根癌农杆菌拮抗真菌的筛选、发酵及有机锡新化合物抗TMV、抗癌机制研究

【作者】 李祝；

【导师】 宋宝安； 梁宗琦；

【作者基本信息】 贵州大学 ， 农药学， 2008， 博士

【摘要】 由致病性根癌农杆菌和植物病毒引起的植物病害分布于世界各地,给农业生产造成严重经济损失,引起世界各国的高度重视。根癌农杆菌（Agrobacterium tumfaciens）通过侵染植物的根茎部形成冠瘿瘤,使感病植株生长迟缓,树势弱,产量少,寿命短,直接影响了果树产品的生产,从而影响了农业经济的发展。烟草受烟草花叶病毒（TMV）侵染后,其代谢受到严重影响,光合速率下降,叶片会不均匀地褪绿、枯黄,使植株生长矮小,干物质积累减少,损害烟叶质量,是我国目前最主要的农业病害之一。本文分为两个部分。第一部分是针对目前根癌病的生防细菌不耐旱,寿命短的缺点,首次从果树根系土壤中筛选拮抗根癌农杆菌的生防真菌,并确定生防真菌的发酵条件,为植物根癌病生防菌株的开发及应用扩大菌种资源;另一部分是针对目前防止TMV的一般药剂很难在不伤害寄主的情况下抑制病毒增殖,首次对南开大学李正名院士提供的有机锡新化合物04-W0319进行了抗TMV活性及机制的研究,并在此基础上对04-W0319的抗肿瘤机制进行了系统研究,为开发低毒、高效的有机锡新化合物,拓展其应用范围提供参考依据。在第一部分的研究中,从我国12个省市采集的土样中分离纯化出152株真菌,采用琼脂柱初筛,得到2株抗根癌农杆菌Agrobacterium tumefaciens T-37（简称T-37）的真菌菌株,通过滤纸片法进行对比复筛,证实了此2株菌株对根癌农杆菌具有拮抗作用。根据Raper（1965）关于曲霉属的分类标准,拮抗1号真菌菌株的主要形态特征与他所描述的黑曲霉（Aspergillus niger V.Tieghen 1867）的标准形态基本相同,鉴定该菌株属于曲霉属黑曲霉群的一株黑曲霉,记为黑曲霉xj（Aspergillus niger xj）,并将该菌种保藏在中国典型培养物保藏中心。根据Brown & Smith（1957）的分类标准,拮抗2号菌株鉴定为轮枝拟青霉,记为轮枝拟青霉49-01（Paecilomyces verticillatus 49-01）,为新种。对xi菌株进行单孢子分离,获得8个单孢子株,分别记为xj-1、xj-2、xj-3、xj-4、xj-5、xj-6、xj-7、xj-8。通过对菌落生长情况、产孢量、分生孢子的萌发率、对T-37抑制活性的综合比较,认为xj-5为优势单孢子株,并对其继代培养后的培养性状及对T-37的抑制活性进行了考察,xj-5单孢子株所表现出的遗传性状较为稳定并且所产生的抑菌圈直径（20.13±0.09 mm）高于对照xj菌株（18.28±0.21 mm）。以xj-5单孢子株作为出发菌株,对其发酵工艺条件进行探索。对培养成分中2%的麦芽糖、葡萄糖、淀粉、蔗糖、乳糖、甘油6种碳源和3%的蛋白胨、牛肉膏、酵母膏、硝酸铵、氯化铵、脲素6种氮源进行单因素筛选,选择麦芽糖、蔗糖为碳源,酵母膏、蛋白胨为氮源,并设计L₉（3⁴）正交实验对C/N进行优化。确定xj-5菌株的发酵培养基组成为:麦芽糖2%,蔗糖2%,酵母膏3%,蛋白胨1%。对培养条件中的不同接种量、装液量、培养温度及发酵培养基起始pH值分别进行了筛选。确定xj-5菌株的发酵培养条件为:接种15%、摇瓶装液量10%、培养温度25℃、发酵培养基起始pH自然。根据以上筛选出的培养基成分和培养条件,将xj-5菌株接种摇瓶培养,每隔24h定时取样,测定生物量和抑菌圈直径,绘制发酵动态曲线。结果显示:xj-5菌株直接进入对数生长期,无生长延迟期,这可能是因为加入的液体母种活力强,并且培养条件的差异不大,菌株能很快适应发酵环境,直接进入快速生长期,并且这段时间较长（1-8d）,生物量在第8天的时候达到了最大（161.24g/L）,同时抑菌成分也在不断分泌与积累。随后延迟期（9-12d）持续了较长的时间,并在当在第10天的时候,代谢产物的累积达到了高峰,抑菌圈直径达到最大（54.48 mm）。随着发酵培养基营养的耗尽,衰老期的出现（13-15d）,毒素的累积及菌体自溶导致生物量急剧下降,而细胞内容物的流出和细胞碎片的产生导致发酵培养基pH值的变化,直接影响着xj菌株的抑菌成分的活性,以致抑菌圈直径也急剧下降。因此,根据xj-5菌株的发酵动力学的模型,确立xj-5菌株抑菌活性成分的发酵工艺为:制备麦芽糖2%,蔗糖2%,酵母膏3%,蛋白胨1%,pH自然的发酵培养基,将xj-5菌株按15%的接种量,接种到装液量为10%的液体发酵培养基中,25℃,120rpm,摇瓶培养10d,收集菌丝体制备发酵浓缩液,滤纸片法测xj-5菌株的抑菌圈直径达54.48 mm。第二部分的研究以心叶烟为材料,得到有机锡新化合物04-W0319对烟草花叶病毒侵染的治疗效果为49.38%;钝化效果为73.23%;保护效果为48.82%。500μg/mL 04-W0319与TMV体外作用30min,电镜下观察,显示病毒粒体出现断裂现象,粒体结构受到了破坏;琼脂糖凝胶电泳显示04-W0319对TMV-RNA具有体外降解作用;TMV-CP的UV测定结果反映了04-W0319对烟草花叶病毒的体外聚合过程具有抑制作用。对普通烟K326的PAL、POD和SOD的活性进行了测定。PAL活性测定结果表明:500μg/mL 04-W0319处理的烟苗在第7天达到酶活高峰值,第9天降到最低,并且烟草在接种TMV后再用04-W0319处理可使酶活性增加。POD活性测定结果表明:单独用04-W0319处理的酶活在第7天达到酶活高峰值,烟草在接种TMV后再用04-W0319处理也可使酶活性增加,在第9天达到酶活高峰值,并高于其它处理。SOD活性测定结果表明:TMV处理的酶活于第5天达到一个高峰值,随后酶活在第9天又达到另一个高峰值,接种TMV后再用04-W0319处理的与发病对照的酶活趋势一致,单独用04-W0319处理的烟苗酶活在第5天达到高峰值,并高于其它处理组。K326的胞间蛋白经SDS-PAGE不连续电泳分析表明:化合物04-W0319对K326的胞间蛋白影响不大,并且对PR蛋白的诱导也不强。04-W0319对寄主叶绿素含量的影响实验结果显示:TMV感染后使得烟草叶片中叶绿素的含量大大降低,04-W0319处理的烟草叶片叶绿素含量有所提高,说明04-W0319能够减少病毒对烟草叶片中叶绿体的破坏作用,增加了叶绿素的含量,从而提高寄主的抗病性;但药剂处理烟草叶片的叶绿素含量仍然低于健康对照,这也说明04-W0319不能完全抑制病毒对叶绿素的破坏作用。因此,04-W0319一方面可在体外与TMV作用,降低TMV的侵染率;另一方面可提高寄主防御酶系活性和增加叶绿素含量,提高寄主抗性,抵御TMV的侵染。以PC3、Bcap-37和BGC823肿瘤细胞为材料,04-W0319作用72h对PC3细胞的IC₅₀为0.13μg/mL;作用48h对Bcap-37细胞的IC₅₀为0.18μg/mL;作用48h对BGC823细胞的IC₅₀为0.17μg/mL。细胞毒性实验初步判断:04-W0319诱导3株细胞死亡主要不是通过细胞毒性。划痕标记法实验表明04-W0319可降低3株细胞的运动能力而抑制肿瘤细胞的生长。细胞形态学观察及细胞培养液上清中LDH活力测定表明:72h内凋亡始终是04-W0319诱导PC3和Bcap-37细胞死亡采用的主要方式;48 h内凋亡始终是04-W0319诱导BGC823细胞死亡采用的主要方式。流式细胞分析术检测结果进一步揭示:0.15μg/mL 04-W0319作用48h,将PC3或Bcap-37细胞阻滞在G₂/M期而抑制细胞的生长、繁殖;当作用时间延长或作用浓度增高时,以诱导细胞发生凋亡为主;对BGC823细胞,0.15μg/mL 04-W0319作用48h,将细胞阻滞在G₀/G₁期,并伴随诱导凋亡而抑制细胞的生长、繁殖;但随着作用时间的延长,04-W0319将BGC823细胞周期阻断在G₂╱M期,并伴随着细胞发生坏死。免疫印迹（Western Blot,WB）实验结果显示:04-W0319可激发PC3、Bcap-37细胞p21蛋白的表达,从而阻碍了细胞通过细胞周期G₂╱M期的限制点,抑制细胞的生长。而对BGC823细胞,04-W0319通过抑制CyclinD蛋白的表达,阻碍了细胞通过细胞周期G₀/G₁期的限制点,抑制细胞的生长。总的来说,04-W0319对3株细胞中的CDK₂和CDK₄都没有影响,提示04-W0319的作用靶标相对专一。更多还原

【Abstract】 Plant diseases caused by virus are of common occurrence and cause serious threats and economic loss to the farmers dependent heavily on the production of agricultural crops. Pathogenic Agrobacterium tumfaciens is known for its ability to adversely infect the rhizome by forming crown galls and thereby weakening and retarding the growth of the diseased plant.This eventually affects the economy,shortens the life of the fruit crop and lowers the output and crop yield.In tobacco leaves infected by TMV,the metabolism is seriously affected,leaves become dry and yellow,chlorosis gets uneven leading to photosynthetic rate decline,plant dwarfing and decrease of accumulation of dry matter.Over the last few years,it has become a major agricultural issue to tackle with in China.The present investigation is mainly divided in two parts.Owing to the disadvantages associated with low drought tolerance and shot life of biocontrol bacterium,in the first part of the study,the biocontrol fungi strains of antagonism A.tumfaciens from root soil were screened and the fermentation conditions were established.Again it is well known that by using the commonly employed reagents it is difficult to inhibit the proliferation without hurting the host.Therefore,in order to develop reference compound with high activity and low toxicity, the second part involved studies on the mechanism,anti-tumor and anti-TMV activities of 04-W0319,an organotin compound provided by Academician Li Zhengming of Nankai University.For the first part of the work,we isolated 152 strains of fungi from the soils of 12 different provinces in China,and could obtain 2 strains of antagonism A.tumefaciens T-37 from our collection by employing agar column.At the same time,the antagonism were confirmed by filter paper method.According to the classification standard of Raper（1965）,NO.1 strain is generally in good agreement with the standard of holotype Aspergillus niger V.Tieghen 1867,therefore,we identified and named the type culture as Aspergillus niger xj.According to the classification standard of Brown & Smith（1957）,NO.2 strain is a new species,we identified and named the type culture as Paecilomyces verticillatus 49-01.We obtained 8 monoconidium strains from xj with single spore separation technique.They were marked as xj-1、xj-2、xj-3、xj-4、xj-5、xj-6、xj-7、xj-8、the xj-5 was considered as the dominant monoconidium strain after comprehensively comparing the situation of colony growth,sporulation, the germination of conidia and the inhibition activity.Subculture characters were found to be relatively more stable and the diameter of inhibition（20.13±0.09mm） was longer than the contrast （18.28±0.21mm）.Subsequently,fermentation process condition was studied with xj-5 serving as the test strain.Six different kinds of carbon and nitrogen sources were subjected to the single factor screening study.They were 2%of maltose,glucose,starch,cane sugar,lactose,glycerol,and 3% of peptone,beef extract,yeast extract,ammonium nitrate,ammonium chloride and urea in culture medium.Maltose,a sugar as the carbon source,yeast extract and peptone as the nitrogen source were designed for orthogonal experiment L₉（3⁴） in order to optimize the C/N ratio.2%maltose, 2%cane sugar,3%yeast extract and 1%peptone were considered as the fermentation media.Different liquid inoculums were inoculated into fermentation medium,15%（v/v） was selected from an economic point of view.The effect of different liquid volume in the flask,10% （v/v） was found to be the optimum.Anti-bacterial activities at various temperatures were also studied.Results showed that the longest diameter was at 25℃.For the production and accumulation of antibacterial substances,natural pH was convenient for media preparation of xj-5.Based on our experimental results,the flask was inoculated with xj-5 and the dynamic fermentation curve was drawn after testing the biomass and diameter of inhibition per every 24h. Results showed that xj-5 followed a direct exponential growth phase without displaying any lag phase.Perhaps due to the strong activity of the liquid inoculum and lack of variation in the culture condition,the strain could quickly adapt the fermentation and go into the growth phase as fast as possible.The time was long（1-8d） and corresponding biomass could reach the peak（161.24 g/L） on the 8^th day.The antibacterial components were secreted and accumulated at the same time. Then the lag phase（9-12d） lasted for an appreciably longer time,the metabolites reached the peak and the diameter was 54.48 mm on the 10^th day.With the exhaustion of the nutrient,aging phase started to appear（13-15d）,the biomass sharply decreased and toxicity enhanced with the accumulation of autolysis.The pH value varied due to the outflow of the cellular content and production of cellular fragments,which affected the antibacterial activity and caused dramatic decrease in the value of the diameter.Thus,in accordance with the dynamic fermentation data,the optimal condition to produce active antibacterial active substance was set as follows:2%maltose,2%sugar,3%yeast extract, 1%peptone,natural pH with the addition of 15%inoculation volume into 10%（v/v） flask at 25℃under 120rpm,cultured for 10 days.After collecting the concentrated fermentation liquid,the diameter was found to be 54.48 mm by filter paper method.The second part of the study concerned with Nicotiana glutinosa as the material.We determined the living therapy,protection and inactivation effect of 04-W0319 against TMV,the treatment effect was 60.53%,inactivation effect was 75.97%and protection effect was 54.85%.Electron microscope studies revealed that the virus particles were fractured when 500μg/mL 04-W0319 was treated with TMV for 30 minutes in vitro.TMV-RNA showed degradation in vitro which was detected by agarose gel electrophoresis.The results indicated that 04-W0319 could inhibit the polymerization of TMV-CP in vitro.The activity of PAL,POD and SOD of Nicotiana tabacum K326 was enhanced by at different degrees when treated with 04-W0319 at 500 mg/L.The results of PAL activity showed that PAL was significantly increased by 04,and achieved the peak on the 7^th day,but fell to the lowest value on the 9^th day.PAL activity was also found to increase when K326 was vaccinated with TMV before being treated with 500 mg/L 04-W0319.The results of POD activity showed that POD achieved the peak on the 7^th day when K326 was treated with 04-W0319,but the peak was attained on the 9^th day when K326 was vaccinated with TMV before treatment with 04-W0319,and the value was higher as compared to other treatments.The results of SOD activity on the other hand showed that SOD achieved its first peak on the 5^th day when K326 was treated with TMV（Control）,then attained a second peak on the 9^th day.Treatment with 04-W0319 before being vaccinated with TMV and the control,revealed the same pattern in the SOD activity of K326.SOD activity also achieved the peak on the 5^th day when K326 was treated with 04-W0319, but the peak was higher than those observed with other treatments.The results of SDS-PAGE showed that the induction of PR protein of K326 was relatively low by 04-W0319,and intercellular protein was not affected by 04-W0319.The role of PR protein to induce plant resistance was perhaps remarkable with their coexistence.It should also be noted that the chlorophyll content of the tobacco leaf infected by TMV has been found to decrease sharply due to the destruction of chloroplast by the virus.However,the issue can be addressed partially by treatment with 04-W0319 which helps to develop disease resistance of the host thereby enhancing the chlorophyll content. Further studies using 04-W0319 on different cancer cells e.g.PC3,Bcap-37 and BGC823 showed that 04-W0319 had different inducing effects towards various cells.The IC₅₀ of 04-W0319 was 0.13μg/mL when the PC3 cells were incubated for 72h;0.18μg/mL when the Bcap-37 cells were incubated for 48h and 0.17μg/mL when the BGC823 cells were incubated for 48h.04-W0319 could reduce the mobility of three cancer cells and inhibit the growth of cancer cells.From the results derived from cellular morphology and LDH assay,we noticed that apoptosis in 72h was the main method of inducing the death of PC3 and Bcap-37 cells and apoptosis in 48h was primarily responsible for inducing the death of BGC823 cells.In addition, the results of FCM further indicated that 0.15μg/mL 04-W0319 could arrest PC3 or Bcap-37 cells at G₂/M stage in 48h.However,with the increase of time and concentration,04-W0319 mainly induced the apoptosis of cells.BGC823 cells could be arrested by 0.15μg/mL 04-W0319 at G₀/G₁ phase of cell cycle in 48h.This was followed by the inhibition of the growth of the cells by induction apoptosis.However,with the elapse of time,04-W0319 could arrest BGC823 cells at G₂/M phase,with the death of BGC823 cells.The results of western blot indicated that 04-W0319 could up-regulate the expression of p21 in PC3 and Bcap-37,in addition to hindering the cell to overpass G₂/M restriction point and inhibit its growth.However,the concentration of 04-W0319 required to up-regulate the expression of p21 in PC3 was 0.1μg/mL,and only 0.05μg/mL for Bcap-37.But for BGC823,04-W0319 at a concentration of 0.2μg/mL could mainly down-regulate the expression of CyclinD.The cells were then hindered through the G₀/G₁ restriction point,and growth of BGC823 cells was also inhibited. As a matter of fact,04-W0319 had practically no influence on CDK₂ and CDK₄ of PC3,Bcap-37 and BGC823 cells,suggesting that the target of 04-W0319 is relatively specific.更多还原