甘草来源内生真菌多样性和抗菌活性研究

甘草作为使用广泛的根茎入药中药材,其自身复杂的微环境蕴藏了丰富的微生物资源,是未被充分发掘的新结构、新机制抗菌活性天然产物资源库.本文主要聚焦内蒙、甘肃和宁夏甘草中内生真菌,经过表观形态学特征分析,结合ITS-rDNA共获得134株真菌,其归属于16个属,显示出较好的真菌群落多样性,同时还发现青霉和曲霉属为各地甘草内生...     

墨西哥仙人掌植物内生真菌的研究Ⅰ.内生真菌分离及其抗菌活性筛选

从一种墨西哥仙人掌[Opuntiamicrodasys(Lehm .)Pfeiff]的肉质茎中分离获得31株内生真菌,经形态观察分类鉴定为3个目、3个科、14个属。同时选择2 2种病原微生物作为指示菌进行抑菌试验,研究其抗菌活性。结果表明:3株仙人掌内生真菌分别对细菌、植物病原真菌和皮肤致病真菌多种病原微生物有较为明显的抑制生长作用。     

木本曼陀罗内生真菌抗菌活性的筛选研究

从木本曼陀罗植物内生真菌中筛选高效抗菌活性的菌株。选择与人类和植物相关的36株病原微生物,分别对分离自木本曼陀罗(Datura arborea L.)植物的内生真菌77株进行了发酵代谢产物的抗菌活性筛选研究。结果显示:对细菌病原菌、皮肤致病真菌、植物致病真菌有抑制作用的内生真菌分别有24,9,17株,其中5株内生真菌对10种以上的供试病原菌有明显的抑制作用,活性最好的1株对20种病原菌有较强抑制活性,最大抑菌圈直径达48mm。这说明木本曼陀罗植物内生真菌抗菌能力较强,抗菌谱较广。     

芦荟植物内生真菌的研究Ⅱ．抗菌活性初探

对从中华芦荟 [AloeveraL .varChinese (HawBerg) ]、元江芦荟 (A .yuanjiangensisXiong&Zhengsp .nov .)及库拉索芦荟 (A .barbadsisMill.)的根、叶、花柄、花中 ,分离的 88株内生真菌 ,作了革兰氏阳性和阴性抑菌试验 ,研究了其抗菌活性。结果表明 ,芦荟植物不同种类、不同部位内生真菌的抗菌活性存在有差异。     

白苞蒿内生真菌的分离及其抗肿瘤抗菌活性筛选

重视药用植物内生真菌的研究,实现利用内生真菌代替药用植物生产活性物质,具有重要的生态学和经济学意义。白苞蒿(Artemisia lactifloraWall.ex DC.)是一种重要的药用植物,已收载于《中国药典》2010年版一部附录,是中药刘寄奴的基原植物之一,具有清热、解毒、止咳、消炎、活血、散瘀、通经等功效,用于肝、肾疾病的治疗。该研究以白苞蒿的根、茎、叶和花为试验材料进行内生真菌的分离,共分离得到白苞蒿内生真菌54株;以人骨髓白血病细胞系(HL-60)、人乳腺癌细胞系(MCF-7)和人前列腺癌细胞系(PC-3)为细胞模型,采用MTT法对54株白苞蒿内生真菌发酵物粗提物的体外抗肿瘤活性进行筛选,发现对3种肿瘤细胞增殖的抑制率在90%以上的菌株共有5株,占总菌株数的9.3%;以白色念珠菌、金黄色葡萄球菌和大肠埃希菌3种病原细菌为靶标菌,采用滤纸片法对54株白苞蒿内生真菌发酵物粗提物的抗菌活性进行筛选。结果表明:所有菌株对白色念珠菌均未表现明显的抑菌活性,而对金黄色葡萄球菌和大肠埃希菌有一定抑菌活性的菌株共有17株,占总菌株数的31.5%,其中有2株对金黄色葡萄球菌表现较强的抑菌活性。     

天山花楸内生真菌的分离及其抗菌活性物质的初步研究

采用组织块分离法从天山花楸茎、叶、果实中分离出17株内生真菌,分别运用对峙培养法和菌丝生长速率法进行初筛、复筛和抑菌活性试验,为抗菌活性菌株的应用以及抗菌物质的分离提纯奠定基础。结果表明:(1)菌株L18对棉花枯萎病菌和棉花黄萎病菌的抑菌作用较强。(2)形态学特征观察和rRNA ITS序列系统发育分析结果初步鉴定菌株L18为波兰青霉(Penicillium polonicum)。(3)该菌株发酵液中抑菌活性物质对温度较敏感,40℃处理后抑菌活性降低至对照的59.1%;对日光不稳定,而对紫外光较稳定;能被蛋白酶降解。(4)乙酸乙酯萃取该抑菌活性物质效果较佳,表明其极性较小,初步推测此活性物质属于蛋白质或肽类物质。     

海南粗榧内生真菌抗肿瘤抗菌活性的筛选

对72株海南粗榧(Cephalotaxus hainanensis Li)内生真菌进行了抗肿瘤和抗菌活性筛选。结果显示, 有9株内生真菌至少对一种指示瘤株具有细胞毒活性, 5株内生真菌对金黄色葡萄球菌有较强的抑菌活性, 1株内生真菌对辣椒疫霉有抑制作用。这表明海南粗榧内生真菌是寻找有价值的生物活性成分的潜在资源, 其生物活性成分值得进一步研究。     

杜仲内生真菌的分离鉴定及抗菌活性研究

采用组织分离法从秦岭药用植物杜仲茎部共分离到内生真菌38株,经形态学鉴定分属于9属,其中青霉属为杜仲内生真菌中的优势菌群,拟青霉属、交链孢属和无孢群属为常见属。对分离得到的杜仲内生真菌发酵液进行了抑菌实验,其中61%的菌株对测试细菌具有抗菌活性,表明杜仲内生真菌的抗菌活性十分普遍;34%的菌株对烟草赤星病菌、苹果炭疽病菌及辣椒炭疽病菌等多种植物病原菌具有较强的抑制活性,表明杜仲内生真菌在植物病害的生物防治领域具有进一步开发的价值。     

虎杖内生真菌的分离及产抗菌活性物质的筛选

从植物虎杖(Polygonum cuspidatum Sieb.et Zucc.)的根状茎中分离出24株内生真菌,分别对其液体发酵液进行抗菌活性检测,结果筛选出3株内生真菌具有抗菌活性,它们分别属于曲霉属、青霉属和无孢菌类。     

云南重楼植物内生真菌的分离及抗菌活性筛选

从云南重楼[Paris polyphylla Smithvar.yunnnanensis(Franch.)Hand.Mazz.]块状茎中分离出166株内生真菌,对其进行形态分类鉴定归于4目,6科,20个属,体现了云南重楼植物内生真菌的生物多样性特征。同时,选择与人类和植物相关的37株病原微生物作为抗菌活性筛选指示菌,进行了云南重楼植物内生真菌抗菌活性的初步研究。结果表明,4株内生真菌对细菌、植物致病真菌、皮肤致病真菌多种病原微生物具有显著抑制生长的作用。     

Ultrastructure of Transfer Cells in Endosperm of Coix lacryma-jobi

Special attention was paid to the ultrastructure of transfer cells (TCs) in different locations of basal endosperm in Coix lacryma-jobi at 10 and 25 days after pollination. At 10 days after pollination. TCs of the outermost layer had long wall ingrowths (WIs) whereas those of the second layer possessed fewer and shorter Wis. In both layers TCs had a lobed nucleus, abundant mitochondria, rough endoplasmic reticulum (RER), ribosomes, and a certain number of dictyosomes and vesicles which contained dense substance connected with plasma membrane of WIs. Mitochondria were located near or between WIs. The distribution of organelles in TCs of the second layer was similar to that of the outermost layer. Mitochondria had well defined cristae and dictyosomes and RER seemed more numerous than in TCs of the outermost layer. At 25 days after pollination, TCs of the outermost and the second layer were almost filled with Wis but the organelles were recognizable. TCs of the fourth layer had branched and network-like WIs, many mitochondria, starch grain within plastids and lipids locating near WIs and in the interstices of WIs. Dictyosomes were frequently found but less RER fragments were seen. TCs of the fifth layer with short WIs contained large starch grains and small protein bodies. Plasmodesmata were not observed in the walls of TCs of the outermost and second layer at both 10 and 25 days after pollination but were found in the walls of TCs of the fourth and upper layers and also in the network-like WIs at 25 days after pollination. The roles of the organelles and functions of TCs of different layers were discussed.     

Studies on Endosperm Development and Deposition of Storage Reserves in Coix lacryma-jobi

The transition from free nuclear to cellular endosperm of Coix lacryma-jobi was eompleted 2 days after pollination. By 3 days after pollination the central cell was filled with endosperm cells. At first all cells of endosperm underwent division, later cell division was limited mainly in the peripheral region. 10 days after pollination the epidermal layer ceased its periclinal division and became the aleurone layer. Cell division persisted in the subepidermal 'cambium-like layers until the caryopsis nearly matured. Ceils of the inner region of endosperm became enlarged. Several layers of transfer cells were formed at the basal part of the endosperm. Starch grains appeared in endosperm cells on the 9th day after pollination. 10 days after pollination, lipid bodies occurred in the aleurone layer and the underlying layers. 13 and 15 days after pollination, the small vacuoles of aleurone cells contained protein and 20 days after pollenation they became aleurone grains. By 15 days after pollination pro tein bodies were formed in starch endosperm. Storage reserve deposition continued until the grain ripened. A correlation between endosperm and emoryo development was also observed.     

通过图像分析方法作出的薏苡定量染色体图

薏苡染色体数目为2n=20,由于中期染色体的长度差异不大,相邻染色体难以区分,而薏苡前中期的染色体相对较长,经DAPI染料染色后染色体上显示明显不同的深染区和浅染区,深染区和浅染区分别相当于染色体上染色质纤维较浓缩和较伸展的区域。前中期染色体上染色深浅不一的染色区可以作为识别薏苡特定染色体的重要标志,也可用于辨别同源染色体。用MetaMorph软件定量分析了薏苡每条前中期染色体上(从短臂到长臂)DAPl信号强度的变化,结合染色体的长度和臂比作为辅助参数,构建了薏苡前中期染色体的定量染色体图。该定量染色体图不仅描述了每条染色体不同区域浓缩程度的不同,而且也反应出不同浓缩区域所占染色体长度的比例。因此该定量染色体图是实际的前中期染色体的一种直观模式,可作为识别薏苡基因组中每条染色体的有力依据。     

Microsporogenesis,Megasporogenesis and the Formation of Male and Female Gametophyte in Coix lacryma-jobi L.

The development of the anther wall follows the monocotyledonous type. During meiotic stages of prophase I, some cytoplasmic channels are observed on the walls between meiotic cells. which divide synchronously. Cytokinesis in the microspore mother cell is of the successive type and gives rise to iscbilateral tetrad. The cell wall between the generative cell and the vegetative cell in early stage shows PAS positive reaction. The mature pollen grain is of 3-celled type. The development of the female gametophyte follows the polygonum-type; the antipodal cells proliferate to form a multicellular tissue mass. Many starch grains are present in the central cell. The nucleus of the mature egg cell is located at the micropylar end; a great deal of starch grains are in the cytoplasm surrounding the nucleus, while vacuoles of various size distribute throughout its cytoplasm but are more and larger at the chalaza,1 end. The nucleus of the synergid cell is located at the micropytar end where a filiform apparatus is formed and many small vacuoles are present at the chalazal part.     

基于SRAP标记的薏苡种质资源遗传多样性及DNA指纹图谱构建

利用SRAP分子标记对从各主要产地收集到的90份薏苡种质进行遗传多样性分析,其中68份收集于福建省,6份来自中国台湾,16份来自浙江、辽宁、山东、河南、云南、江苏、湖南、广东、上海等省(市)。结果表明,从88对SRAP引物组合中筛选出26对引物进行SRAP扩增,共扩增出185条带,其中具有多态性的有157,占总数的84.86%,表明90份薏苡种质表现出丰富的遗传多样性。基于SRAP标记利用系统聚类法将90份薏苡种质资源分为4大类,与形态性状分类结果有一定的相似性;利用16对SRAP引物构建了73份薏苡种质资源的DNA指纹图谱,为薏苡遗传研究、品种选育与资源保护提供了依据。     

薏苡种子形态性状多样性评价

薏苡是优质的药食兼用作物。本研究对我国36份薏苡种质的种子形态性状进行了主成分分析和聚类分析。结果表明,36份薏苡种质具有较高的形态性状多样性。不同薏苡种质的4个种子质量性状间存在差异。单因素方差分析显示11个种子数量性状在不同薏苡种质间呈极显著差异。数量性状的变异系数在8.43%~68.01%之间,种壳与外种皮重量的变异系数最大而种仁长度的变异系数最小。主成分分析将36份薏苡种质分为中部及南部栽培种质、北部栽培种质和野生种质3大类,3类种质的形态特征存在明显差异。聚类分析将36份薏苡种质分为5大类群,即中部及南部栽培种质、北部栽培种质和3个类群野生种质。初步筛选出6份可能具有较高抗肿瘤活性的薏苡种质FJNJ、TJ-BK-1、TJ-BK-2、MY-BK-1、MY-BK-2和JYSD。     

水稻程序性细胞死亡抑制基因Dad-1在薏苡中的定位

基因Dad-1是普遍存在于动物和植物中一个高度保守的程序性细胞死亡抑制基因。以水稻Dad-1基因为探针,采用Southern杂交在薏苡中检出了Dad-1基因的同源序列,并利用荧光原位杂交的方法对其进行了染色体物理定位。在薏苡第9染色体短臂上检测到Dad-1基因的杂交信号,信号与着丝粒的百分距离为67.44±1.45。     

茅苍术挥发油对三种内生真菌及七种外源真菌的抑菌活性

为了研究宿主植物次级代谢产物对内生真菌生长的抑制作用,以药用植物茅苍术及其内生真菌为材料,采用有机溶剂萃取法提取茅苍术挥发油,应用滤纸片扩散法和平板涂布法研究茅苍术挥发油对茅苍术内生真菌及其他外源真菌的抑菌作用.结果表明: 挥发油对3种内生真菌生长具有抑制作用;对酵母、水霉有很强的抑菌活性;对根霉、犁头霉抑菌作用不明显;对绿色木霉、黑曲霉的产孢具有抑制作用;对疫霉生长无抑制作用.在高浓度挥发油胁迫下,内生真菌菌丝分支增多,分支间距离变短,气生菌丝生长受到抑制.两种内生真菌对挥发油具有代谢转化能力,降低了其主要成分的含量.表明茅苍术挥发油对内生真菌生长具有限制作用,但内生真菌能够通过自身分解代谢适应茅苍术挥发油.     

中国南海部分深海沉积物真菌多样性及其抗菌活性

【目的】从南海4个站位的深海沉积物中分离真菌,揭示其多样性并测定抗菌活性。【方法】使用4种培养方法和8种培养基,从12个深海沉积物样本中分离培养真菌,通过菌落形态观察和ITS序列系统发育分析进行鉴定。采用滤纸片扩散法和生长速率法分别测试真菌小量发酵液粗浸膏的抗细菌和抗真菌活性。【结果】共分离到125株纯培养真菌,基于形态和ITS序列分析,排重后得到18个种类型,这些真菌可以划分到12个属,大多数属于子囊菌门(Ascomycota),只有2株属于担子菌门(Basidiomycota)。4个站位可培养真菌多样性具有差异性。抑菌活性筛选显示,大多数真菌具有较好的抑菌活性;链格孢属(Alternaria)、青霉属(Penicillium)、匐柄霉属(Stemphylium)这几个属的真菌表现出对多种指示细菌有抑制作用,尤其是Alternaria tenuissima DN09、Alternaria alternata DN14和Penicillium chrysogenum DN16对G~+和G~–细菌均表现出抑制作用。【结论】本研究揭示了南海深海沉积物可培养真菌多样性和抑菌活性,为进一步利用深海沉积物来源真菌奠定了基础。