一株表面活性剂产生菌的分离及抑菌活性

【目的】研究分离得到的表面活性剂产生菌的产表面活性剂能力、分类地位和抑菌活性。【方法】采用血平板、油平板进行表面活性剂产生菌的分离,以排油圈法进行表面活性的测定;通过生理生化特性和16S rDNA序列相似性分析对BS1菌株进行初步鉴定;利用对峙培养法和菌丝生长、孢子囊形成、孢子萌发的抑制率测定研究其抑菌活性。【结果】从石油污染土壤中分离到的BS1菌株可产生表面活性剂,在分类学地位上属于假单胞菌属(Pseudomonas sp.)。BS1菌体、发酵上清液、挥发性物质对12种供试病原真菌均表现出一定的抑制作用。BS1菌体、发酵上清液对大豆疫霉菌(Phytophthora sojae)的抑制率最大,分别达到65.31%和95.93%。发酵上清液通过影响大豆疫霉菌菌丝生长、孢子囊形成、孢子萌发等方式抑制病原菌的正常生长,稀释20倍的发酵上清液依然具有明显的抑制作用。BS1菌株产生的挥发性物质对大豆菌核菌(Sclerotinia sclerotiorum)的抑菌效果最好,抑制率达到84.25%。【结论】BS1菌株在产生表面活性剂的同时,还具有生物防治作用潜力。     

炭团菌提取物对樟子松枯梢病菌的抑菌活性及稳定性研究

采用水提和酯提2种方式提取炭团菌液体培养菌液中高效抑制樟子松枯梢病菌活性物质。采用生长速率法测定提取物对病原菌菌丝生长的抑制率,采用悬滴法测定其对病原菌孢子萌发的抑制率。炭团菌液体培养菌液乙酸乙酯提取物稳定性试验选用紫外线、温度、pH、氧化剂与还原剂以及贮存时间5个因子。结果表明:炭团菌液体培养菌液的5种提取物对樟子松枯梢病菌均有一定的抑菌活性,乙酸乙酯提取物对病原菌菌丝生长的抑制率最高,达到73.20%;乙酸乙酯提取物、超声波提取物、正丁醇提取物对病原菌孢子萌发抑制率均超过90%。乙酸乙酯提取物对自然环境下的紫外线、温度具有很高的稳定性,并且有一定的抗氧化还原能力和良好的耐热性,长期贮存不影响其抑菌活性,具有很高的开发价值和很好的应用前景。     

植物提取物对大豆疫霉菌抗菌活性的初步研究

采用Paper Disk法测定了云南省200种植物的提取物对大豆疫霉(Phytophthora sojae)的抑菌活性。结果表明,光叶合欢(Albizia lucidior)、白钩藤(Uncaria sessilifructus)及黄豆树(Albizia procera)的提取物在浓度为10μg/μL时有较好的抗菌活性,而在2.5μg/μL时只有黄豆树提取物显示出抗菌活性。并从黄豆树中分离出其抗菌的主要活性成分3-O-[α-L-arabinopyranosyl-(1→2)-β-D-fucopyranosyl-(1→6)-2-acetamidO-2-deoxy-β-D-glu-copyranosyl]echinocystic acid。     

不同生育期菊芋叶片过氧化物酶同工酶表达特点的研究

用聚丙烯酰胺凝胶电泳(PAGE)法,分离了不同生育期菊芋叶片过氧化物酶(Helianthus tuberosus.1eves peroxidase,HLP)同工酶,并测定了HLP的酶活性;比较了菊芋叶及其他4种植物POD的特点。结果表明:(1)同一植株不同部位的HLP酶活性大小顺序为,中部成熟叶(6.89×103U/g)>底部衰老叶(5.38×103U/g)>上部嫩叶(2.82×103U/g);由嫩叶→成熟叶→衰老叶的发育过程中,HLP同工酶酶带共有9条,出现的方式有稳定型(Rf 0.81带)、酶活性先弱后强型(Rf0.52、0.67、0.74、0.85带)、酶带先有后无型(Rf 0.70、0.72带)、酶带先无后有型(Rf 0.77)4种。(2)几种植物POD的酶活性的大小顺序是:甘蔗叶>竹笋壳>菊芋叶>大豆壳;POD同工酶电泳最大迁移率(Rf)大小顺序是:菊芋叶(0.85)>甘蔗叶(0.70)>竹笋壳(0.61)>大豆壳(0.57)>辣根(0.55),HLP同工酶属阴离子型。探讨了菊芋POD的表达特点。     

香茅精油对番茄早疫病菌的抑菌作用及抑菌机制

为探明香茅精油的抑菌作用及其在植物病害生物防治中的应用价值,采用平板抑菌法和熏蒸法测定了香茅精油对番茄早疫病菌的抑菌活性,及其对菌丝体内电解质渗漏、可溶性蛋白质含量、丙二醛(MDA)含量、营养物质的吸收和超氧化物歧化酶(SOD)活性的影响.结果表明: 香茅精油对番茄早疫病菌有较强的抑菌作用,且该作用具有时间-剂量依赖性,但无杀菌作用.采用熏蒸法处理的抑制效果较平板抑菌法更好,处理48 h后,半抑制浓度(IC₅₀)分别为13.40 μL·L^-1和103.23 mg·L^-1;处理144 h后,IC₅₀分别为33.81 μL·L^-1和145.16 mg·L^-1.125 mg·L^-1香茅精油处理12 h后,菌丝体的电导率和MDA含量分别为对照的2.7和2.2倍,SOD活性和可溶性蛋白质含量分别提高88.5%和21.9%,还原糖的吸收减少11.3%.香茅精油可通过破坏病原菌细胞膜完整性和抑制菌体对营养物质的吸收来抑制病原菌菌丝的生长.香茅精油在植物病害生物防治中有一定的开发潜力.     

炭团菌提取物对樟子松枯梢病菌的抑菌活性及稳定性

摘要 【目的】获得炭团菌液体培养菌液中高效抑制樟子松枯梢病菌的活性物质。【方法】炭团菌液体培养菌液中活性物质的提取采用水提和酯提2种方式。水提方式采用直接提取法和超声波提取法2种方法,酯提方式选择正丁醇、乙酸乙酯、乙醇3种萃取剂。各提取物对病原菌的抑菌活性研究选用菌丝生长抑制率和孢子萌发抑制率2个指标,对病原菌菌丝生长的抑制测定采用生长速率法,对病原菌孢子萌发的抑制试验采用悬滴法。炭团菌液体培养菌液乙酸乙酯提取物稳定性的研究选用紫外线、温度、pH值、氧化剂与还原剂以及贮存时间5个因子。乙酸乙酯提取物中抑菌活性成分的分离采用柱层析、纯化采用薄层层析、结构鉴定采用紫外光谱（UV）、红外光谱（IR）、气质联用（GC-MS）和核磁共振（NMR）等分析测试技术。【结果】炭团菌液体培养菌液5种提取物对樟子松枯梢病菌均有一定的抑菌活性,其中乙酸乙酯提取物对病原菌菌丝生长的抑制率最高,达到73.20%;乙酸乙酯提取物、超声波提取物、正丁醇提取物对病原菌孢子萌发抑制率均超过90%。乙酸乙酯提取物对自然环境下的紫外线、温度具有很高的稳定性,并且具有一定的抗氧化还原能力和良好的耐热性,长期贮存不影响其抑菌活性。乙酸乙酯提取物中有3个组分,组分I的抑菌率最高（72.94%）。组分I含3种化合物,其中得率最高（57.952%）的为对甲氧基肉桂酸甲酯,化学式为C11H12O3。其他两种化合物为邻苯二甲酸二丁酯和邻苯二甲酸二异丁酯。本研究首次发现上述3种化合物的天然存在。【结论】炭团菌液体培养菌液乙酸乙酯提取物对樟子松枯梢病菌具有较高的抑菌活性,在自然环境下具有很高的稳定性,具有很高的开发价值和应用前景,其主要抑菌活性成分为对甲氧基肉桂酸甲酯。对甲氧基肉桂酸甲酯作为医药中间体主要用于化妆品中,作紫外防护吸收剂;邻苯二甲酸二丁酯和邻苯二甲酸二异丁酯均是聚氯乙烯最常用的增塑剂。上述3种化合物天然存在的发现,对于医药工业和化学工业具有极为重要的意义。     

小麦内生细菌的分离及其对小麦纹枯菌的拮抗作用*

利用涂布平板法从小麦根系中分离出8株内生细菌,从中筛选出1株对小麦纹枯菌(Rhizoctoniacerealis)具有拮抗作用的内生菌。室内测定该菌株培养液对小麦纹枯病菌的抑制作用,结果发现,小麦纹枯病菌在培养液中生长缓慢,培养6d后菌丝量与对照相比下降了89%,同时发现病菌菌丝生长畸形,出现断裂和细胞壁瓦解。双抗标记法测定该拮抗菌在小麦根系中的定殖情况,发现该菌能够在根系中长期定殖。初步的鉴定结果表明该菌为蜡样芽孢杆菌。     

灰毛豆叶片的化学成分研究

从灰毛豆叶片中分离得到10个化合物,通过波谱数据和理化性质分别鉴定为:stigmast-1,5-dien-3β-ol(1),clemaphenol A(2),松脂醇(3),桉素(4),3-吲哚甲醛(5),色氨酸(6),(S)-4-苄基-2-噁唑烷酮(7),4-羟基苯基丙酸(8),buteaspermanol(9),bovatachalcone(10)。其中化合物1、2、4、5、7、8、9首次从该植物中分离得到。     

甘草提取物的抑菌活性研究

目的:研究甘草提取物的体外抑菌活性。方法:以甘草为原料,用氯仿、乙酸乙酯、正丁醇、95%乙醇和水五种不同极性的溶剂对其进行提取。以金色葡萄球菌和大肠杆菌为代表菌种,通过纸片扩散法考察这些提取物对的抑制效果。结果:甘草提取物中不同极性成分的物质对两种菌均有一定的抑制效果,而且对金黄色葡萄球菌的抑菌活性比大肠杆菌的抑菌活性强。     

七星瓢虫成虫石油醚提取物的化学成分及抑菌活性

【目的】确定七星瓢虫Coccinella septempunctata成虫石油醚提取物的化学成分及其抑菌活性。【方法】用甲醇室温提取, 石油醚、 乙酸乙酯和正丁醇梯度萃取, 利用气相色谱-质谱连用仪（Gas Chromatograph-Mass Spectrometer, GC-MS）对七星瓢虫成虫石油醚提取物成分进行分析, 通过硅胶、 凝胶Sephadex LH-20、 ODS-C₁₈柱层析法对正丁醇提取部分的成分进行分离, 采用质谱和核磁共振等手段鉴定结构。采用纸片扩散法对七星瓢虫成虫石油醚提取物的抑菌活性进行测试。【结果】经GC-MS联用技术从石油醚相中鉴定出14个化合物, 从正丁醇相中分离鉴定了7个化合物。石油醚提取物对肠道沙门氏菌Salmonella enterica有抑制作用, 在浓度为100和50 mg/mL时, 抑菌率分别为52.78%和27.05%, 在该浓度下对绿脓杆菌Pseudomonas aeruginosa、 玉米大斑病菌Exserohilum turcicum和水稻纹枯病菌Rhizoctonia solani没有表现出明显的抑制作用。【结论】七星瓢虫C. septempunctata成虫石油醚提取物对肠道沙门氏菌具有抑制作用。     

Chemical Composition of the Tuber Essential Oil from Helianthus tuberosus L. (Asteraceae)

Helianthus tuberosus L. (Jerusalem artichoke) is cultivated in Europe and other parts of the world as a food crop and ornamental plant. The volatile oils of the aerial parts of H. tuberosus were investigated more than 30 years ago, but no study could be found to date on the constituents of the tuber essential oil. Herein, the first characterization by GC‐FID, GC/MS, and ¹³C‐NMR analyses of a hydrodistilled essential oil of Jerusalem artichoke tubers was reported. Fresh plant material collected in Serbia (Sample A) and a commercial sample (Sample B) yielded only small amounts of oil (0.0014 and 0.0021% (w/w), resp.). In total, 195 constituents were identified, representing 88.2 and 93.6% of the oil compositions for Samples A and B, respectively. The main constituents identified were β‐bisabolene ( 1 ; 22.9–30.5%), undecanal (0–12.7%), α‐pinene (7.6–0.8%), kauran‐16‐ol ( 2 ; 6.9–9.8%), 2‐pentylfuran (0.0–5.7%), and (E)‐tetradec‐2‐enal (0.0–4.9%). Several rare compounds characteristic for Helianthus ssp. were also detected: helianthol A ( 6 ; 2.1–1.9%), dihydroeuparin ( 10 ; 0.0–2.3%), euparin ( 9 ; 0.0–0.4%), desmethoxyencecalin ( 7 ; traces – 0.2%), desmethylencecalin ( 8 ; 0.0–0.4%), and an isomer of desmethylencecalin (0.0%‐traces). The essential oils isolated from the tuber and the aerial parts share the common major component 1 .     

HPLC法测定不同品种不同产地菊芋叶片中绿原酸的含量

为建立同时测定大丰地区3个菊芋品种及大庆地区2个菊芋品种叶片绿原酸含量的方法,采用高效液相色谱法对不同品种不同产地菊芋叶片中绿原酸含量进行测定。结果表明:1)在检测条件流动相为1%磷酸溶液、乙腈(87∶13)、流速1 mL/min和检测波长327 nm时,绿原酸在0.01～0.1 mg/mL呈良好的线性关系,精密度良好,平均回收率为98.21%,RSD=0.68%;2)大丰地区南芋1号、青芋2号、南芋10号叶片中绿原酸含量分别为干重的0.431%、0.040%和0.933%;3)大庆地区南芋1号、南芋9号叶片中绿原酸含量分别为干重的1.245%、2.139%。研究结果表明菊芋叶片中绿原酸含量在不同品种不同生态区域间有差异,HPLC方法简便、快速、准确,可用于菊芋叶片中绿原酸含量的测定。     

菊芋组织培养快繁技术的建立

以菊芋带芽点的薯盘及带节的幼嫩茎段为外植体,MS为基本培养基,附加不同种类和浓度的生长调节物质,研究菊芋组织培养和快速繁殖的技术环节,最终筛选出最优技术参数组合,建立菊芋再生体系。试验结果表明：茎段外植体是理想的快速繁殖材料,正接（形态学下端向下）是最佳的接种方式。芽诱导最佳培养基为MS＋1.0mg·L-16-BA＋0.2mg·L-1IBA,诱导率为95%;继代增殖最适宜培养基为MS＋2.0mg·L-16-BA;壮苗培养最佳培养基为MS＋0.1mg·L-16-BA;生根适宜培养基为MS＋0.2mg·L-1NAA,生根率达100%;移栽成活率达95%,大田种植成活率达95%以上。     

碱胁迫对菊芋幼苗生长及其光合作用和抗氧化作用的影响

采用砂培试验研究了碱胁迫(0、12.5、25.0、37.5和50.0 mm o l.L-1N a2CO3)对2种菊芋(H elianthustuberosus L.)南芋8号(H t 1)和南芋1号(H t 2)幼苗生长及其光合作用和抗氧化作用的影响。结果表明,2种菊芋耐碱的极限浓度为50.0 mm o l.L-1;碱胁迫,尤其是高浓度(37.5 mm o l.L-1)的胁迫,显著降低菊芋幼苗总鲜重、净光合速率(Pn)、气孔导度(G s)、蒸腾速率(T r)、水分利用效率(WUE)及叶绿素总量(Ch l a Ch l b)、类胡萝卜素(C aro)含量与叶片和根POD活性,而显著提高细胞间隙CO2浓度(C i)、叶片和根SOD活性以及丙二醛(M DA)含量。此外,H t 1叶绿素a/b(Ch l a/Ch l b)在碱胁迫下没有明显变化,而H t 2却明显降低。这些结果表明非气孔限制是碱胁迫下2种菊芋幼苗Pn降低的主要原因。菊芋品种H t 1幼苗耐碱能力大于菊芋品种H t 2,这可能与H t 1碱胁迫下维持了较高的Pn、G s、T r、WUE、Ch l a/Ch l b、C aro含量、SOD和POD活性和较低的M DA含量有关。     

菊芋试管苗的初代培养及愈伤组织不定芽诱导

菊芋（Helianthus tuberosus Linn.）为菊科（Asteraceae）向日葵属（Helianthus Linn.）多年生草本植物,耐寒、耐旱、耐贫瘠、耐盐碱[1];其地下块茎富含菊糖,还可通过发酵生产乙醇,在功能性食用多糖及生物能源方面的开发潜力巨大。菊芋主要通过块茎进行无性繁殖,其种子成活率和发芽率均很低[2],严重阻碍了菊芋的杂交育种。近年来以植物组织培养为基础的一系列现代育种技术为菊芋的种质改良提供了新途径,但由于菊芋的愈伤组织难以诱导不定芽或体胚发生,导致以农杆菌转化为主的转基因育种技术的应用受到限制。     

Cloning and Functional Analysis of the Bifunctional Agglutinin/Trypsin Inhibitor from Helianthus tuberosus L.

In order to find new insect resistance genes, four homologous cDNAs, hta-a, hta-b, hta-c and hta-d with lengths of 775, 718, 784 and 752 bp, respectively （GenBank accession numbers AF477031-AF477034）, were isolated from a tuber cDNA expression library of Helianthus tuberosus L. Sequence analysis revealed that all four cDNAs contain an open reading frame of 444 bp, coding a polypeptide of 147 amino acid residues, and that the sequences of the cDNAs are very similar to those of the mannose-binding agglutinin genes of the jacalin-related family. In hemagglutination reactions and hapten inhibition assays, affinity-purified HTA （Helianthus tuberosus agglutinin） from induced Escherichia coli BL21（DE3） expressing GST-HTA shows hemagglutination ability and a higher carbohydrate-binding ability for mannose than other tested sugars. Trypsin inhibitory activity was detected in the crude extracts of induced E. coli BL21（DE3） expressing HTA, and was further verified by trypsin inhibitory activity staining on native polyacrylamide gel. The mechanism of interaction between HTA and trypsin was studied by molecular modeling. We found that plenty of hydrogen bonds and electrostatic interactions can be formed between the supposed binding sites of HTA-b and the active site of trypsin, and that a stable HTA/trypsin complex can be formed. The results above imply that HTA might be a bifunctional protein with carbohydrate-binding activity and trypsin inhibitory activity. Moreover, Northern blotting analysis demonstrated that hta is predominantly expressed in tubers of H. tuberosus, very weakly expressed in stems, but not expressed at all in other tissues. Southern blotting analysis indicated that hta is encoded by a multi-gene family. The insect resistance traits have been described in another paper.     

Overexpression of a Monocot Acyl‐CoA‐Binding Protein Confers Broad‐Spectrum Pathogen Protection in a Dicot

Plants are continuously infected by various pathogens throughout their lifecycle. Previous studies have reported that the expression of Class III acyl‐CoA‐binding proteins (ACBPs) such as the Arabidopsis ACBP3 and rice ACBP5 were induced by pathogen infection. Transgenic Arabidopsis AtACBP3‐overexpressors (AtACBP3‐OEs) displayed enhanced protection against the bacterial biotroph, Pseudomonas syringae, although they became susceptible to the fungal necrotroph Botrytis cinerea. A Class III ACBP from a monocot, rice (Oryza sativa) OsACBP5 was overexpressed in the dicot Arabidopsis. The resultant transgenic Arabidopsis lines conferred resistance not only to the bacterial biotroph P. syringae but to fungal necrotrophs (Rhizoctonia solani, B. cinerea, Alternaria brassicicola) and a hemibiotroph (Colletotrichum siamense). Changes in protein expression in R. solani‐infected Arabidopsis OsACBP5‐overexpressors (OsACBP5‐OEs) were demonstrated using proteomic analysis. Biotic stress‐related proteins including cell wall‐related proteins such as FASCILIN‐LIKE ARABINOGALACTAN‐PROTEIN10, LEUCINE‐RICH REPEAT EXTENSIN‐LIKE PROTEINS, XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE PROTEIN4, and PECTINESTERASE INHIBITOR18; proteins associated with glucosinolate degradation including GDSL‐LIKE LIPASE23, EPITHIOSPECIFIER MODIFIER1, MYROSINASE1, MYROSINASE2, and NITRILASE1; as well as a protein involved in jasmonate biosynthesis, ALLENE OXIDE CYCLASE2, were induced in OsACBP5‐OEs upon R. solani infection. These results indicated that upregulation of these proteins in OsACBP5‐OEs conferred protection against various plant pathogens.     

Stimulated Accumulation of Lectin mRNA and Stress Response in Helianthus tuberosus Callus by Methyl Jasmonate

Callus from Helianthus tuberosus expresses a mannose-specific lectin (HTA). The level of HTA mRNA significantly increased one hour after treatment of the callus with 20 mg/l methyl jasmonate. Following this, fragmentation of the callus DNA at regular intervals was observed together with strong self-fluorescence emission in the callus cells.     

水杨酸对NaCl胁迫下菊芋幼苗光合作用及离子吸收的影响

为探明水杨酸(SA)对NaCl胁迫下菊芋耐盐生理的调控作用,研究了100μmol·L-1水杨酸对不同浓度NaCl胁迫下菊芋幼苗光合响应特征及离子吸收运输的影响.结果表明:施用水杨酸不仅能够有效缓解NaCl胁迫对菊芋光合作用的抑制,促进NaCl胁迫下菊芋幼苗各种光合色素含量、净光合速率、蒸腾速率、气孔导度和水分利用效率的增加,降低细胞间隙CO2浓度,同时也能明显降低NaCl胁迫下菊芋体内Na+的积累,促进菊芋幼苗对K+和Ca2+的吸收和向上运输,其中在100 mmol·L-1 NaCl处理下施用水杨酸处理的菊芋叶片中K+和Ca2+含量分别比未施用水杨酸处理增加了12.9％和14.7％,而Na+含量则降低了30.6％.由此证明,一定浓度外源水杨酸的施用有利于促进NaCl胁迫下菊芋幼苗光合功能的改善,以及有效维持菊芋幼苗体内矿质营养元素含量平衡,从而增强菊芋对NaCl胁迫的抗性,提高NaCl胁迫下的生产力.     

Cloning and Expression Pattern of a Gene Encoding a Putative Plastidic ATP/ADP Transporter from Helianthus tuberosus L.

Herein, we report the cloning and molecular characterization of a full cDNA encoding a putative plastidic ATP/ADP transporter, designated HtAATP, for Helianthus tuberosus L. The ATP/ADP translocator protein was isolated from the tuber-cDNA library of H. tuberosus for the first time. The predicted HtAATP protein was judged as a plastidic ATP/ADP translocator protein from its high homology at the amino acid sequence level to the two Arabidopsis thaliana plastidic ATP/ADP translocator proteins AATP1 and AATP2 （84.8% and 79.9% identity, respectively）. Amino acid sequence analysis of the primary structure of HtAATP revealed that it belonged to the plastidic ATP/ADP transporter family. Hydropathy prediction indicated that HtAATP gene product is a highly hydrophobic membrane protein that contains 10 transmembrane domains to form a spanning topology. Southern blotting analysis showed that the HtAATP gene is a single-copy gene in the H. tuberosus genome. Tissue distribution analysis showed that the HtAATP gene is prominently expressed in sink tissues. A stable expression pattern in tubers at different developmental stages implies an active involvement of HtAATP during carbohydrate formation.