关键词: 南方红豆杉, 草木樨中华根瘤菌, 定殖, 绿色荧光蛋白基因

Abstract:

[Objective]Taxus chinensis var. mairei is an endemic, endangered and first-class protected tree species in China. This species is considered as an important botanical resource of taxol which is an effective medicinal compound against various cancers. Phosphate solubilizing bacteria (PSB) Sinorhizobium meliloti CHW10B,which was isolated from rhizosphere of T. chinensis var. mairei, showed significant growth-promoting effect for T. chinensis var. mairei. Survival in the environment and efficient colonization are a necessary and vital step for plant-beneficial bacteria to playing a role in plants. In order to effectively take advantage of S. meliloti CHW10B, the colonization of this strain in rhizosphere and roots of the seedlings of T. chinensis var. mairei was studied via the green fluorescent protein (GFP) gene label technique. [Method]The green fluorescent protein (GFP) technique is widely used to study the colonization of target bacteria in rhizosphere and host plants. The shuttle vector pGFP4412, containing one copy of the constitutively expressed GFP, neomycin and ampicillin resistance genes in tandem, was transformed into CHW10B strain by using the repeated freezing and thawing methods with modification. Then the transformants with strong fluorescence expression and stable hereditary were further screened using fluorescence microscopy. The morphological characteristics of the cell and colony for CHW10B and CHW10B -GFP were identified and the phosphate-dissolving abilities of the two bacterial strains were determined with the molybdenum-antimony anti-spectrophotometric method. After inoculation by pouring the bacteria to root surface of T. chinensis var. mairei, the survival and colonization of strain CHW10B labelled with GFP were investigated by combining antibiotics plate recovery with fluorescence microscopy. [Result]The transformants (CHW10B-GFP2) were successfully obtained, and the stain had strong fluorescence expression and stable hereditary. The cells of CHW10B-GFP2 and CHW10B were both Gram-positive, short rod-shaped. The colonies of the two strains were all circular, entire margin and a sticky. But the colors of colonies were not same, strain CHW10B was milky white, and CHW10B-GFP was light brown. Phosphate-dissolving abilities of CHW10B-GFP2 and CHW10B were similar and the content of soluble P in the supernatant was 639.12 mg·L^-1 and 656.57 mg·L^-1 in 4 days after inoculation. The colonization amount of the strain CHW10B-GFP in the rhizosphere soil of T. chinensis var. mairei had big variations throughout the experiment after inoculation. At the first day after inoculation, the number of the labelled cells reached 6.08×10⁷cfu·g^-1 in the rhizosphere soil. Thereafter the bacterial population decreased sharply and then began to increase in 15 days after inoculation. In 25~40 days after inoculation, the bacterial population subsequently increased and leveled off. In 40 days after inoculation, many cells of this strain could be observed in the surface and inside of seedling roots by a Fluorescent microscope. [Conclusion]This study demonstrated that the marked strain could survive stably in the rhizosphere of T. chinensis var. mairei. Moreover, CHW10B-GFP could colonize in the root surface and internal of the seedlings, indicting strain CHW10B was an endophte. The study would provide a scientific basis for the exploitation and utilization of strain CHW10B.

Key words: Taxus chinensis var. mairei, Sinorhizobium meliloti, colonization, GFP