胁迫下鹰嘴豆防御相关基因的表达分析

为挖掘获得新的抗性基因,培育鹰嘴豆Cicer arietinum抗壳二孢叶枯病(病原菌为Ascochyta rabiei)新品种,以项目组前期获得的102个差异表达的新基因为基础,随机选取29个基因进行同源性分析,以鹰嘴豆Actin(EU529707)和Ef-1α(AJ004960)作为参考基因,利用实时荧光定量PCR技术检测这29个基因在宿主植物鹰嘴豆抗性品种系选03中的表达规律。结果显示,基于同源性分析结果可将29个基因大致分成4类,涉及信号传导机制、细胞运输、转录和细胞拯救、防御、毒性;功能分析结果显示,功能未知的基因数目最多,达到了11个,其中多数为鹰嘴豆未定性基因。这29个基因在A. rabiei胁迫下都发生了不同程度的差异表达,表达差异时间点集中在胁迫后72 h,并在96 h恢复至正常表达水平。其中解毒相关基因474在72 h时相对表达水平最高,是对照处理0 h的19.773倍,抗氧化修复相关基因1137的相对表达水平最低,约为对照处理0 h的1/3。筛选获得的差异表达基因中,表达上调的基因有10个,表达下调的基因有16个,其余3个基因的表达差异不明显。上调表达基因可能与鹰嘴豆应对A. rabiei侵染的应答机制有关,其中与免疫应激相关的蛋白基因如谷胱甘肽S-转移酶、咖啡酰辅酶A、泛素蛋白基因等可能直接参与了鹰嘴豆应对A. rabiei的免疫识别和防御。     

白叶枯病菌与非亲和水稻IRBB4悬浮细胞互作中蛋白类激发子的纯化和鉴定

 本研究对水稻白叶枯病菌与水稻悬浮细胞非亲和互作中蛋白类激发子进行了分离纯化和鉴定.白叶枯病菌JXOV与水稻IRBB4和IR24悬浮细胞互作36 h后的上清液,经Q-Sepharose阴离子交换层析柱分离,对分离的各组分进行抗病性诱导测定,结果表明JXOV与IRBB4非亲和互作的上清液中存在蛋白类激发子.有活性的蛋白组分经阴离子交换层析柱Mono-Q进一步纯化后,SDS-PAGE分析鉴定出2个具激发活性的蛋白,其分子量分别为17.2 kD和49.2 kD,等电点分别为5.8和6.2.利用上述激发子处理水稻能减少病斑长度并诱导水稻防卫酶活性的增加.     

小地老虎2种不同类型肌动蛋白基因的克隆与序列分析

本研究以鳞翅目夜蛾科昆虫小地老虎［Agrotis ipsilon（Hüfnagel）］3龄幼虫整个虫体为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术（RACE）,分别扩增得到2条肌动蛋白的cDNA序列Aiactin1和Aiactin2。GenBank数据库搜索及序列比对结果表明,克隆的2条肌动蛋白基因应属于2种不同类型的肌动蛋白,Aiactin1为肌肉特异型肌动蛋白,Aiactin2为细胞质特异型肌动蛋白。Aiactin1的cDNA序列含有1 469个碱基,Aiactin2的cDNA序列含有1 408个碱基。2条基因的cDNA序列均包括一个1 131个碱基的开放阅读框,编码一个含376个氨基酸的蛋白。Aiactin1肌动蛋白分子量约为41.77 ku,等电点5.22;Aiactin2肌动蛋白分子量约为41.83 ku,等电点5.47。Prosite软件分析结果表明,Aiactin1和Aiactin2肌动蛋白氨基酸序列中都存在3个肌动蛋白特征片段。2个基因的cDNA序列已经登录GenBank并获得登录号。     

多粘类芽孢杆菌BRF-1抗菌蛋白的分离纯化

通过硫酸铵分级沉淀、Sephadex G-50柱层析并采用抑菌活性和SDS-PAGE跟踪检测，从多粘类芽孢杆菌BRF-1菌株代谢产物中分离纯化到一种对大豆立枯丝核菌具有拮抗活性的抗菌蛋白，分子量约35．4kD。     

水稻纹枯病菌PG的分离纯化及其理化性质研究

水稻纹枯病菌产生的多聚半乳糖醛酸酶(Polygalacturonase,PG)是其重要的致病因子之一。用丙酮法提取PG粗蛋白,分别经DEAE-Sepharose Fast Flow离子交换柱、Phenyl-Sepharose 6 Fast Flow疏水柱、Sephadex G-75凝胶柱和DE52离子交换柱层析纯化得到一种具有较高活性的PG纯蛋白。该蛋白分子量为39.81 kD;等电点为4.58;含有糖基,含糖量为1.48%;含有α氨基酸,但不含芳香族氨基酸;不含脂基。这种蛋白在pH4~12范围内均具有活性,pH5时活性最大;对热不稳定,100℃下水浴20 min,活性完全丧失;对胰蛋白酶和蛋白酶K敏感,酶处理后其活性只有对照的35.0%和35.2%;对紫外线和氯仿亦敏感,处理后活性仅为对照的40.0%和51.7%。     

Identification of novel sources of resistance to Pea enation mosaic virus in chickpea germplasm

Chickpea can be seriously affected by Pea enation mosaic virus (PEMV) in the Pacific Northwest region of the USA and other areas of the world. Use of pesticides to manage PEMV vector transmission is ineffective and PEMV-resistant chickpeas have not been identified. Therefore, the Cicer core collection consisting of 499 wild Plant Introduction (PI) accessions and improved cultivars representing 25 countries and two chickpea phenotypes (Desi and Kabuli) was evaluated for resistance to PEMV based on a 1 to 5 scale where 1 = symptomless and 5 = severe mosaic and stunting. Eight accessions were considered resistant to PEMV based on mean disease severity values equal to or less than 1·9 in repeated greenhouse tests. All of the resistant accessions to PEMV were of the Desi type and all originated from Iran or India. PI 450763 was the only Kabuli accession that demonstrated tolerance to PEMV. PEMV resistance was not detected in commercial chickpea cultivars or advanced breeding lines currently grown in the USA and Canada. This is the first report of chickpea germplasm with resistance to PEMV.     

稻瘟病菌66 kDa糖蛋白激发子的纯化及性质研究

 采用离心、超滤、柱层析等方法对稻瘟病菌(Magnaporthe grisca) ZC₁₃菌株97-151a细胞壁来源的水溶性糖蛋白激发子进行了纯化;经SDS-PAGE检测,GP66达到电泳纯;凝胶过滤法及SDS-PAGE测定表明,GP66分子量为64~66 kDa;经蒽酮-硫酸法检测,糖/蛋白比例为3.84;可诱导水稻叶片过氧化物酶与苯丙氨酸解氨酶活性升高。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,GP66的活性位点为糖基部分。     

Purification and characterization of a Bacillus cereus collagenolytic/proteolytic enzyme and its effect on Meloidogyne javanica cuticular proteins

A novel collagenolytic/proteolytic enzyme isolated from the bacterium Bacillus cereus was purified and characterized. The extracellular enzyme was secreted into the growth medium only after induction by collagen. It was purified by two-step chromatography, consisting of gel filtration through a Sephadex G-100 column and then through an anion-exchange column. Molecular mass, as determined by SDS-PAGE was 42.8. The 42.8-kDa collagenase band was eluted from the gel to obtain the purified enzyme. The enzyme was found to have a very wide range of optimal pHs for activity (5.4 – 8.2), and was stable at temperatures between 4 and 40 °C. In addition to its collagenolytic property, the enzyme revealed very strong proteolytic activity, demonstrated by its ability to digest bovine serum albumin. The enzyme's ability to damage nematode cuticles was demonstrated by the digestion of collagens extracted from intact cuticles of second-stage juveniles of the root-knot nematode Meloidogyne javanica.     

稻瘟菌诱导性稻叶脂氧合酶的进一步纯化及抗体制备

 采用DEAE-Toyopearl离子交换、Buty l-Toyopear l疏水层析、CM-Toyopearl离子交换、Sephacryl S100凝胶过滤和FPLCMono Q等步骤,从非亲和性稻瘟菌侵染稻叶中纯化了两种诱导性脂氧合酶,即CM-Loxl和CM-Lox2。SDS-PAGE检测结果表明:CM-Lox1和CM-Lox2为单链多肽,它们的分子量分别为98 kd和102 kd。利用微量免疫法制备了CM-Lox1和CM-Lox2的抗体,制备的抗体效价达到1:1000倍,能检测0.5~2.0 ng的抗原。免疫印迹杂交证明CM-Lox1和CM-Lox2之间存在密切的血清学关系。此外,Anti-CM-Lox1和Anti-CM-Lox2与水稻中另一稻瘟菌诱导性脂氧合酶RLL以及发芽种子出现的RL-2也存在交叉反应。     

棉花黄、枯萎病拮抗菌的筛选及抗菌蛋白B110-a的初步测定

从分离自中药上的307株芽孢杆菌中,筛选到3株对棉花黄、枯萎病菌均有强烈抑制作用的菌株,B₁₁₀、B₁₈₄和B₂₁₆,其中B₁₁₀拮抗作用尤为显著。B₁₁₀分离自中药白豆蔻,根据其形态特征和生理生化特征初步鉴定为枯草芽孢杆菌,其蛋白粗提液经Sephadex^TMG-75后得到两个峰,其中峰a有较强抑菌活性,命名为B_110-a,峰b基本无活性;SDS-PAGE分析结果显示B₁₁₀-a主要由两种蛋白组成,含量较高的分子量为50kD,含量较低的分子量为29kD。     

Purification and partial characterization of xylanase from the fungal maize pathogenHelminthosporium turcicum (Pass)

The fungal pathogenHelminthosporium turcicum was found to secret xylanase when grown on minimal medium containing xylans, wheat straw or isolated maize cell walls. The highest xylanase activity occurre when the fungus was grown on maize cell walls. When glucose was added to this medium xylanase activity was suppressed. The xylanase enzyme was purified from the culture filtrate by subsequent anion exchange chromatography, cation exchange chromatography and gel filtration. The purified xylanase gave a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis corresponding to an apparent molecular weight of 22.5 kDa. It is determined to have a pI of 7.4, specific activity of 11300 nanokatals mg^–1, pH optimum between pH 5.5 and 6.5 and optimal temperature between 50 °C and 60 °C. The half-life of the enzyme at pH 6.0 and 50 °C was found to be 35 min. For primary structure comparison with other xylanases, the protein was digested with trypsin and the resulting peptides were separated by reversed phase chromatography and selected peptides were sequenced. The determined amino acid sequence showed high homology with xylanase fromCochliobolus carbonum and three other fungal xylanases.     

Production of a proteinaceous phytotoxin by Ascochyta rabiei grown in expressed chickpea sap

Production of the solanapyrone toxins by Ascochyta rabiei is nutrient dependent. When grown on a medium consisting entirely of expressed sap from the aerial parts of young chickpea plants (PSM). only low concentrations of the solanapyrones were produced (< 24 μM). However, toxicity of 4-day-old culture filtrates to isolated cells of chickpea leaflets was comparable with that obtained from 12-day-oId culture filtrates on Czapek Dox nutrients supplemented with chickpea seed extract or cations—media that are conducive to solanapyrone production. The additional toxic component which peaked at 4 days in culture was heat labile, losing about 50% of its activity on boiling for 10 min. Affinity for solid-phase extraction media, precipitation with ammonium sulphate and acetone, ionization properties and UV absorption characteristics suggested that the toxin was a polypeptide. The toxin was purified by solid-phase extraction, acetone precipitation and high performance liquid chromatography (HPLC) on a C2 column. Hydrolysis of the purified toxin yielded 14 amino acids, and calculation of the numbers of residues of each amino acid suggested a molecular mass of 7, 551 Da, A band corresponding to this molecular mass was present on SDS-PAGE gels. However, both the native peptide and its hydrolysate contained a compound that reacted with p -anisaldehyde suggesting the possibility of a glycosidic moiety.     

抗菌蛋白磷酸盐结合蛋白前体的分离及其产生菌的鉴定

以抑菌活性生物测定结果为指导,采用硫酸铵沉淀、离子交换柱层析、凝胶柱层析和SDS-聚丙烯酰胺凝胶电泳等方法,从采自广西土壤的1株放线菌菌株GXWl发酵液中分离到1种未见文献报道具有抑菌活性的蛋白,经质谱鉴定并与NCBInr蛋白数据库比对,认为其应为磷酸盐结合蛋白前体 (phosphate-binding protein precursor,pre-PBP),分子质量为39.18 ku。根据其形态特征、培养特征、生理生化特性及16S rDNA序列分析,将菌株GXWl归入链霉菌属,并鉴定为锈赤蜡黄链霉菌Streptomyces rubiginosohelvolus。     

利迪链霉菌E12发酵液及其粗提物的抑菌活性初探

利迪链霉菌E12的发酵液对多种植物病原真菌具有较强的抑制活性。采用琼脂稀释法考察了淀粉和葡萄糖对利迪链霉菌发酵液抑菌活性的影响;采用大孔树脂吸附层析和浓缩沉淀提取发酵液中的抑菌活性物质;利用聚丙烯酰胺凝胶(SDS-PAGE)与含β-1,3-葡聚糖的琼脂糖凝胶直接反应测定粗提物的分子质量及β-1,3-葡聚糖酶活性并采用琼脂扩散法测定其抑菌活性。结果表明:当以淀粉作为惟一碳源时发酵液的抑菌率为89.2%,当以葡萄糖作为惟一碳源时发酵液无抑菌活性;在SDS-PAGE上仅出现1条大小为3 kDa左右的条带,且该条带具有抑菌活性,与其位置对应的琼脂糖凝胶上出现透明带。以上结果表明,利迪链霉菌E12可能产生一种具有β-1,3-葡聚糖酶活性的抑菌活性物质。