Cloning, expression and functional analysis of longan DlSWEET1 gene

Abstract: 【Objective】Longan (Dimocarpus longan L.) is one of the important economic fruit crops in southern China. Longan has the function of nourishing the heart, spleen and blood, and calming the mind of peaple. It has been regarded as a precious supplement since ancient times. The sugar content in fruits is a key factor affecting fruit quality, and improvement of fruit sugar content is of great significance for promoting the high- quality and efficient development of China’s longan industry. The SWEET sugar transporters protein (SWEETs) not only plays an important role in plant stress and hormone response, but also plays a crucial role in the normal growth and development of plants, especially in promoting sugar accumulation. However, there has been limited research on the DlSWEETs in longan, especially on sugar accumulation. The purpose of this study is to screen and validate the functions of the candidate DlSWEETs that may be involved in sugar accumulation processes.【Methods】The CDS sequence of the DlSWEET1 was cloned using the cDNA of the preserved Songfengben fruit in the laboratory as a template. The DNAMAN software was used to translate the correctly sequenced DlSWEET1 gene nucleotide sequence into amino acid sequence, and its conserved domain was predicted by NCBI. The protein transmembrane domains were analyzed using the TMHMM2.0. We extracted the total RNA from different tissues (roots, stems, leaves, and fruits) of longan and leaf samples after different treatments, reversed the transcribe to obtain cDNA, and then used real-time fluorescence quantita-tive PCR (qRT-PCR) to detect the expression level of DlSWEET1 in different tissues and organs of longan, as well as its expression level under hormone, cold, heat, and drought stress. All the experiments were repeated three times in terms of biology and technology, and the relative expression levels of the genes were calculated using the 2^–∆∆CT method, and then statistically analyzed by t test. p＜0.05 indicated significant difference, and the error line represented the standard deviation of three biological repeats. the CDS sequence of the DlSWEET1 was cloned and connected to the pMD18-T vector. Using this plasmid as a template, PCR amplification of the DlSWEET1 was performed using primers. The pH7LIC5.0-ccdBrc-N-eGFP vector enzyme was cleaved using Stu Ⅰ enzyme cleavage, the pSAK277 vector enzyme was cleaved +using EcoR Ⅰ and Hind Ⅲ enzyme cleavage, and the pDR196 vector enzyme was cleaved using Pst Ⅰ and Spe Ⅰ enzyme cleavage. The amplification product was inserted into the multi clone sites of each vector. The first two recombinant vectors were transformed into GV3101 strain, and the last recombinant vector was transformed into EBYVW4000 yeast strain. The lower epidermis of tobacco leaves was injected with Agrobacterium and cultured in the dark room (25 ℃) for 2 days. The distribution of green fluorescence was observed by confocal laser microscope. The strawberries injected with Agrobacterium were cultured under 16 h light/8 h darkness in a greenhouse at 25 ℃ for 9 days. The relative expression of the DlSWEET1 in strawberries and the determination of soluble sugar content were analyzed by qRT- PCR. The bacterial solutions with OD600 of 0.1, 0.01 and 0.001 were taken 5 µL into various sugar substrate media, and the media were cultured in a constant temperature incubator at 28 ℃ for 2-3 days, and the growth was observed and recorded. The function of the DlSWEET1 gene was investigated by subcellular localization, sugar transport activity analysis and transient transformation of strawberry.【Results】The DlSWEET1 contained 750 bp of ORF (open reading frame) and encoded 249 amino acids, which contained a PQ-loop conserved domain and a protein typical conserved domain MtN3_slv. Further analysis indicated that DlSWEET1 protein contained seven transmembrane domains. The qRT-PCR analysis results showed that the DlSWEET1 was expressed in different tissues such as the roots, stems, leaves, and pulp, with higher expression levels in the leaves, followed by in the pulp, and lower expression levels in the stems and roots. After treating the leaves with different concentrations of sucrose, glucose, and fructose, the expression level of the DlSWEET1 showed varying degrees of increase. The expression level of the DlSWEET1 in the leaves treated with sucrose was significantly higher than that in the control group, but there was no significant difference among different concentrations. The expression of glucose increased significantly with the increase of glucose concentration. In the treatment of fructose, lower concentration (0.5 g ·L- 1 ) and higher concentration (5 g · L- 1 ) could significantly increase the expression of the DlSWEET1. The expression of the DlSWEET1 was significantly increased under low temperature, drought and MeJA treatments, and significantly decreased under ABA treatment. However, there was no significant change in its expression after high temperature, 6-BA and GA3 treatments. The fluorescence signals of GFP were mainly concentrated and overlapped in the cell membrane and nucleus. The sugar transport activity analysis showed that the DlSWEET1 protein could transport glucose, fructose, sucrose and mannose. After transient transformation, the contents of sucrose, glucose and fructose in the strawberry were significantly higher than those in the control treatment, and the expression level of the DlSWEET1 was significantly increased.【Conclusion】The DlSWEET1 gene was cloned from longan fruit, and its expression level could be induced by different sugar components (sucrose, glucose, and fructose), stress (low temperature and drought), and hormones (MeJA and ABA). The subcellular localization revealed that the gene is localized on the cell membrane and nucleus. The analysis of sugar transport activity showed that it could transport various sugar components, such as sucrose, glucose, fructose and mannose, but could not transport the toxic substrate deoxyglucose. The transient overexpression of the DlSWEET1 resulted in increased soluble sugar content in the transgenic strawberry fruits. The transient conversion of this gene in the strawberry significantly increased the relative expression of the DlSWEET1. These results indicated that the DlSWEET1 has the function of promoting sugar accumulation in fruits of longan. The article would provide a theoretical reference for improving fruit quality of longan.