溶磷真菌ATMT转化条件优化及其对矮牵牛的促生效果评价

Optimization of a phosphate-solubilizing fungus ATMT system and evaluation of its growth-promoting effect on Petunia hybrida

摘要:

[目的] 建立和优化一株溶磷真菌咖啡果小蠹青霉菌Penicillium brocae的转化体系，并利用分子标记观察其在根部的定殖；检测接种咖啡果小蠹青霉菌对植物的促生作用，为菌肥的研发奠定基础。[方法] 利用农杆菌介导的转化体系（Agrobacterium tumefaciens-mediated transformation，ATMT）获得转化子并优化转化条件，结合插入片段携带的分子标记进行转化子验证和根部定殖研究，同时利用TAIL-PCR （thermal asymmetric interlaced-PCR）锚定插入位点序列信息；通过矮牵牛盆栽实验，设置T1（10 mL水）、T2（10 g/L磷矿粉，10 mL）、T3（10⁶个/mL孢子，10 mL）和T4（10 g/L磷矿粉+10⁶个/mL孢子，10 mL）4个处理，探究P.brocae对植物的促生作用。[结果] 利用优化后的ATMT体系可获得44个转化子/10⁶个孢子，通过标记基因hyg和gfp的克隆以及GFP荧光观察和GUS染色结果验证了转化子，并利用GUS标记观察到了菌株在矮牵牛根部的定殖情况；利用TAIL-PCR克隆了一株溶磷能力有较显著降低的转化子上插入位点一端的侧翼序列，并确定了插入位点的位置；T4处理组鲜重较T1、T2和T3差异显著（P ≤ 0.05），且分别高出35.4%、35.6%和21.4%，T1、T2和T3相互之间无显著差异。T4处理组干重较T1、T2以及T3也有显著差异（P ≤ 0.05），且分别高出25.1%、37.8%和26.0%，T1、T2和T3之间亦无显著差异。[结论] 本研究优化了P.brocae的ATMT体系并观察到了转化子在矮牵牛根表的定殖；磷矿粉和P.brocae孢子共施能够显著促进矮牵牛幼苗的生长，但二者分别单独施用则无此效果，推测在二者共施情况下P.brocae可能提高了磷矿粉的肥效。

Abstract:

[Objective] To optimize the transformation system of a phosphate-solubilizing fungus Penicillium brocae and modify the strain with molecular tag for root colonization analysis; to evaluate the growth promotion effects of P. brocae. [Methods] We optimized ATMT (Agrobacterium tumefaciens-mediated transformation) parameters to get P. brocae transformants, we confirmed the transformants and its root colonization with molecular tags; we used TAIL-PCR (thermal asymmetric interlaced-PCR) to characterize T-DNA insertion site; we evaluated the growth promotion effects of P. brocae by Petunia hybrida pot experiments with treatments of T1 (10 mL water), T2 (10 g/L phosphate rock, 10 mL), T3 (10⁶ spores/mL, 10 mL) or T4 (10 g/L phosphate rock and 10⁶ spores/mL, 10 mL).[Results] We got 44 transformants per 10⁶ spores by parameters optimization and confirmed the transformants with gfp and hyg gene cloning, GFP fluorescence detecting and GUS staining. We observed the colonization of P. brocae on P. hybrida root surface by GUS staining. The insertion site flanking sequence of a transformant characterized with phosphate solubilization capacity significantly decreasing was obtained by TAIL-PCR. P. hybrida fresh weight of T4 were 35.4%, 35.6% and 21.4% higher than that of T1, T2 and T3, the dry weight of T4 were 25.1%, 37.8% and 26.0% higher than that of T1, T2 and T3, respectively. There was no significant difference among the fresh or dry weight of T1, T2 and T3 (P ≤ 0.05). [Conclusion] We optimized the ATMT system and observed the root colonization of P. brocae. The application of P. brocae spores and phosphate rock together but not alone, could promote the growth of P. hybrida and this gave a hint that P. brocae may enhance the fertilizer efficiency of phosphate rock in soil.