巨大芽胞杆菌WY4的GFP标记及其在小白菜上的定殖  

Bacillus megatherium WY4 Labeled by GFP and Its Colonization in Chinese Cabbage(Brassica chinensis)

作　　者：王珍[1] 曹翠玲[1] 库永丽 徐国益 林雁冰[1] 柳晓东[1] WANG Zhen CAO Cui-Ling KU Yong-Li XU Guo-Yi LIN Yan-Bing LIU Xiao-Dong(College of Life Science, Northwest A ＆ F University, Yangling 712100, China)

机构地区：[1]西北农林科技大学生命科学学院,杨凌712100

出　　处：《农业生物技术学报》2016年第12期1925-1934,共10页Journal of Agricultural Biotechnology

基　　金：陕西省科学技术研究发展计划项目(No.2013K01-38)

摘　　要：解磷菌(phosphate solubilizing bacteria,PSB)可以通过提高土壤有效磷含量而增加作物产量,目前已有许多解磷菌被分离并应用于农业生产中,但关于解磷菌在植物根际中的定殖情况仍缺乏系统性的研究。WY4为本实验室前期从小白菜(Brassica chinensis)根际分离得到的一株高效解磷菌,本研究利用绿色荧光蛋白(green fluorescent protein,GFP)标记技术研究了WY4在小白菜根际及土壤中的定殖规律。与原菌株相比,GFP标记对菌株WY4-GFP生长及解磷活性具有较小影响,同时在促进小白菜生长上WY4-GFP与WY4无显著性差异;WY4-GFP具有持久的定殖能力(接种21 d的自然土及30 d的小白菜根际土中,WY4-GFP的定殖数量分别为105和104CFU/g左右),同时随时间的增加WY4-GFP在土壤中定殖数量逐渐减少;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,同时表皮细胞间隙上也有较多的标记菌株。研究结果表明,WY4-GFP在小白菜根际及土壤中具有良好的定殖能力,这为后期深入研究解磷菌与植物间的关系提供了重要参考。Phosphate solubilizing bacteria（PSB） can increase available phosphorous in soil and improve yields in several crop species like rice（Oryza sativa）, maize（Zea mays）, potato（Solanum tuberosum）,sunflowers（Helianthus annuus） etc. In previous studies, many phosphate solubilizing bacteria had been isolated and widely used in agricultural production. However, the colonization of phosphate solubilizing bacteria in plant rhizosphere have not been completely characterized. Phosphate solubilizing bacteria WY4 is a bacterium isolated from the soil of Brassica chinensis rhizosphere soil. WY4 is a gram- positive bacterium which is tested by Gram staining and its cell morphology is rod shape. The sequence analysis of 16 S r RNA gene in the NCBI database using a Blast N analysis showed 99% similarity to Bacillus megaterium strain. To evaluate colonization in soil and Chinese cabbage rhizosphere by this microorganism, generated WY4-GFP, a green fluorescent protein（GFP） labeled derivative of WY4, and inoculated the marked strain into Chinese cabbage seedlings. Compared with the original strain, GFP-labeled had a smaller influence on the growth and phosphate solubilizing activity. While, the results demonstrated that WY4- GFP could survive in the natural soil to achieve 105CFU/g（wet soil） at 21 days; WY4- GFP increased the plant height, root length, leaf numbers, fresh weight and dry weight of Chinese cabbage plants. Phosphorus could promote root growth of Chinese cabbage, WY4 could increase content of available phosphorus as PSB, so the root system was developed after application of WY4： The root volume that reached 1.72 m L was significantly higher than water treatment（0.38 m L）. After the treatments of WY4-GFP and WY4, the fresh weight of the plant reached17.48 and 17.52 g/plant respectively. There was no significant difference between WY4 and WY4- GFP in growth promotion. The results further indicated that WY4- GFP flourishing colonizes in Chinese cabbage plants and rhizosphere soils, colonizat

关 键 词：解磷细菌 绿色荧光蛋白 小白菜 定殖 

分 类 号：S154.3[农业科学—土壤学]