Cloning And Function Analysis Of The Promoter Of Sulfate Transporter Gene GmSULTR1;2b And The Preliminary Screening Of Yeast One-hybrid

Following the nitrogen,phosphoras and potassium,sulfur is the fourth nutrients necessary for the plants.The sulfate(SO42-)in the external environment enters the plants to proceed with assimilatory sulfate reduction by plant roots,and generates a series of important sulfur-containing compounds.These sulfur-containing organic compounds involved in many important physiological and biochemical reactions in plants.They play an important role to withstand environmental stress and in growth and development.The absorption and reload of sulfate is not only the first step in the pathway of assimilatory sulfate reduction,but also is one of the main regulated step in the sulfur metabolism.The sulfate transporters(SULTR)are necessary for absorption and reload of sulfate.Soybean(Glycine Max(L.)Merr.)is one of important economical crops,which is the main source of vegetable protein.Soybean protein amino acid composition is complete,but the low contents of sulfur amino acids(Cys and Met)limit the nutritional value of soybean.Studying Gmultr1;2b gene functions showed GmSultr1;2b had sulfur transfer activity,specifically expressed in the root,and was induced by low concentration sulphate.Here,we study the functions of GmSultr1;2b promoter to know about the regulatory mechanism of GmSULTR1;2b.We carry out yeast one-hybrid to find out proteins interacted with GmSultrl;2b promoter,which contributes to discovering sulfur metabolism and the molecular mechanisms.By knowing that,we hope to provide theoretical basis for improving the content of sulfur-containing amino acids in soybean by genetic engineering methods and regulating the distribution of sulphate(SO42-)flow in sulfur metabolic pathways.Using GmSultr1;2b sequence information in the databases of NCBI and Phytozome,we predict 2259bp upstream as the candidate promoter.Through online prediction analysis of regulatory elements,we could know the promoter contained not only TATA-box,which was the necessary component of eukaryote,but also hormone response element ERE(ethylene response element),ABRE(abscisic acid response element),stress response element TC-rich repeats(diseases and insect pests stress and drought stress),the AT-rich element(the DNA of AT-rich and protein binding sites)and MYB,etc.The promoter was cloned by PCR,and a series of expression vectors were constructed.To certificate the activity of the promoter,transient expression of soybean was conducted.The X-gluc dyeing conducted on the transient expression of soybean showed blue where the soybean was infected by recombinant vectors.It indicated the GmSULTR12b promoter could drive GUS expression downstream.Through deleting 5'and 3'of the promoter,we demonstrated the TSS of the predicted promoter was correct.The recombinant vectors p2179+69::GUS and 35S::GUS were transformed into soybean,after staining the transformed hairy roots,the transverse section of positive hairy roots was analyzed under the stereoscope.The GUS was mainly found in the root hair,root epidermis and the stele,which manifested the promoter mainly expressed in the root hair,root epidermis and the stele.The transformed hairy roots were conducted with inductive experiment of sulfate,and the consequences displayed the deeper stain of GUS in lower concentration of sulfate.To detect the activity of the promoter quantificationally,the GUS activity test of the transformed hairy roots and the inductive hairy roots were carried out.According to the results analysis,the GmSULTR12b promoter was attested weaker activity than the promoter of CaMV35S.The inductive experiment demonstrated the higher activity of the GUS in lower concentration of sulfate than higher concentration of sulfate,which illustrated the promoter could response abduction of lower concentration of sulfate,and it was consistent with the expression of gene GmSULTR12b.It was suggesting that the promoter belonged to inducible promoter.We discovered several transcription factors interacted with GmSULTR1;2b promoter by yeast one-hybrid,which could pointing out directions of knowing about the regulation of sulfur,and providing an reliable theory basis for the further study at the same time.