Cloning And Functional Validation Of The SWEET Gene In Sorghum Dochna

Sugars are involved in various growth and development processes of plants as a source of carbon skeleton,substrate of respiratory reactions,intermediate metabolites in biochemical reactions,nutrients,permeate and signals in biotic and abiotic stresses.Carbohydrate compounds cannot be transported independently across membranes,and corresponding sugar transporters are required to complete the transport and biological metabolism of sugars in plants.SWEETs(sugars will eventually be exported transporters)proteins participate in the growth and development of plants,and also play an important role in plant response to abiotic stress and biological stress.Known for its high sugar,short growth time,high environmental adaptability and high biomass,sweet sorghum(Sorghum dochna)is considered one of the most promising yield crops.In this study,the sequence information,gene structure and kinship of 18 SdSWEETs were analyzed through bioinformatics.Its functions were preliminarily explored by specific expression,subcellular localization,yeast functional complementarity,and Arabidopsis heterologous expression verification.The main findings are as follows:1.Cloning and identification of 18 SdSWEETs genes from sweet sorghum,analysis found that the amino acid numbers encoded by 18 SdSWEETs genes were all in the range of 231-336,the relative molecular mass was between 25.15-35.69 KDa,and the isoelectric point was between 6.41-9.69,all of which had the typical Mt N3 domain of the SWEET family and seven transmembrane helixes.By constructing an evolutionary tree with Arabidopsis thaliana and rice SWEET proteins,18 SdSWEETs proteins can be divided into 4 subfamilies.2.The expression level of SdSWEET gene in different tissues of sweet sorghum growth cycle was different by RT-PCR.SdSWEET03,04,05,06,09,11,14,15 and 17 are more pronounced in stems and leaves,and lower in roots.SdSWEET01 and Sd WEET07 have high levels of expression in roots and are not significantly expressed in stems and leaves.The expression of SdSWEET1-18 gene under abiotic stress was analyzed by q RT-PCR,and it was found that the expression levels of SdSWEET03 and SdSWEET07 in the 3 h root treated at low temperature were significantly upregulated,and the expression levels of SdSWEET2,8,11,15,17 in the stem and SdSWEET04 in the leaf were induced by low temperature and upregulated at 6 h.SdSWEET01 and 05 in roots and stems at 3 h,6 h in stems,SdSWEET06,09,13,14,16,17 and 18 in stems,and SdSWEET03 and SdSWEET15 in leaves are all induced by high temperatures and the expression levels are upregulated.Under the treatment of 20%PEG6000,the expression levels of SdSWEET06 in 3 h roots and 12 h stems and leaves were significantly upregulated,and the expression levels of SdSWEET13 and 16 in stems increased significantly at 3 h and 12 h,respectively.Under the treatment of 200 mmol Na Cl,the expression levels of SdSWEET02,07,09,10,12,13 and SdSWEET01,02,04,09,11,12,14,16 and 18 in the 6 h and 12 h leaves were significantly regulated.Therefore,it can be speculated that the SWEET gene responds to abiotic stress.3.Yeast functional complementarity experiments showed that SdSWEET01 localized on the plasma membrane had the ability to transport galactose and glucose,SdSWEET03 on the vacuole membrane could restore the growth of defective yeast in a medium with fructose as a carbon source,and the transporters of SdSWEET06 and SdSWEET09 localized on the plasma membrane had glucose and fructose transport capabilities.4.Transgenic Arabidopsis thaliana showed that the SdSWEET01-OE,SdSWEET06-OE and SdSWEET09-OE strains had yellowed leaves and higher sugar content than WT plants at 1.5% sugar content.It grows better than the wild type at0.15% sugar.In addition,the rosette leaves of the SdSWEET03-OE and SdSWEET06-OE strains under normal soil cultivation are larger than those of WT plants.It is speculated that SdSWEET01,SdSWEET03,SdSWEET06 and SdSWEET09 are involved in the growth of Arabidopsis.Under salt treatment,the transgenic strains of SdSWEET01,SdSWEET06 and SdSWEET09 grew better than WT strains,and the rare importance was higher than that of the wild type.The growth of SdSWEET01,SdSWEET03,SdSWEET06 and SdSWEET09 transgenic lines was better than that of wild type under drought treatment,and less important than that of WT.This suggests that SdSWEET01,SdSWEET03,SdSWEET06 and SdSWEET09 play a role in responding to drought and salinity stress.According to the research progress of plant SWEET,the SWEET gene of sweet sorghum was studied and analyzed from the aspects of evolutionary tree,protein structure,function and function analysis,which laid a theoretical foundation for sugar utilization and coping with abiotic stress in sweet sorghum.