Functional Analysis Of A Tomato Chloroplast DnaJ Protein SlCDJ2

Tomato is a kind of thermophilic vegetable crops originated from tropic, but its resistance to heat is not strong, and the high temperature is usually accompanied by the drought, and the production of tomato will decline when suffered with heat and drought stresses. Therefore, it is important to study the resistance of tomato to heat and drought stresses. In addition, the tomato is easily infected by pathogen when the environment changes. In severe cases, the tomato fruit will show extensive rot and crop failure. Also, the study of tomato disease has practical significance. When plants are suffered from adversity stresses, intracellular protein homeostasis will be destroyed, and a large number of ROS accumulate, and then photosynthesis is restrained. Dna J proteins are stress response factors which are ubiquitous in the cell. Dna J proteins play important roles, alone or in association with Hsp70 partners, in protein folding, refolding and protein complex stabilization under stress conditions. Therefore, it is of great theoretical significance and practical value to explore the physiological function of tomato Dna J proteins under adversity stresses.In this study, a tomato chloroplast-targeted Dna J protein gene SlCDJ2 was cloned from tomato leaves. The roles of SlCDJ2 in biotic and abiotic stress tolerance were investigated here using sense and antisense transgenic tomatoes and transgenic tobaccos. The major points are as follows:(1) We isolated the SlCDJ2(Gen Bank number, AK323942.1) gene from tomato leaves. The full length of SlCDJ2 is 1159 bp. This gene contains an 834 bp open reading frame(ORF), and encodes 277 amino acids. The prediction of the molecular weight is 32.7k Da, the isoelectric point is 9.30. This gene is located in the tomato chromosome 12.(2) q RT-PCR showed that SlCDJ2 specifically expressed in the tomato root, stem, leaf, flower and fruit, and the highest expression in the leaf. The expression of SlCDJ2 was obviously induced by heat, drought, SA and pathogen. The western blot results futher showed that SlCDJ2 was obviously induced by 42°C.(3) SlCDJ2-GFP fusion protein was constructed and transiently expressed in Arabidopsis protoplasts and observed with confocal microscopy. The results showed that SlCDJ2 is located in chloroplast. The western blot results futher showed that SlCDJ2 was uniformly distributed in thylakoids and stroma.(4) Prokaryotic expression vector p ET- SlCDJ2 was constructed and transformed into E.coli BL21. The proteins induced by IPTG was purified and used to immunize the mice to obtain the antiserum, which was used in the following western blot analysis. The titer of antibody was 1:10000.(5) The yeast two-hybrid assays indicated that SlCDJ2 interacted with a chloroplast heat-shock protein 70(cp Hsp70).(6) The ORF of SlCDJ2 was subcloned into the expression vector p BI121 under the control of the 35S-Ca MV promoter to form sense and antisense constructs. The Agrobacterium tumefaciens-mediated leaf disk method was used to generate transgenic tomato and tobacco plants. Kanamycin-resistant plants were detected by PCR, RT-q PCR and western blot. The results suggested that over-expression lines and antisense lines were obtained.(7) Under heat stress, Rubisco activity, Rubisco large subunit(Rbc L) content, and CO2 assimilation capacity were all higher in sense plants and lower in antisense plants compared with wild-type plants. After heat treatment, the expression of genes(SENU3, Sl SGR1, LX, Rbc L, Rbc S, and Psb A) up- and down-regulated by senescence showed different degrees of increases and decreases, respectively. The proteolytic activity of antisense plants increased significantly compared with that in WT and sense plants. Thus, SlCDJ2 contributes to maintenance of CO2 assimilation capacity mainly by protecting Rubisco activity under heat stress. SlCDJ2 probably achieves this by keeping the levels of proteolytic enzymes low, which prevents accelerated degradation of Rubisco under heat stress.(8) Under drought stress, ectopic expression of SlCDJ2 in transgenic tobacco reduced the accumulation of superoxide anion radical(O2·-) and hydrogen peroxide(H2O2), compared with Vec plants, the maximum photochemical efficiency of photosystem II(PSII)(Fv/Fm), net photosynthetic rate(Pn), and content of D1 protein were relatively higher in transgenic plants. Overexpression of SlCDJ2 enhanced tolerance to drought stress.(9) When Vec and transgenic leaves were inoculated with Pseudomonas solanacearum E.F. Smith for 2 weeks, the leaves of Vec plants exhibited prominent chlorosis symptoms. By contrast, the disease symptoms on the leaves of SlCDJ2-overexpressing plants were difficult to observe. Both Trypan blue staining and DAB staining indicated that overexpression of SlCDJ2 enhanced resistance to P. solanacearum of transgenic plants. Moreover, the expression of disease-related genes was investigated in Vec and transgenic lines before and after inoculation with P. solanacearum for 2 days. The results showed higher m RNA levels of PR1 a and PR2 in transgenic line than in Vec plants under normal conditions and when inoculated with P. solanacearum. These results confirmed that SlCDJ2 was involved indefense responses, and SlCDJ2 overexpression enhanced the resistance to P. solanacearum of transgenic plants.