解磷细菌WY4的GFP标记及其在小白菜根系的定殖研究

发布时间：2018-08-15 16:34

【摘要】：磷(phosphorus,P)是自然生态系统和农业生态系统中一种非常重要的大量元素。全国土壤普查统计,约有2/3的土壤依旧缺磷,土壤缺磷成为限制作物高产的主要影响因素。随着粮食危机及环境危机的日益加剧,如何在保护环境的基础上提高农作物产量已成为全球乃至全人类面临的一个主要问题。解磷细菌(phosphate-solubilizing bacteria,PSB)是人们较早发现并被广泛利用的一类植物促生菌(plant growth-promoting bacteria,PGPB),能促进土壤难溶性磷的溶解,提高土壤磷素的利用效率,对环境危害极小,在促进作物生长方面具有极好的效果。本试验选用实验室前期分离的一株有机无机双解解磷细菌WY4,经16s rRNA鉴定为巨大芽孢杆菌(Bacillus megaterium),利用绿色荧光蛋白标记(green fluorescent protein,GFP)技术对WY4进行荧光标记,以期研究解磷细菌在小白菜根系及根系土壤中的定殖动态,同时研究解磷细菌WY4在大田条件下对小白菜(Brassica rapa L.Chinensis Group.)生长、品质及土壤养分的影响。主要研究结果如下:1.绿色荧光蛋白标记解磷细菌WY4,获得具有较为稳定遗传性的标记菌株WY4-GFP。标记菌株WY4-GFP与野生型菌株WY4在生长动态、有机酸分泌及解磷活性均无显著性差异,说明GFP标记不影响WY4菌株的生理活性,可以替代野生型菌株。2.WY4-GFP在小白菜根际土壤及根系组织中均可定殖。(1)在小白菜根际土中接种30 d后,WY4-GFP定殖数量为5.80×104 CFU/g,说明WY4-GFP在小白菜根际土壤中具有持久稳定的定殖能力;(2)WY4-GFP在小白菜根冠及分生区大量定殖,在伸长区及侧根根毛处数量较少,同时根表皮细胞间隙上也有较多的标记菌株,表明WY4-GFP在小白菜根系组织中具有良好的定殖能力;试验结果同时表明WY4-GFP与WY4均能促进小白菜的生长,且二者之间无显著差异。这为后期深入研究解磷细菌在土壤中的定殖奠定了基础。3.大田条件下,解磷细菌WY4仍具有较好的解磷能力。施用解磷细菌WY4后显著提升土壤速效磷含量,降低土壤pH,且土壤速效钾、有机质、硝态氮、铵态氮含量也显著提高;小白菜根长、株高、叶片数、生物量与对照差异显著增加;小白菜叶片硝态氮含量显著降低,可溶性糖、维生素C、纤维素等含量显著增加,小白菜品质明显改善。表明解磷细菌WY4能够显著提高土壤磷素利用效率,改善土壤品质,促进小白菜生长。
[Abstract]:Phosphorus (P) is a very important element in natural and agricultural ecosystems. According to the national soil census, about two thirds of the soils are still deficient in phosphorus, and phosphorus deficiency is the main factor limiting the high yield of crops. With the increasing of food crisis and environmental crisis, how to improve crop yield on the basis of environmental protection has become a major problem facing the whole world and all mankind. Phosphate-solubilizing bacterium PSB is a kind of plant promoting bacteria (plant growth-promoting bacterium PGPB), which has been discovered and widely used by people earlier. It can promote the dissolution of insoluble phosphorus in soil, improve the utilization efficiency of soil phosphorus, and do little harm to the environment. It has excellent effect in promoting crop growth. In this experiment, a strain of organic-inorganic biolysis bacteria WY4 was isolated in the laboratory and identified as Bacillus megaterium (Bacillus megaterium), by using green fluorescent protein labeling (GFP) technique to label WY4 by 16s rRNA. The purpose of this study was to study the colonization dynamics of phosphorus releasing bacteria in Chinese cabbage root system and root system soil, and to study the effect of phosphorus releasing bacteria WY4 on (Brassica rapa L.Chinensis group of Chinese cabbage under field conditions. Effects of growth, quality and soil nutrients. The main results are as follows: 1. A stable genetic marker strain WY4-GFPwas obtained by labeling phosphobacteria WY4 with green fluorescent protein. There was no significant difference in growth dynamics, organic acid secretion and phosphorus-releasing activity between the labeled strain WY4-GFP and wild-type strain WY4, indicating that GFP labeling did not affect the physiological activity of WY4 strain. WY4-GFP can be substituted for wild-type strain .2.WY4-GFP can colonize in rhizosphere soil and root tissue of pakchoi. (1) after inoculating in rhizosphere soil of pakchoi for 30 days, the colonization quantity of WY4-GFP is 5.80 脳 10 ~ 4 CFU / g, which indicates that WY4-GFP has lasting and stable colonization in rhizosphere soil of pakchoi. (2) colonization of WY4-GFP in root crown and meristem of pakchoi, At the same time, there were more labeled strains in the intercellular space of root epidermis, which indicated that WY4-GFP had a good colonization ability in the root tissue of pakchoi (Brassica campestris). The results also showed that both WY4-GFP and WY4 could promote the growth of Chinese cabbage, and there was no significant difference between them. This laid a foundation for further study on the colonization of phosphorus releasing bacteria in soil. Under the field conditions, the phosphorus releasing bacteria (WY4) still had good phosphorus releasing ability. The content of available phosphorus, soil pH, available potassium, organic matter, nitrate nitrogen and ammonium nitrogen in soil were significantly increased after WY4 application, and the root length, plant height, leaf number, biomass and biomass of pakchoi increased significantly. The contents of nitrate nitrogen, soluble sugar, vitamin C and cellulose in leaves of Chinese cabbage were significantly decreased, and the quality of Chinese cabbage was improved obviously. The results showed that WY4 could significantly improve the utilization efficiency of phosphorus in soil, improve soil quality and promote the growth of pakchoi.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S182;S634.3

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