Screening of differentially expressed miRNAs and target genes in two potato varieties under nitrogen stress
Authors: Yue Lu , Zhijun Han , Jiayue Zhang , Haoran Ma , Yuzhu Han , Jingying Zhang , Zhongcai Han , Shuang Li 
Published: April 2022

Abstract
Background: Nitrogen is an important element for potato growth and development, and improving nitrogen utilization efficiency is an effective way to reduce the amount of nitrogen fertilizer. Transgenic breeding is an effective means to solve the problem that traditional breeding is difficult to solve, it is of great significance to excavate genes that improve nitrogen utilization for breeding nitrogen-efficient potato. It was found that the key enzyme genes in potato nitrogen metabolism pathway were highly responsive to nitrogen stress, and the activity of the key enzyme gene NiR (nitrite reductase) was significantly regulated with the increase of nitrogen supply. With the continuous in-depth study of the functions of miRNAs, it has been found that key enzyme genes can regulate their spatiotemporal expression through specific miRNAs, thereby regulating plant phenotypic traits. For example, miR159 regulates MYB, miR169 regulates NFYA. miRNAs are endogenous non-coding RNAs with regulatory functions that recognize target genes through complementary base pairing. In recent years, some mechanisms have been elucidated through the correlation between the expression levels of potato miRNAs target genes and regulated genes under stress conditions, but the role of miRNAs in the inhibition/expression of key genes regulating nitrogen metabolism under N stress is still unclear.Results: Through the extension of nitrogen-efficient potato, Yanshu4 ("Y") and nitrogen-inefficient potato Atlantic ("D"), the roots and leaves at the seedling and budding stages after different nitrogen fertilization treatments were collected. miRNAs sequencing, degradome analysis, qRT-PCR and luciferase activity measurement to analyze the expression changes of miRNAs under different N treatment conditions. The study found that with the increase of nitrogen application, NiR activity increased significantly from seedling stage to budding stage, and NiR responded significantly to different nitrogen application treatments. miRNAs sequencing predicted 48 families of conserved miRNAs, mainly involved in nitrogen metabolism, carbon metabolism and amino acid biosynthesis. The following differences of miRNAs were screened out (high expression and P < 0.05): stu-miR396-5p, stu-miR408b-3p_R-1, stu-miR3627-3p, stu-miR482a-3p, stu-miR8036-3p, stu-miR482a-5p, stu-miR827-5p, stu-miR156a_L-1, stu-miR827-3p, stu-miR172b-5p, stu-miR6022-p3_7, stu-miR398a-5p, stu-miR166c-5p_L-3. Degradome analysis showed that most miRNAs had many-to-many relationships with target genes. The main target genes involved in nitrogen metabolism include NiR, NiR1, NRT2.5 and NRT2.7. qRT-PCR verification found that stu-miR396-5p, stu-miR8036-3p and stu-miR482a-3p were significant differences in leaves and roots between Yanshu4 and Atlantic at seedling and budding stages under the conditions of no nitrogen application and excess nitrogen application, which are induced in response to N stress. The correlation between differential stu-miR396-5p and its corresponding target gene NiR was further verified by luciferase activity, and it was found that there was a strong negative correlation, which was conducive to further functional research.Conclusion: From seedling stage to budding stage, the activity of NiR was significantly positively correlated with nitrogen application. Differential miRNAs and target genes showed a many-to-many relationship. The expression of stu-miR396-5p, stu-miR482a-3p and stu-miR8036-3p in Yanshu4 and Atlantic, seedling and budding stage at roots and leaves were most different under two N stresses. Under two N stresses, in Yanshu4, stu-miR396-5p was down-regulated in seedling leaves and shoot-stage roots, and up-regulated in seedling stage roots and shoot-stage leaves; stu-miR482a-3p was up-regulated in seedling stage and shoot stages; The expression of stu-miR8036-3p was up-regulated in leaves and roots at the seedling and budding stages, and down-regulated in roots under both nitrogen stresses. The N metabolism key enzyme gene StNiR and the stu-miR396-5p luciferase activity assay reporter detected that they have a strong regulatory relationship.