黑翅土白蚁肠道放线菌菌株BYC-18及其抗菌代谢产物的分离鉴定

【目的】测定黑翅土白蚁肠道放线菌发酵产物的抗菌活性,并对其抗菌活性成分进行分析,以发现新颖的抗菌先导化合物。【方法】采用涂布平板法对黑翅土白蚁肠道放线菌进行分离;通过牛津杯法测试菌株发酵液提取物对4种致病菌(金黄色葡萄球菌Staphylococcus aureus、大肠杆菌Escherichia coli、枯草芽孢杆菌Bacillus subtilis和白色念珠菌Candida albicans)的抗菌活性,筛选出活性菌株BYC-18;通过形态学特征和16S rRNA序列分析确定BYC-18的分类学地位;采用滤纸片法测定BYC-18发酵液在不同极性溶剂萃取物的抗菌活性;运用多种色谱方法从乙酸乙酯粗提物中分离纯化抗菌活性化合物,利用质谱和核磁共振谱鉴定其化学结构;采用滤纸片法和最低抑制浓度法测定分离的化合物的抗菌活性。【结果】BYC-18被鉴定为链霉菌属Streptomyces sp.菌株,该菌发酵液对4种致病菌均有抗菌活性且其乙酸乙酯萃取物对金黄色葡萄球菌的抑制作用明显,抑菌圈直径达11.1 mm。从乙酸乙酯萃取物中分离得到1个单体化合物BYC-18-1,经鉴定为β-玉红霉素(β-ru...     

白蚁巢拮抗放线菌BYC01代谢产物的分离和鉴定

【目的】从白蚁巢中分离出具有抗菌活性的放线菌,并在其代谢产物中寻找具有抗菌活性的先导化合物。【方法】通过形态学观察和16S rRNA序列分析初步确定目标菌株BYC 01的分类地位。利用生长速率法和琼脂扩散法测定其代谢产物的抗菌活性。并通过大量发酵提取浸膏,运用多种色谱方法对发酵产物进行分离、纯化,利用质谱和核磁共振谱分析鉴定出化合物的结构。【结果】通过形态学观察和16S rRNA序列分析,菌株BYC 01被鉴定为紫红链霉菌(Streptomyces violaceoruber)。BYC 01发酵液不同极性溶剂萃取物的抗菌活性结果表明其有效抑菌物质主要存在于中等极性的乙酸乙酯部位。在供试浓度为100μg/mL时,BYC 01发酵液乙酸乙酯萃取物对苹果树腐烂病菌具有强烈的抑制作用,抑制率大于90%;对水稻纹枯病菌和杨树溃疡病菌具有较好抑制活性,抑制率均大于60%;在供试浓度为30μg/滤纸片时,与阳性对照相比,乙酸乙酯提取物对白色念珠菌、金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌和水稻白叶枯病菌有中等的抑制作用,其抑菌圈直径范围为11.3 mm-16.5 mm。经质谱和核磁共振谱分析,从BYC 01发酵产物中分离到的单体化合物被鉴定为fogacin;在供试浓度为30μg/滤纸片时,化合物fogacin对白色念珠菌生长的抑制作用与阳性对照两性霉素相当,其抑菌圈大小分别为19.3 mm和20.1 mm。【结论】菌株BYC 01具有开发为微生物杀菌剂的潜力。     

决明草内生真菌拮抗菌株筛选鉴定及桔青霉ZH-11抗菌物质分析

[背景] 由于抗生素的滥用，导致“超级细菌”出现，寻找新的抗菌药物将有效地应对细菌耐药问题，因为大多数抗菌药物都是从微生物中发现的，所以药用植物内生真菌的研究拓宽了药用资源，并且具有巨大的应用价值。[目的] 对采自江西九江庐山植物园的决明草进行内生真菌分离，筛选出拮抗菌株，并对拮抗菌株的次级代谢产物进行分离，分析其抗菌物质理化性质，为新型抗菌物质的研究提供基础数据。[方法] 用管碟法筛选拮抗菌株，并根据形态学特征和分子生物学的方法鉴定菌株，采用硅胶柱层析、葡聚糖凝胶LH-20柱层析和RP-C18柱层析对其次级代谢产物进行分离，并用液质联用（高分辨飞行时间质谱）和能谱仪分析所得抗菌物质的分子量和分子式。[结果] 筛选到一株广谱拮抗活性菌株桔青霉ZH-11，通过管碟法显示其对大肠杆菌、金黄色葡萄球菌、苏云金芽孢杆菌、水稻黄单胞菌、枯草芽孢杆菌、地衣芽孢杆菌、白色念珠菌、蜡样芽孢杆菌这8株指示菌均有较好的抑菌效果。从桔青霉ZH-11次级代谢产物中分离得到纯化合物Y3，其分子量为410.169 1，分子式为C₂₄H₂₆O₆。当Y3浓度为10 μg/mL时，其对大肠杆菌和苏云金芽胞杆菌的抑菌圈直径分别为16.23 mm和17.27 mm。[结论] 菌株桔青霉ZH-11的活性物质与已知的来源于桔青霉类的抗菌活性物质不同，该研究结果为进一步挖掘桔青霉属的活性产物奠定基础，同时丰富了人们对决明草内生真菌的认识。     

短头熊蜂肠道拮抗菌株的分离筛选鉴定及其生物特性评价

【背景】近年来,由于栖息地减少、农药的大量使用及病原菌侵染等综合因素,导致全世界的熊蜂种类与数量逐年减少,病原菌的侵染可通过微生物在自身生长过程中会产生的抑菌物质进行有效抑制或杀灭。【目的】短头熊蜂(Bombus breviceps)长期生存在野外环境中,其肠道内存在着大量微生物资源。从短头熊蜂肠道内筛选拮抗菌株,并对其抑菌特性进行研究。【方法】采用牛津杯双层法筛选拮抗菌株,测定抑菌活性最佳菌株发酵液的抑菌物质稳定性与抑菌广谱性等抑菌特性,并借助细胞膜通透性、流式细胞仪检测等试验探究其抑菌机制。【结果】得到了5株具有明显抑菌作用的拮抗菌株,其中果杆菌(Fructobacillus tropaeoli)CZ01对金黄色葡萄球菌(Staphylococcus aureus)、沙门氏菌(Salmonella choleraesuis)、大肠杆菌(Escherichia coli)、福氏志贺氏菌(Shigella flexneri)和无乳链球菌(Streptococcus agalactiae)这5种病原指示菌都具有高度抑菌效果。菌株CZ01对金黄色葡萄球菌的抑菌效果最佳,抑菌圈直径可达到(21.21±0.25) mm,在121 ℃处理后仍具有67.36%以上的抑菌活性,调整pH值为10.0时仍具有78.16%的抑菌活性。【结论】短头熊蜂肠道微生物资源较丰富,尤其是果杆菌(F.tropaeoli)CZ01具有抑菌活性高、稳定性好、抑菌谱广等特性,对金黄色葡萄球菌具有良好的杀灭效果,显示出良好的应用潜能。     

家蝇共生真菌的抗菌活性筛选及菌株CY-03的初步鉴定

利用平板分离法从家蝇中分离出14株家蝇共生真菌,活性筛选表明CY-03有较好的抗菌活性,在供试浓度为0.1 mg/m L时,CY-03发酵液乙酸乙酯萃取物对番茄早疫病菌和苹果树腐烂病菌具有较好的抑制作用,抑制率约60%;在供试浓度为30μg/滤纸片时,与阳性对照相比,乙酸乙酯提取物对枯草芽胞杆菌有中等的抑制作用,抑菌圈直径为21.0 mm。进一步研究CY-03发酵液不同极性溶剂萃取物的抗菌活性,结果表明其活性物质主要集中在中等极性部位,在供试浓度为0.1 mg/m L时,CY-03发酵液乙酸乙酯提取物对苹果树腐烂病菌、杨树溃疡病菌、番茄早疫病菌和苦瓜枯萎病菌等具有较好的抑制作用,抑制率均大于45%;在供试浓度为30μg/滤纸片时,乙酸乙酯提取物对金黄色葡萄球菌、枯草杆菌和大肠埃希菌有中等的抑制作用,抑菌圈直径范围为14.8~23.0 mm。通过形态学特征观察和5.8S r DNA测序分析,初步确定该菌株为尖孢镰刀菌(Fusarium oxysporum)。CY-03作为微生物源杀菌剂值得进一步研究。     

老鼠艻内生曲霉HU0206的抗细菌活性代谢成分

为阐明老鼠艻内生曲霉Aspergillus sp.HU0206发酵物的抗细菌物质基础，采用色谱分离法从其玉米发酵物中分离得到7个化合物，分别鉴定为6个甲基苯酚类化合物：sydowiol F (1)、5-methoxy-3,5''-dimethyl-2,3''-oxybiphenyl-1,1'',2''-triol (2)、corynesidone A (3)、鹿蹄草素(4)、3-羟基鹿蹄草素(5)、4-乙氧基-3-羟甲基苯酚(6)和1个甲基环己酮二聚体epoxydine A (7)，其中化合物1为新化合物。载样量为40 μg时，化合物1、2和4对金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌、黑胫病菌和青枯病菌抑菌效果较好，抑菌圈半径≥2 mm，最小抑菌浓度(MIC)为50或100μg/mL；化合物3对这4种菌虽有1~2 mm的抑菌圈，但MIC大于400 μg/mL。所有化合物对大肠杆菌均无抑制活性。     

桐花树活性内生真菌筛选及其抗菌化学成分的研究

为了探究桐花树内生真菌在抑菌方面的价值,该文以内生真菌发酵产物的抑菌作用为评价指标筛选活性菌株,采用生物活性跟踪方法结合多种色谱技术分离活性菌株的化学成分,通过波谱与文献数据比对鉴定单体化合物结构,并利用微孔板法测定单体化合物的抑菌活性。结果表明:(1)从桐花树分离得到的16株内生真菌分属2纲7目10科10属,镰刀菌属(Fusarium)为优势菌属。内生真菌GXIMD02029和GXIMD02039的发酵产物对枯草芽孢杆菌、表皮葡萄球菌、耐甲氧西林金黄色葡萄球菌、藤黄微球菌、粘性放线菌和金黄色葡萄球菌有不同程度的抑制作用,GXIMD02038发酵产物对耐甲氧西林金黄色葡萄球菌、藤黄微球菌和金黄色葡萄球菌有抑制作用。(2)7个化合物从内生真菌Phomopsis sp. GXIMD02029中被分离并鉴定为(15R)-acetoxydothiorelone A(1)、cytosporone B(2)、pestalotiopsone H(3)、pestalotiopsone B(4)、4-Hydroxybenzaldehyde(5)、p-Hydroxybenzoic acid(6)、N-(2-phenylethyl)acetamide(7)。(3)化合物1和2有不同程度的抑菌作用,化合物1对枯草芽孢杆菌、表皮葡萄球菌、耐甲氧西林金黄色葡萄球菌的MIC值为16.25 SymbolmA@ g·mL^-1,对藤黄微球菌和粘性放线菌的MIC值为7.812 5 SymbolmA@ g·mL^-1,对金黄色葡萄球菌的MIC值为31.25 SymbolmA@ g·mL^-1。化合物2对藤黄微球菌的MIC值为62.5 SymbolmA@ g·mL^-1,对枯草芽孢杆菌、表皮葡萄球菌、耐甲氧西林金黄色葡萄球菌、粘性放线菌的MIC值为125 SymbolmA@ g·mL^-1,对金黄色葡萄球菌的MIC值为250 SymbolmA@ g·mL^-1。该文筛选了3株活性菌株,首次报道化合物1具有抗菌活性,为桐花树内生真菌在抗菌价值方面提供了依据。     

南海东海岛沉积物分离细菌的鉴定及其抑菌活性

[背景] 海洋微生物在活性物质开发方面具有巨大的应用前景,而目前有关南海东海岛微生物的研究鲜少。[方法] 对从东海岛沉积物中分离纯化的海洋细菌,采用形态学观察、生理生化以及16S rRNA基因序列的系统发育分析方法进行鉴定;以大肠杆菌（Escherichia coli）、枯草芽孢杆菌（Bacillus subtilis）和金黄色葡萄球菌（Staphylococcus aureus）作为指示菌,测定其抑菌活性;对具有抑菌活性的菌株扩增聚酮合酶（Polyketide synthase I,PKSI）基因,并与已知的PKSI氨基酸序列比对;选择具有PKSI基因的代表菌株,检测菌株及其发酵抑菌物的稳定性。[结果] 分离纯化到25株海洋细菌,分属于不动杆菌属（Acinetobacter）、交替单胞菌属（Alteromonas）、芽孢杆菌属（Bacillus）、嗜冷杆菌属（Psychrobacter）、假交替单胞菌属（Pseudo-alteromonas）、海洋单胞菌属（Oceanimonas）、葡萄球菌属（Staphylococcus）、微球菌属（Micrococcus）和海杆菌属（Marinobacter）。12株菌株通过基因筛选检测到PKSI编码基因,其中6株菌株具有抑菌活性和PKSI编码基因,并分属于芽孢杆菌属和交替单胞菌属;PKSI氨基酸序列同源性分析推测菌株DHD-15和DHD-a可能产生新的I型聚酮合酶结构。菌株DHD-15和DHD-L生长温度范围为15-40℃,可耐受10% NaCl高盐以及pH 3和pH 11的酸碱条件,但不耐高温;菌株DHD-15产生的抑菌物质可耐受100℃和pH 11的高温碱性条件,在50℃、pH 9条件下制备和室温保藏条件下抑菌活性较高,其稳定性较好。[结论] 南海东海岛沉积物筛选的细菌种具有抑菌活性,具有产聚酮类活性物质的潜力。     

中华真地鳖共附生放线菌DB2C2的筛选和鉴定

利用稀释平板法从中华真地鳖体内分离得到52株共附生放线菌,活性筛选表明菌株BD2C2具有较好的抗菌活性,其发酵液对金黄色葡萄球菌的抑菌圈直径超过23mm,对枯草芽胞杆菌的抑菌圈直径约17.5mm,对大肠杆菌也有一定的抑制作用。进一步研究表明其活性物质主要集中在中等极性部位。在供试浓度为每张滤纸片30μg时,菌株BD2C2发酵液的乙酸乙酯提取物对金黄色葡萄球菌、枯草芽胞杆菌和大肠杆菌的抑菌圈直径分别约27.5、22.4和10.7mm,其抑制活性与阳性对照硫酸庆大霉素相当。利用形态学观察和分子生物学方法初步鉴定该菌为Streptomyces sp.,为一个潜在新种。作为微生物源杀菌剂,DB2C2具有一定开发潜力,值得进一步研究。     

海南产木薯茎化学成分研究

从木薯(Manihot esculenta Crantz)茎的乙醇提取物中分离得到6个化合物,通过波谱分析,分别鉴定为呋喃(1)、肥牛木素(2)、3-吲哚甲酸(3)、3,9,13-megastigmanetriol (4)、穗花杉双黄酮(5)、yucalexin P-21 (6),其中化合物1~5为首次从该植物中分离得到。用滤纸片琼脂扩散法测定了这些化合物的抗菌活性,结果表明化合物1、3、4和6对耐甲氧西林金黄色葡萄球菌(MRSA)和金黄色葡萄球菌均有抑制作用。     

过表达或沉默棉花GhPCBER基因株系的获得及其茎叶木质素和木脂素含量研究

编码苯基香豆满苄基醚还原酶(phenylcoumaran benzylic ether reductase,PCBER)的基因PCBER属于PIP亚家族,是苯丙烷代谢途径中参与木脂素合成的关键基因。该研究构建了棉花GhPCBER基因的植物过表达载体并转化拟南芥,同时构建了VIGS(virus induced gene silencing,病毒诱导的基因沉默)载体转化棉花,采用实时荧光定量PCR技术对GhPCBER基因在不同组织中的表达进行分析;对野生型和转基因植株茎叶组织中的木质素和木脂素含量进行测定分析。结果表明:(1)成功构建了GhPCBER植物过表达载体pGWB17-GhPCBRE以及基因沉默重组载体pTRV2-GhPCBER;经遗传转化获得6株转棉花GhPCBER基因抗性拟南芥植株,同时获得15株GhPCBER基因沉默棉花植株(5株为一组)。(2)PCR检测表明,6株转基因拟南芥均为过表达株系,其中株系1、2、3相对表达量更高,且在茎、叶组织中的表达量分别较野生型提高了7～14倍和6～16倍,表明GhPCBER基因成功在拟南芥中过表达;GhPCBER基因沉默棉花植株的茎、叶组织中的表达量分别比野生型棉株约下降12%和26%,表明烟草脆裂病毒(TRV)体系(pTRV2-GhPCBER)成功抑制了GhPCBER基因的表达。(3)转GhPCBER基因拟南芥茎、叶中木质素和木脂素含量较野生型均显著降低;GhPCBER基因沉默棉花植株茎、叶中木质素和木脂素含量较野生型均极显著降低;组织化学染色观察发现GhPCBER基因沉默棉花植株茎秆颜色明显比野生型染色浅,也证明沉默基因棉花植株茎秆中的木质素含量减少。(4)苯丙烷代谢通路中8个相关基因的实时荧光定量PCR分析发现,过表达或抑制GhPCBRE基因均会导致苯丙烷代谢途径发生重新定向。     

Effect of chitinase antisense RNA expression on disease susceptibility of Arabidopsis plants

Chitinases accumulate in higher plants upon pathogen attack are capable of hydrolyzing chitin-containing fungal cell walls and are thus implicated as part of the plant defense response to fungal pathogens. To evaluate the relative role of the predominate chitinase (class I, basic enzyme) of Arabidopsis thaliana in disease resistance, transgenic Arabidopsis plants were generated that expressed antisense RNA to the class I chitinase. Young plants or young leaves of some plants expressing antisense RNA had <10% of the chitinase levels of control plants. In the oldest leaves of these antisense plants, chitinase levels rose to 37–90% of the chitinase levels relative to vector control plants, most likely because of accumulation and storage of the enzyme in vacuoles. The rate of infection by the fungal pathogen Botrytis cinerea was measured in detached leaves containing 7–15% of the chitinase levels of control plants prior to inoculation. Antisense RNA was not effective in suppressing induced chitinase expression upon infection as chitinase levels increased in antisense leaves to 47% of levels in control leaves within 24 hours after inoculation. Leaves from antisense plants became diseased at a slightly faster rate than leaves from control plants, but differences were not significant due to high variability. Although the tendency to increased susceptibility in antisense plants suggests that chitinases may slow the growth of invading fungal pathogens, the overall contribution of chitinase to the inducible defense reponses in Arabidopsis remains unclear.     

The Saccharomyces cerevisiae chitinase,encoded by the CTS1-2 gene,confers antifungal activity against Botrytis cinerea to Transgenic tobacco

The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene has recently been confirmed by in vitro tests to possess antifungal abilities. In this study, the CTS1-2 gene has been evaluated for its in planta antifungal activity by constitutive overexpression in tobacco plants to assess its potential to increase the plant's defence against fungal pathogens. Transgenic tobacco plants, generated by Agrobacterium-mediated transformation, showed stable integration and inheritance of the transgene. Northern blot analyses conducted on the transgenic tobacco plants confirmed transgene expression. Leaf extracts from the transgenic lines inhibited Botrytis cinerea spore germination and hyphal growth by up to 70% in a quantitative in vitro assay, leading to severe physical damage on the hyphae. Several of the F₁ progeny lines were challenged with the fungal pathogen, B. cinerea, in a detached leaf infection assay, showing a decrease in susceptibility ranging from 50 to 70%. The plant lines that showed increased disease tolerance were also shown to have higher chitinase activities.     

菘蓝IiEMF基因的克隆与功能分析

该研究以菘蓝(Isatis indigotica Fort.)转录组数据为基础,克隆得到菘蓝EMF基因的cDNA全长,命名为IiEMF。(1)序列分析表明,IiEMF基因开放阅读框长度为1896 bp,编码631个氨基酸。进化树分析表明,菘蓝IiEMF蛋白与甘蓝(Brassica oleracea)EMF蛋白亲缘关系最为接近。(2)实时定量PCR结果显示,IiEMF在菘蓝不同器官中均有表达,且在叶中表达量最高,果实中表达量最低;IiEMF基因在菘蓝抽薹开花过程中叶内的表达量呈先升后降的趋势,并于初花期表达量达到最高后逐渐降低回落;在花/果期IiEMF基因表达量较花蕾中明显降低。(3)成功构建了超表达载体pCAMBIA1300-EMF,经农杆菌介导侵染拟南芥,PCR鉴定表明,有7株为超表达转IiEMF基因植株。(4)表型观察发现,在长日照和短日照条件下,与野生型相比2个转IiEMF基因拟南芥株系的开花时间都明显较早(提前6~10 d),且转IiEMF基因株系的莲座叶数比野生型多10片以上,叶片也比野生型大而肥厚。(5)qRT-PCR检测结果显示,在拟南芥营养生长过程中,过表达IiEMF显著抑制了拟南芥AtAP1、AtCO和AtLFY的表达,而促进了AtFLC的表达;当拟南芥开花时,转基因株系中的AtAP1和AtFLC表达量均高于野生型,AtCO和AtLFY的表达量显著低于野生型。研究表明,过量表达IiEMF基因能够促使拟南芥提前开花,且IiEMF可能是通过影响多种开花途径来共同调节促进拟南芥的早花。     

The role of phytohormones in basal resistance and Trichoderma-induced systemic resistance to Botrytis cinerea in Arabidopsis thaliana

Thirty-six phytohormone-affected mutants of Arabidopsis thaliana (L.) Heynh. and their parental ecotypes were tested for resistance/susceptibility to Botrytis cinerea Pers.; Fr. and ability to develop Trichoderma-mediated induced systemic resistance (ISR). Ecotype Colombia-0 (Col-0) was relatively resistant to B. cinerea, and Trichoderma harzianum Rifai T39 application at sites spatially separated (roots) from the B. cinerea inoculation (leaves) resulted in reduction of grey mold symptoms. Ecotypes Wassilewskija-4, Nossen-0 and Landsberg-0 had low levels of basal resistance to B. cinerea and were unable to express ISR. Mutants derived from ISR-non-inducible ecotypes displayed ISR-non-inducible phenotypes, whereas the ISR inducibility of mutants derived from the ISR-inducible genotype Col-0 varied according to the type of mutant. Thus, salicylic acid (SA)-impaired mutants derived from Col-0 were ISR-inducible, while ethylene/jasmonic acid (ethylene/JA)-impaired mutants of the same origin were ISR-non-inducible. SA-impaired mutants retained basal level of resistance to B. cinerea, while most ethylene/JA-impaired mutants were highly susceptible. Abscisic acid- and gibberellin-impaired mutants were highly susceptible to B. cinerea and showed ISR-non-inducible phenotypes irrespective of their lines of origin. Auxin-resistant mutants derived from Col-0 were ISR-inducible; mutant originating from Landsberg-0 and mutants which were resistant to both auxin and ethylene were ISR-non-inducible. Most of the arabidopsis genotypes which were unable to express Trichoderma-mediated ISR against B. cinerea exhibited enhanced susceptibility to this pathogen. T. harzianum treatments enhanced the growth of arabidopsis plants regardless of genotype or ISR inducibility.     

Overexpression of Maize IAGLU in Arabidopsis thaliana Alters Plant Growth and Sensitivity to IAA but not IBA and 2,4-D

Overexpression of the IAGLU gene from maize (ZmIAAGLU) in Arabidopsis thaliana, under the control of the CaMV 35S promoter, inhibited root but not hypocotyl growth of seedlings in four different transgenic lines. Although hypocotyl growth of seedlings and inflorescence growth of mature plants was not affected, the leaves of mature plants were smaller and more curled as compared to wild-type and empty vector transformed plants. The rosette diameter in transgenic lines with higher ZmIAGLU expression was also smaller compared to the wild type. Free indole-3-acetic acid (IAA) levels in the transgenic plants were comparable to the wild type, even though a decrease in free IAA levels might be expected from overexpression of an IAA-conjugate–forming enzyme. IAA-glucose levels, however, were increased in transgenic lines compared to the wild type, indicating that the ZmIAGLU gene product is active in these plants. In addition, three different 35SZmIAGLU lines showed less inhibition of root growth when cultivated on increasing concentrations of IAA but not indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D). Feeding IAA to transgenic lines resulted in increased IAA-glucose synthesis, whereas the levels of IAA-aspartate and IAA-glutamine formed were reduced compared to the wild type. Our results show that IAA homeostasis can be altered by heterologous overexpression of a conjugate-forming gene from maize.     

The grapevine polygalacturonase-inhibiting protein (VvPGIP1) reduces Botrytis cinerea susceptibility in transgenic tobacco and differentially inhibits fungal polygalacturonases

Polygalacturonase-inhibiting proteins (PGIPs) selectively inhibit polygalacturonases (PGs) secreted by invading plant pathogenic fungi. PGIPs display differential inhibition towards PGs from different fungi, also towards different isoforms of PGs originating from a specific pathogen. Recently, a PGIP-encoding gene from Vitis vinifera (Vvpgip1) was isolated and characterised. PGIP purified from grapevine was shown to inhibit crude polygalacturonase extracts from Botrytis cinerea, but this inhibitory activity has not yet been linked conclusively to the activity of the Vvpgip1 gene product. Here we use a transgenic over-expression approach to show that the PGIP encoded by the Vvpgip1 gene is active against PGs of B. cinerea and that over-expression of this gene in transgenic tobacco confers a reduced susceptibility to infection by this pathogen. A calculated reduction in disease susceptibility of 47–69% was observed for a homogeneous group of transgenic lines that was statistically clearly separated from untransformed control plants following infection with Botrytis over a 15-day-period. VvPGIP1 was subsequently purified from transgenic tobacco and used to study the specific inhibition profile of individual PGs from Botrytis and Aspergillus. The heterologously expressed and purified VvPGIP1 selectively inhibited PGs from both A. niger and B.␣cinerea, including BcPG1, a PG from B. cinerea that has previously been shown to be essential for virulence and symptom development. Altogether our data confirm the antifungal nature of the VvPGIP1, and the in vitro inhibition data suggest at least in part, that the VvPGIP1 contributed to the observed reduction in disease symptoms by inhibiting the macerating action of certain Botrytis PGs in planta. The ability to correlate inhibition profiles to individual PGs provides a more comprehensive analysis of PGIPs as antifungal genes with biotechnological potential, and adds to our understanding of the importance of PGIP:PG interactions during disease and symptom development in plants.Dirk A. Joubert and Ana R. Slaughter contributed equally to this work.     

砂藓RcPLD基因的抗旱功能分析

砂藓(Racomitrium canescens)是一种具有极强耐脱水性的苔藓植物,编码磷脂酶D的基因RcPLD能够在砂藓的脱水和复水过程中产生显著的表达响应,它可能参与了砂藓的强耐脱水性功能。该研究使用已克隆的RcPLD编码序列构建拟南芥(Arabidopsis thaliana)过量表达转基因株系rcpld-oe,初步考察过表达株系的干旱胁迫耐受能力及其相关的生理生化指标,分析RcPLD增强拟南芥抗旱性的机制。结果表明:(1)利用已克隆的RcPLD编码序列构建了植物中的过表达载体,成功构建了RcPLD的过表达转基因拟南芥株系rcpld-oe,并获得了多个T_3代rcpld-oe纯合体株系。(2)在正常生长条件下,rcpld-oe株系T_3代纯合体植株比野生型拟南芥植株体积小,但营养生长期较长,抽薹较晚,莲座叶衰老速率较慢;在干旱处理条件下,rcpld-oe株系表现出比野生型拟南芥更强的干旱耐受能力。(3)在干旱胁迫处理过程中,rcpld-oe株系莲座叶的水分散失速率降低,可能在一定程度上降低了干旱对膜完整性的损伤和光合作用的抑制,但其渗透调节物质含量的变化相对较小。研究发现,在干旱胁迫条件下,rcpld-oe植株莲座叶的水分散失速率和光合作用抑制程度显著降低,从而表现出明显强于野生型的干旱耐受能力,这为后续RcPLD功能的深入研究和更多砂藓抗旱功能基因的挖掘奠定了基础。     

Inhibition of CAT enzyme activity in Arabidopsis thaliana

Previously it was shown that transient chloramphenicol acetyltransferase (CAT) marker gene expression in Arabidopsis thaliana and Nicotiana tabacum resulted in significant differences in the accumulation of the CAT reaction products in radioactive CAT assays. Compared to Nicotiana tabacum, conversion of chloramphenicol to the acetylated products in Arabidopsis thaliana extracts was rather low. Here we report that the low CAT enzyme activity can be attributed in part to a heat sensitive CAT inhibitory effect in extracts of Arabidopsis thaliana. CAT enzyme activity in transgenic tobacco is inhibited by extracts from Arabidopsis. This inhibitory effect diminishes when Arabidopsis extracts were heat incubated. CAT activity in transgenic Arabidopsis lines was very low and was only detected in heat incubated extracts. Alternatively, enzyme-linked immunosorbent assays (ELISAs) can be used to detect the CAT protein in transgenic Arabidopsis.Abbreviations CAT chloramphenicol acetyltransferase - CAM chloramphenicol - ELISA enzyme linked immunosorbent assay     

Constitutive expression of an inducible lipoxygenase in transgenic tobacco decreases susceptibility to Phytophthora parasitica var. nicotianae

Plant lipoxygenases (LOXs) are key enzymes involved in the generation of fatty acid derivatives, called oxylipins. In tobacco, LOX gene expression and activity are very low in healthy tissues and are highly enhanced in response to infection by Phytophthora parasitica nicotianae and to elicitor treatment. We previously showed, using antisense-LOX1 plants, that expression of the tobacco LOX1 gene is required for the race-cultivar specific resistance of tobacco to Phytophthora parasitica nicotianae. In order to investigate the effect of over-expressing a LOX gene on plant resistance, we transformed tobacco plants with the LOX1 coding sequence fused to the CaMV 35S promoter. Four transgenic lines with enhanced levels of LOX protein and specific activity over control plants were selected for further analysis. These plants were macroscopically indistinguishable from WT plants. Upon stem inoculation, the sense-LOX1 plants displayed a significantly decreased susceptibility to virulent races of Phytophthora parasitica nicotianae, stem lesions being 2- to 3-fold shorter in the transgenic lines than in WT plants. Using a root inoculation assay, the survival rate of sense-LOX1 seedlings was increased about 4-fold compared to their WT counterparts, with 60 to 80% of transgenic plants vs 15 to 20% of WT controls remaining healthy following inoculation with Phytophthora parasitica nicotianae. This is the first demonstration that the over-expression of a LOX gene is sufficient to reduce the susceptibility of a host plant to an oomycete pathogen.