Identification and target gene analysis of miRNAs related to NACl stress alleviated by ALA in wild jujube

【Objective】The objective of this study was to identify miRNAs related to the alleviation of NaCl stress in wild jujube by ALA (5-aminolevulinic acid), thereby providing a theoretical basis for elucidating the molecular mechanism by which miRNAs and core target genes participate in the alleviation of NaCl stress by ALA.【Methods】The leaves of plantlets propagated by tissue culture of wild jujube (Ziziphus jujuba var. spinosa) were used as the research materials. Four treatments were set up: spraying distilled water on the leaf surface as the control (CK), spraying ALA on the leaf surface (ALA), adding NaCl solution and spraying distilled water on the leaf surface (NaCl), and adding NaCl solution and spraying ALA on the leaf surface (ALA + NaCl). The differences in physiological indicators such as plant height, root length, root surface area, and root volume were measured and analyzed. High-throughput sequencing was performed of the leaves of the four treatments to identify key miRNAs responding to ALA alleviating NaCl stress in wild jujube seedlings, and to determine the functions of their target genes.【Results】The study showed that NaCl treatment led to water lost and wilt of the seedling leaves, significantly reduced the plant height, root length, root surface area, and root volume of wild jujube seedlings. The damage caused by NaCl stress to jujube seedlings was alleviated after exogenous applica-tion of ALA. Twelve sRNA libraries were constructed from the leaves of the four treatments, and the Q30 value of each sample was ≥ 93%, with the reads aligned to genes accounting for 86.46%-93.12% of the total reads, indicating good data quality. A total of 149 known miRNAs belonging to 31 miRNA families and 11 novel miRNAs were identified from the 12 libraries. The length of sRNAs was mainly between 21-24 nt, with 24 nt sRNAs being the most abundant in all libraries. Compared with the CK, there were 152 differentially expressed miRNAs in the NaCl treatment group, including 76 down-regulated genes and 76 up-regulated genes; in the ALA+NaCl treatment group, there were 149 differentially expressed miRNAs, including 78 down-regulated genes and 71 up-regulated genes. The functional enrichment analysis of differentially expressed miRNAs based on the Kyoto Encyclopedia of Genes and Genomes database showed that the target genes of differentially expressed miRNAs were involved in metabolic pathways such as TMV resistance proteins, membrane proteins containing SPX domains, sodium-potassium transporters, ribosomal RNA processing proteins, transcription factors such as auxin response factors (ARF) family, MYB transcription factors, WRKY transcription factors. Many miRNAs responding to salt stress, such as zju- miR172d, zju- miR395i, and zju- miR172a, were significantly down-regulated, indicating that these miRNAs might be inhibited by single salt stress. Further analysis revealed that ALA significantly inhibited the expression of zju-miR408a. The target gene annotation of zju-miR408a showed that it targeted enzymes such as the peroxidase, protoporphyrinogen oxidase, mitochondrial anion peroxidase, lignin- forming anion peroxidase, calcium-dependent protein kinase 1, and laccase. The qRT- PCR analysis results were basically consistent with sequencing results. The expression level of the target gene POD and POD enzyme activity increased more significantly after exogenous application of ALA than after NaCl stress.【Conclusion】The results of this study showed that exogenous application of ALA alone had no significant effect on wild jujube seedlings. The growth of jujube seedlings was inhibited by NaCl stress, and exogenous application of ALA could alleviate NaCl stress in wild jujube seedlings. The construction of sRNA libraries from wild jujube seedlings revealed that ALA significantly induced the expression of zju-miR408a. The target gene annotation analysis of zju-miR408a mainly targeted the enzymes such as oxidases and protoporphyrinogen oxidase. The results were further verified by measuring the expression level of target gene POD and POD enzyme activity. In summary, ALA would alleviate NaCl stress in wild jujube seedlings by inhibiting the expression of zju-miR408a to upregulate the expression level of target gene POD, thereby scavenging reactive oxygen species and improving salt tolerance. This result would provide new information for a more comprehensive analysis of miRNAs involved in alleviating salt stress in wild jujube by ALA.