关键词: 青檀, 氮素, 形态和光合生理响应, 全长转录组, 差异表达基因

Abstract:

【Objective】Our objectives are to study the morphological and photosynthetic physiological changes of Pteroceltis tatarinowii under different nitrogen application levels, and screen nitrogen response related genes in order to provide reference for further research on the molecular mechanisms how nitrogen application trigger changes in P. tatarinowii. 【Method】A pot experiment was conducted with the cuttings of P. tatarinowii fertilized with Hocking’s complete nutrient solution with 0 (limiting, N₀), 2 (intermediate, N₂) and 50 mmol/L (luxuriant, N₅₀) NH₄NO₃, respectively. The physiological and photosynthetic morphological parameters were determined at day 28. SMRT-seq, performed with the pooled sample of P. tatarinowii treated with different nitrogen application levels, provided full length transcriptome data as a reference for the RNA-seq. Differentially expressed genes responding to different nitrogen levels were screened using bioinformatics methods. Then, the KEGG pathway enrichment analysis was conducted. The expression of the differentially expressed genes enriched in roots and leaves was quantified by RT-qPCR. 【Result】The physiological and morphological observations, determined at day 28 after being fertilized with different nitrogen application levels, showed that the values of height, stem diameter, number of leaves, leaf area, specific leaf area, biomass of leaves, biomass of stems, N concentration in roots, leaves and stems increased with the increase of nitrogen concentration, while the values of the total root length, total root surface area, total root volume, specific root length, root to shoot ratio, and biomass of roots, all decreased with the increase of nitrogen concentration. The chlorophyll a, chlorophyll b, carotenoid content, net photosynthetic rates and stomatal conductance were significantly promoted by the luxuriant nitrogen level. Of the differentially expressed genes among different nitrogen levels based on the global transcriptomic profiling by RNA-seq, 76 genes were up-regulated following the increase of nitrogen application levels, while 32 genes were opposite. The KEGG pathway enrichment analysis of the 108 genes showed that the top two pathways were photosynthesis and nitrogen metabolism. The photosynthesis pathway was enriched with OEE2, OEE3, PSBR, PSB27, PSAF and PSAK, while the nitrogen metabolism pathway was enriched with two NR genes and one NiR gene. The expression analysis showed that the nine genes were with similar expression patterns in roots and leaves, lowest expression levels in limiting nitrogen level while highest expression levels in luxuriant nitrogen. The expression levels of six genes of photosynthesis pathway in leaves were higher than that in roots under each nitrogen level. 【Conclusion】 Following the increase of nitrogen application levels, photosynthesis pathway genes were up-regulated, which promoted photosynthesis, while, nitrate reductase genes and nitrite reductase genes were up-regulated, which improved the nitrate assimilation efficiency. In the absence of a reference genome sequence, our results provided a basis for exploring the molecular mechanisms and discovering key candidate genes how nitrogen trigger changes in P. tatarinowii.

Key words: Pteroceltis tatarinowii, nitrogen, morphological and photosynthetic physiological response, full length transcriptome, differentially expressed gene