Cloning and expression analysis of Binzi LOX2a

Abstract:【Objective】Binzi (Malus pumila × M. asiatica), an old local fruit species in Beijing, belongs to the Rosaceae family. Binzi is one of the varieties, and its fruit can give forth full aroma. Lipoxygen-ase (LOX), catalyzing the oxygenation of unsaturated fatty acids, contributes to the formation of fruit aroma. However, the LOX has not been reported in Binzi fruit, and the molecular mechanism of aroma formation remains unknown. In the present study, we firstly cloned the LOX2a gene from the Binzi, and identified its expression pattern in different tissues and at different fruit developmental stages. More-over, the effect of phytohormone on LOX2a gene expression level was analyzed.【Methods】The Binzi fruits were collected on 17 May, 16 June, 17 July, 15 August and 14 September in 2020. We screened one LOX gene based on the results of total volatiles and transcriptome data of Binzi fruit after harvest.The LOX2a protein sequences of different plants with higher homology were downloaded from the NC-BI database, and phylogenetic tree was constructed by MEGA 5.1 software through NJ method (Neigh-bor-Joining). The protein conserved domain, protein secondary and tertiary structure, molecular weight, molecular formula, isoelectric point, and subcellular localization of Binzi LOX2a protein were analyzed using online software. In addition, cis-acting elements in LOX2a promoter region were predicted with  PlantCARE software. Binzi LOX2a was cloned into the pCAMBIA-Super1300-GFP vector to construct the 35S::LOX2a-GFP recombinant vector. The recombinant vector and one control GFP vector were re-spectively transformed into Agrobacterium tumefaciens GV3101 competent cells, and then were trans-formed into tobacco (Nicotiana × tabacum) leaf cells. After 3 days, the subcellular localization of LOX2a was observed under a Confocal laser scanning microscope. The expression characteristics of LOX2a in different plant tissues (root, stem, leaf, flower and seed) and fruit at different (30, 60, 90, 120 and 150 d after flowering period) development stages were detected through the Real- Time fluores- cence quantitative PCR. Finally, the fruit at 90 d after flowering period was chosen for further hormone treatment research. Fruit discs (10 mm in diameter and 1 mm in thickness) were prepared with a cork borer. The discs were immediately immersed in equilibration buffer (200 mmol·L^-1 Mannitol, 50 mmol·L^-1 MES pH 5.5, 10 mmol · L^-1 MgCl2, 10 mmol · L^-1 EDTA, 5 mmol · L^-1 CaCl2 and 5 mmol · L^-1 Vc). The discs were respectively incubated in equilibration buffer with 100 mmol·L ^-1 ABA, 100 mmol·L^-1 MeJA,100 mmol·L^-1 SA and 200 mmol·L^-1 GA3. Meanwhile, the fruits were treated with 200 mmol·L^-1 ethyl-ene and 1 mL·L^-1 1-MCP. Then the expression levels of LOX2a gene after phytohormone treatment were determined by RT-qPCR.【Results】We screened out one LOX gene (named LOX2a) for further re-search based on the results of Binzi total volatiles and transcriptome. Binzi LOX2a contained 2721 bp of open reading frame and encoded 906 amino acids, which contained a conserved PLN02264 (Lipoxy-genase) domain. Binzi LOX2a sequence has been uploaded to the NCBI database, and its GenBank No.is ON952464. The physicochemical property results of Binzi LOX2a showed that its protein molecular formula was C4612H7222N1254O1341S27 and isoelectric point was 6.97. The protein secondary and tertiary structure prediction results showed that the LOX2a protein mainly contained a-helix, extended strand, and random coil with the proportion of 33.33%, 15.56% and 51.10%, respectively. The phylogenetic analysis showed that the Binzi LOX2a had the highest similarity with M. domestica LOX2a. The subcel-lular localization result showed that the green fluorescence in Nicotiana tabacum leaves transformed with 35S::LOX2a-GFP recombinant vector was only accumulated in the cytoplasm, which was consis-tent with the result from the Cell-PLoc 2.0 prediction. The results of RT-qPCR showed that LOX2a gene was expressed in roots, stems, leaves, flowers, fruits and seeds. And the expression of LOX2a was high-er in roots, followed by the stems, leaves, and the lowest in seeds. The above results indicated the LOX2a gene expression had tissue expression specificity. During Binzi fruit development period, the highest expression level of LOX2a was detected from the 30 d after flowering period, and then in- creased at 150 d, which accompanied with the Binzi fruit aroma accumulation. The cis-acting elements of the LOX2a promoter consisted of the photo responsive element, hormone response element and stress related component. And the hormone response element included ABA, MeJA, GA and SA respon-sive element. Combining with fruit disc tissue incubation and hormone treatment, we determined the ex-pression level of LOX2a using the RT-qPCR. It was found that the expression level of LOX2a was sig-nificantly higher after treatment with hormone than that in controls (p＜0.05), indicating that the LOX2a gene expression was induced by ABA, MeJA, GA, SA and ethylene.【Conclusion】LOX2a gene may be involved in aroma metabolism of Xiangbizi fruit through multiple hormone signaling pathways.