关键词: 南高丛蓝莓, VcLon1蛋白酶, 基因功能, 植物抗旱

Abstract: [Objective] Lon1 protease plays a critical role in degrading oxidized proteins in chloroplast and mitochondria and maintaining the normal cellular metabolism. Up to now, its function related to abiotic stress tolerance has attracted little attention. Based on an analysis of the patterns of temporal and spatial expressions of blueberry VcLon1 gene(GenBank number:MF972079), the tobacco genetic transformation technique and drought tolerance assay were applied to elucidate the biological functions of VcLon1 involved in drought stress tolerance in plants. This study would provide a theoretical basis for developing biotechnology of drought-resistant blueberry germplasms and provide basic information for understanding drought resistance mechanism of other plants.[Method] On the basis of cloning VcLon1 full length from Vaccinium corymbosum ‘Sharpblue’,Protparam and CDD software was used to analyze the protein structure of VcLon1. Phylogenetic tree of Lon1 proteins was constructed by MEGA4.0 software. The expression levels of VcLon1 in different tissues and in drought stress condition was analyzed by real-time fluorescence quantitative PCR (qPCR).After drought stress treatment, the differences of chloroplast ultrastructure, photosynthetic physiology and oxidative stress between wild type(WT) and VcLon1 overexpressing Nicotiana benthamiana were investigated.[Result] 1)The ORF of VcLon1 was 2 982 bp, which encoding 993 amino acids. The theoretical isoelectric point was 5.44 and the molecular weight was 109.5 kDa.The protein sequence encoded by VcLon1 contains a typical AAA+ domain and belongs to the AAA+ superfamily. Localization analysis showed that the coding protein was expressed in chloroplast and mitochondria, and had close relationship with Lon1 protease such as grape and apple. 2)The qPCR analysis revealed that VcLon1 was expressed ubiquitously throughout ‘Sharpblue’ plant, with relatively lower expression levels in older tissues. The drought stress induced significantly higher transcription level of VcLon1 in blueberry. 3)The overexpression of VcLon1 in tobacco led to increased drought tolerance. The largest biomass was observed in line of VL-6, which exhibited green leaves and well-developed roots, and the height and dry weight were 36.66% and 114.29% higher than those of the wild type, respectively. 4)Under drought stress,the chloroplast was swollen and some of the lamellar structures were blurred of WT. However, no obvious damage was observed in chloroplast ultrastructure of VcLon1 overexpressed lines except some of the voids in the chloroplast matrix were increased, and the chlorophyll content of VcLon1 overexpressed lines was higher than that of the wild type. Meanwhile, the mitochondria of wild-type were swollen and metabolic, and the crista were fractured, disintegrated and vacuolated. The mitochondria in VcLon1 overexpressed lines still maintain the normal ellipsoid shaped structure. 5)Under drought stress, MDA content of VcLon1 overexpression plants was 34.38%-49.68% lower than WT, while less H₂O₂ accumulation occurred in leaves of VcLon1 overexpression plants, as indicated by their brown areas. Comparatively, the brown area of the WT increased significantly. Furthermore, under drought stress, the content of carbonylated protein in the overexpressed plants was also significantly lower than that of WT, which may result from the generally higher activities of SOD, GR, APX and POD in VcLon1 overexpressed plants, as compared with WT.[Conclusion] Under drought stress, VcLon1 may play a key role in keeping the normal morphology of the chloroplasts and mitochondria and to protect the cell membrane system. Besides, the normal energy metabolism may maintain by degrading carbonylated protein in mitochondrial and in reducing productions of reactive oxygen species (ROS). Moreover, VcLon1 helps to maintain relatively higher activities of antioxidant enzymes to reduce the accumulations of intracellular ROS, ultimately alleviate the intracellular oxidative stress, which facilitate the maintenance of the normal cellular metabolism and improve the drought resistance capacity.

Key words: Vaccinium corymbosum, VcLon1 protease, gene function, plant drought resistance