钩状木霉的绿色荧光蛋白标记体系构建及其对辣椒的促生和疫霉病害的生防作用

发布时间：2018-05-12 01:26

本文选题：钩状木霉 + 根癌农杆菌介导的遗传转化(ATMT)　；参考：《贵州师范大学》2017年硕士论文

【摘要】：木霉菌(Trichoderma spp.)是一类重要的生防真菌资源,在农作物病害的绿色防控中具有重要的发展前景和应用价值。木霉在与宿主植物的互作、促生及生物防治作用的研究将为高效、稳定的木霉生防菌制剂的研发和利用提供重要的理论依据。根癌农杆菌介导(Agrobacterium tumefaciens-mediated transformation,ATMT)的绿色荧光蛋白(Green Fluorescence Protein,GFP)基因的转化方法为微生物互作及与宿主植物互作的研究提供了一个强有力的工具。钩状木霉(Trichoderma hamatum)ACCC31649是一株优秀的生防菌株,前期研究表明该菌株具有显著促进黄瓜植株生长的特性。本研究通过根癌农杆菌介导的遗传转化方法,建立了生防钩状木霉的遗传转化体系,筛选获得了稳定遗传的转化子T.hamatum-GF21。利用荧光显微技术揭示了T.hamatum-GF21在辣椒组织及辣椒根际土壤中的定殖能力和特点,并进一步阐明了钩状木霉菌株对辣椒植株的促生作用和对辣椒疫霉菌(Phytophthora capsici)生防作用的初步研究。本论文的主要研究结果如下:1、根癌农杆菌介导钩状木霉的转化体系的构建及稳定遗传的转化子筛选。本研究通过DAPI(4',6-二脒基-2-苯基吲哚)和卡氏白(Calcoflour White)两种染料对钩状木霉ACCC31649的菌丝及分生孢子的细胞核进行组合染色,结果表明其为单核细胞;优化ATMT转化的乙酰丁香酮浓度、分生孢子悬浮液浓度、诱导培养时间和共培养时间等转化条件;获得了钩状木霉的GFP标记转化子100个,通过连续继代的潮霉素抗性筛选和PCR检测,筛选了10个强绿色荧光和稳定遗传的转化子。此外,比较了GFP标记转化子与野生型菌株其在形态学特征、生长速率、产孢量和生物量等方面的一致性。2、钩状木霉菌株在辣椒组织中定殖特点及其对辣椒幼苗的促生作用。通过木霉孢子液灌根接种4叶期辣椒幼苗,标记菌株T.hamatum-GF21在根际土壤中呈现“先增后降”的数量变化趋势;辣椒组织切片的荧光显微连续观察表明,辣椒根、茎和叶组织中都检测到钩状木霉菌株的定殖。30天后,钩状木霉处理与水处理相比,辣椒植株的株高增长13.52%,根长增长16.19%,鲜重和干重分别增加了43.75%和45.28%,而且野生型菌株与GFP标记菌株对辣椒的促生作用没有显著差异。3、钩状木霉对辣椒疫霉菌的生防作用的初步研究。通过平板对峙实验结果表明,钩状木霉菌株对辣椒疫霉菌生长抑制率为40.87%;抑菌实验结果表明钩状木霉发酵液能够强烈抑制辣椒疫霉菌的生长,并且初步研究表明钩状木霉菌能够通过对辣椒疫霉菌菌丝缠绕和分泌细胞壁裂解酶类而产生抑制作用。通过辣椒果实的病原接种实验结果表明,钩状木霉对辣椒疫病的防治效果为19.01%。
[Abstract]:Trichoderma spp.. It is a kind of important biocontrol fungi resources and has important development prospect and application value in the green control of crop diseases. The research on the interaction, growth promotion and biological control of Trichoderma with host plants will provide an important theoretical basis for the development and utilization of highly efficient and stable Trichoderma biocontrol agents. Agrobacterium tumefaciens mediated transformation of the Green Fluorescence protein (GFP) gene of Agrobacterium tumefaciens-mediated transformationmt provides a powerful tool for the study of microbial interaction and interaction with host plants. Trichoderma hamatum)ACCC31649 is an excellent biocontrol strain. Previous studies have shown that Trichoderma hamatum)ACCC31649 has the characteristics of promoting cucumber plant growth significantly. In this study, the genetic transformation system of Trichoderma hook was established by Agrobacterium tumefaciens mediated genetic transformation, and the stable genetic transformant T.hamatum-GF21 was obtained. The colonization ability and characteristics of T.hamatum-GF21 in capsicum tissue and rhizosphere soil of capsicum were revealed by fluorescence microscopy, and the effects of Trichoderma hook strain on growth and biocontrol of Phytophthora capsiciae were further studied. The main results of this study are as follows: 1. Construction of Agrobacterium tumefaciens mediated Trichoderma hook transformation system and screening of stable genetic transformants. In this study, the mycelium and conidial nuclei of Trichoderma hookiformis ACCC31649 were stained by DAPI4 (DAPI4) -6-diamidine-2-phenylindole (DAPI4) and Calcoflour White (Calcoflour White). The results showed that the cells were monocytes, and the concentration of acetyl eugenone transformed by ATMT was optimized. The transformation conditions, such as conidial suspension concentration, induction culture time and co-culture time, were used to obtain 100 GFP marker transformants of Trichoderma hook, which were screened by successive subculture hygromycin resistance screening and PCR detection. Ten strong green fluorescent and stable genetic transformants were screened. In addition, the morphological characteristics, growth rate, sporulation and biomass of GFP marker transformants were compared with those of wild type strains. The colonization characteristics of Trichoderma hook strain in capsicum tissue and its promoting effect on the growth of pepper seedlings were compared. After inoculated with Trichoderma spore solution to inoculate 4 leaf pepper seedlings, the number of labeled strain T.hamatum-GF21 increased first and then decreased in rhizosphere soil, and the fluorescence microscopic observation of tissue sections of pepper showed that the number of capsicum root increased first and then decreased. 30 days after colonization of Trichoderma hook strain was detected in stem and leaf tissue, the treatment of Trichoderma hook was compared with water treatment. The plant height of pepper plant increased 13.52%, root length increased 16.19%, fresh weight and dry weight increased 43.75% and 45.28%, respectively. There was no significant difference between wild type strain and GFP labeled strain in promoting growth of pepper. Preliminary study on biological control effect of Trichoderma hook on Phytophthora capsici. The results showed that the inhibition rate of Trichoderma hook strain against Phytophthora capsici was 40.87 and that the fermentation broth of Trichoderma hook could strongly inhibit the growth of Phytophthora capsici. Preliminary studies showed that Trichoderma hook could inhibit the cell wall lytic enzymes of Phytophthora capsici by winding and secreting cell wall cleavage enzymes of Phytophthora capsici. The results of inoculation of pepper fruit showed that the control effect of Trichoderma hook on pepper blight was 19.01 kW.
【学位授予单位】：贵州师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476

【参考文献】