Differentially Expressed MicroRNAs That Target Functional Genes in Mature Soybean Nodules

Soybean can establish symbiosis with soil-borne bacteria, rhizobia. Following rhizobium infection, soybean roots undergo organogenesis to form nodules, where rhizobia take shelter and carry out symbiotic nitrogen fixation (SNF). SNF in soybeans can provide the soybean plant with nitrogen and also significantly enrich the bioavailable nitrogen content of the soil which can reduce the nitrogen fertilizer application in agriculture. In recent years, microRNAs (miRNAs) are verified to be important regulators in nodule formation. However, roles of miRNA in mature nodule are not well studied. In this research, I aimed to identify miRNAs in roots and nodules that are important for nitrogen fixation in both cultivated (Glycine max) and wild soybeans (Glycine soja) 28 days post inoculation with rhizobia (Sinorhizobium fredii).Uninfected roots, infected roots with nodules removed and nodules were harvested from cultivated (C08) and wild (W05) soybeans 28 days after inoculation with rhizobia for small RNA. Hierarchical clustering analysis identified miRNA expressions exhibit tissue-specificity, suggesting a universal regulatory role of miRNAs on root and nodule functions in both soybean species. There were also some miRNA expressions showing variety-specificity, indicating a different response to rhizobium infection between two soybean varieties.After miRNA target prediction, differential expressions of miRNAs that are functionally associated with plant hormone signaling, nodulation processes and nutrient transportation in soybean roots and mature nodules were identified. Amongst these miRNAs, miR4407 showed higher expression in C08 and could regulated the soybean nodule number. Except miR4407, members of miR399 family were strongly induced in inoculated roots and nodules upon rhizobium infection. MiR399 was predicted to target PHO2, which encodes a negative regulator of phosphate transporter. It was found that miR399 could be induced by both nodulation and phosphate starvation. Furthermore, miR399 also affects nitrate transportation through regulating transcription of NITRATE TRANSPORTER1.1 (NRT1.1), NRT2.4 and NRT2.5. Three nitrate reductase encoding genes were also down-regulated by miR399. Transgenic alteration of miR399 expression significantly influenced the whole plant phosphate concentration, nitrogen fixation efficiency and plant growth in the rhizobia-infected soybeans. These findings suggested an important role of miRNAs in nutrient allocation in soybean roots and nodules that is essential for effective nitrogen fixation.大豆可以與土壤中的根瘤菌共生。大豆在被根瘤菌侵染後會形成根瘤。根瘤菌在根瘤中生存並同時進行生物固氮。生物固氮可以顯著增強土壤中的氮，提高土壤肥力，因此可以減少農業中的氮肥使用量。近年來，研究發現小RNA是根瘤形成的重要調控因子。但是關於小RNA對於成熟根瘤的作用尚不明確。所以在這次研究中，我的研究目標為小RNA對於中華根瘤菌侵染30天後的栽培和野生大豆的根和根瘤的調控作用。中華根瘤菌侵染28天後的栽培型和野生型大豆的根和根瘤以及兩種大豆未侵染的根被收集起來用作小RNA測序。測序結果顯示小RNA的表達模式存在組織特異性，這意味著小RNA對於共生大豆的根與根瘤有一個通用的調節機制。當然也有一些小RNA在栽培大豆和野生大豆中對於中華根瘤菌的侵染展現出不同的表達量，這意味著小RNA也存在品種特異性。階層式分群研究發現小RNA在不同樣品中變化的表達量與植物激素信號傳導，根瘤形成過程，以及根瘤中營養運輸有關。研究發現小RNA，miR4407，在栽培型大豆中展現出更高的表達量並且能調控根瘤數量。在所有研究的小RNA群體中，根中miR399在侵染前後表達量產生了巨大變化。同時miR399也對低磷脅迫做出響應。轉基因方式改變MiR399在大豆中的表達量可以改變大豆中的磷含量，加強固氮效率。另外我們還發現miR399可以影響硝酸鹽運輸蛋白和硝酸鹽還原酶的轉錄表達量，這也對加強生物固氮有重要作用。這些發現意味著小RNA在共生大豆的根和根瘤中的營養調配對於生物固氮具有重要意義。FAN, Kejing.Ph.D. Chinese University of Hong Kong 2021.Includes bibliographical references (leaves )Abstracts in English and Chinese.Title from PDF title page (viewed on ...