Cloning and function analysis of PR-1 gene in Actinidia

Abstract:【Objective】China is the origin of the kiwifruit (Actinidia spp.), with rich germplasm resources and wide geographical distribution. It is one of the most recently domesticated fruit plants and has become an important horticultural crop. There are 54 species and 21 varieties of the A. Lindl. in the world. Kiwifruit bacterial canker is a devastating disease in kiwifruit industry globally and caused by pathogen Pseudomonas syringae pv. actinidiae (Psa). Psa is highly virulent, and once systemic invade plant may quickly lead plant to death. It has been documented that the Pathogenesis-related 1 protein (PR-1) could resist the spread of viruses, limit the invasion of pathogens and protect plants from adversity stress. In many plant species such as Arabidopsis and tobacco, the overexpression of the PR- 1 gene could enhance plant resistance to P. syringae. However, the PR-1 gene in kiwifruit and its role in responses to abiotic stress remain largely unknown. The objective of this study was to explore the function of kiwifruit Pathogenesis-related 1 gene (PR-1) in response to biological stress. This analysis could contributeto in-depth understanding the function of the PR-1 gene in kiwifruit disease resistance.【Methods】Annual grafted treess of kiwifruit species A. eriantha were used as experimental materials. The full-length sequence of the PR-1 homologous gene AePR-1 of A. eriantha was cloned and analyzed by multiple bioinformatic tools. The DNAMAN software was used to compare and analyze the sequence of the AePR-1 gene. The conserved domain of AePR-1 protein sequence was analyzed by NCBI website. The Expasy ProtParam tool was used to predict the molecular weight, theoretical pI, instability index and grand average of hydropathicity (GRAVY) of AePR-1 protein. The Cell-PLoc 2.0 software was used to predict the subcellular localization of PR-1 protein. The phylogenetic relationship between the AePR-1 protein and PR-1 of other plants was analyzed by the MEGA 11.013 software using neighbor-joining method. The qRT- PCR was performed to analyze the expression level of the AePR-1 in different tissues and flower organ. The expression of the AePR-1 gene in response to P. syringae pv. Actinidiae (Psa) bacterial solution and Jasmonic acid (JA), Salicylic acid (SA), Abscisic acid (ABA), Gibberellin A3 (GA3) treatments was detected by real-time fluorescence quantitative PCR method. The samples were taken at 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, 72 h and 96 h after treatment and immediately frozen in liquid nitrogen and stored at -80 ℃ for RNA isolation. The subcellular localization technology was used to analyze the expression position of the AePR-1 gene in cells. The homologous recombination was used to construct the AePR-1 overexpressed vector and heterologous expression was carried out in the tobacco to validate the function of the gene PR-1 under Psa infection. All the experiments and data in this study involved at least three repeats. The Excel, SPSS and Origin2023 software were used to statistics and analysis of test measurement data. LSD test (p＜0.05) was used to assess significant differences in means. 【Results】The full length of the cloned AePR-1 gene sequence was 522 bp, encoded 173 amino acids and contained CAP_PR- 1 conserved domain. The relative molecular weight (MW) of protein was 19.28 ku and the isoelectric points (PI) was 9.28. The protein instability index was 41.54 and the protein belonged to unstable protein and the grand average of hydropathicity (GRAVY) was -0.261. The subcellular localization of PR-1 showed that the AePR-1 gene was mainly localized in the cytoplasm and cell membrane. The phylogenetic tree results showed that the protein AePR-1 was highly homologous to the AthPR-1 from Arabidopsis and CsaPR-1 from Cucumis. The qRT-PCR results showed that AePR-1 gene was highly expressed in the roots and pistils. And after inoculation with Psa bacterial solution, the expression level of the AePR-1 gene decreased at the early stage, increased rapidly after 6 hours, and reached its peak at 24 hours. With the prolongation of the treatment time of different hormones, the expression of the AePR- 1 gene generally showed two peaks. The AePR- 1 expressed the highest on the second day after overexpression of tobacco. Therefore, Psa was used to infect injected tobacco on the second day of tobacco transient expression. The results showed that on the 14th day after infection, the leaves of the empty control group showed large areas of yellow spots, while a small number of yellow spots appeared on the surface of the leaves overexpressing the AePR-1 gene.【Conclusion】This study explored the anti-disease effect of kiwifruit AePR-1 gene in kiwifruit bacterial canker. The results showed that the AePR-1 gene of kiwifruit was expressed in large quantities under the induction of Psa bacteria and exogenous hormones, participated in the immune response of kiwifruit and enhanced the disease resistance of kiwifruit. This study shows that the AePR-1 gene plays an important role in the disease resistance of kiwifruit and can be valuable for resistance breeding of kiwifruit.