Cloning, Expression And Function Of Soybean Sucrose Transporter Gene GmSUT4

Soybean(Glycine max)is an important economic crop with rich nutritional value.Soybean is sensitive to salt stress.Under high salt stress,the growth of soybean is inhitited,the quality is reduced and the yield is decreased.Sucrase transporter(SUT)is responsible for the transmembrane transport of sugars in plants and plays an important role in response to adversity.Till now,several soybean SUT genes have been cloned and identified,but mainly works were focused on the bioinformation analysis,and their role in response to adversity is rarely reported.Therefore,further study of gene function soybean SUTs has important implications for understanding roles in soybean growth and development,as well as in response to adversity.In this study,the soybean GmSUT4 gene was cloned and identified.The bioinformatics,expression frofile,subcellular localization,and response to under salt stress were analyzed.The VIGS technology was used to initially verify the gene function and construct it for genetics.and the pBI121 overexpression vector and CRISPR/cas9 knockout vector were constructed for genetic transformation,which laid a foundation for the comprehensive study of the function of the gene in the future.the main results are as follows:1.The GmSUT4 gene was cloned by homologous methold.The length of CDS region is 1623 bp,encoding 537 amino acids,with a molecular mass of 57869.35 Da,a theoretical isoelectric point of 9.43.The amino acid sequences contains several conserved GPH and MFS functional domains.GmSUT4 has a low similarity with other soybean SUT genes,belonging to SUT4 subfamily,and is closest to SUT4 protein of lotus and peanut.The results of q RT-PCR showed that GmSUT4 was expressed in roots,stems and leaves,with the highest expression in roots and the least expression in stems.Transient expression in Tobacco showed that GmSUT4 was localized in the plasma membrane.2.Treated with NaCl,the physiological indexes of soybean seedlings and the expression level of GmSUT4 gene were analyzed.The plant height,stem diameter,and fresh weight of soybean seedlings decreased significantly with increasing salt concentration.SOD,POD activity and MDA content in roots and leaves increased significantly,while CAT activity decreased first and then increased with the increase of salt concentration,with the highest activity under 150 mM NaCl stress;GmSUT4 gene expression in roots and leaves increased with the increase of salt concentration.The correlation analysis showed that the expression of GmSUT4 gene was related to physiological indexes,which suggested that GmSUT4 gene might be involved in the response of soybean to salt stress.3.The VIGS vector of GmSUT4 gene was constructed,and the true leaves of soybean were injected by injection.The q RT-PCR results showed that the expression level of GmSUT4 in the silent plants was significantly reduced,indicating that GmSUT4 has been successfully injected into soybean.4.After treatment with 150 mM NaCl for 3 days,the leaves of GmSUT4 silenced plants were significantly wilted compared with the control plants,in which CAT,SOD,GR activity and proline content were significantly reduced,MDA content was increased and POD activity was not significantly changed;The contents were reduced by 16.52 %,9.75 % and 36.81 % respectively.5.Silencing of GmSUT4 caused the expression of sugar synthesis related genes Gm SPS and Gm CInv1 to be significantly down-regulated,but Gm SPP2 was not significantly down-regulated.It is suggested that GmSUT4 may be involved in the regulation of sugar synthesis related gene expression in response to salt stress in soybean.6.The pBI121-GmSUT4 overexpression vector and Cas9-GmSUT4 vector were constructed and successfully transformed into Agrobacterium,which will lay the foundation for future genetic transformation and comprehensive study of the gene function.