Cloning And Functional Analysis Of GhBASS5 Gene In Gossypium Hirsutum

High concentrations of Na⁺in saline soils inhibit cotton growth and reduce agricultural productivity.Three mechanisms can be used to prevent excess Na⁺accumulation in the plant,Na⁺entry into plant cells may be reduced once Na⁺influx transporter genes are identified in plants,Na⁺that enters the cells can be transported and stored in the vacuoles,Na⁺in the cytoplasm can be exported back to external medium or the apoplast via plasma membrane Na⁺/H⁺antiporters.In this study,a pilot experiment by comparing root transcriptome of two upland cotton genotypes with contrasted salinity tolerance indicated a negative relationship between salt tolerance and expression level of a sodium bile acid symporter GhBASS5?bile acid:Na⁺cotransporter,BASS is a type of membrane channel dependent on Na⁺gradient co-transporting small molecule acid Protein?.BASS5 is a sodium-dependent transporter,but its role in salt stress has not been reported yet.In our present work,a GFP fluorescence localization vector was constructed and the synthetic GhBASS5 antibody was immunofluorescently organized.The method was used to determine the subcellular and subcellular localization.The virus-induced gene silencing?VIGS?was used to investigate the biological function of GhBASS5 in Gossypium hirsutum under salt stress and the use of NMT to measure sodium ion flux in the roots.The result is as follows:1.A homolog of BASS in crops,designated GhBASS5 was identified and cloned.The opening reading frame?ORF?of GhBASS5 is 1233 bp,which encodes for a protein containing 413-amino acid.The amino acid sequence of GhBASS5 was predicted to contain conserved domain YfeH,Transducer domains and shared high similarity with T.cacao and rice,which indicates that they might have originated from the same common ancestor.And have two Na⁺binding sites conserved by BASS family.2.Real-time fluorescence quantitative PCR analysis showed that GhBASS5 gene was expressed in all tissues,and the highest expression in plant roots,followed by stems,and leaves at least belong to constitutively expressed genes.While the GhBASS5-GFP fusion protein was localized to the envelope of discreted spherical chloroplasts in cotton mesophyll protoplasts,it was restricted to the envelope of“seemed connected”lamellae leucoplastids in root protoplasts.And the immuno-staining results indicated that GhBASS5 was expressed in the xylem and stele cells.3.The plants treated with GhBASS5 gene silencing and VIGS-GFP control plants were treated with salt stress.Under the salt treatment,the physiological indexes of cotton plants were measured and it was found that the cotton growth potential of the experimental group was better than that of the control group.Under salt stress,VIGS silencing of GhBASS5 significantly reduced the Na⁺level in cotton roots,stems and leaves,and enhanced the salt tolerance of cotton.GhBASS5 was over-expressed in protoplasts of cotton roots,and sodium ion content in cells was determined by SBFI.The results showed that over-expression of GhBASS5 resulted in increased intracellular sodium ions.The NMT technique was used to measure the sodium currents in the roots of the silenced and normal plants.The results showed that silencing of GhBASS5enhanced the sodium efflux ability of cotton roots.Silencing GhBASS5 on the one hand may induce oxidative stress through plastid function,promote cotton under normal growth conditions into stress response,accumulate stress protective agent for stress,and on the other hand,reduce Na⁺in the absorption and transport of Na under salt stress.Accumulation on the ground increases the salt tolerance of cotton.Fluorescent quantitative PCR detected BASS1,BASS2,BASS4,NHX-2,HBI,RD22 and HKT-1upregulation,while BASS3,NHX-6,H⁺/Ca²⁺,SOS3,ABF-1,RD29A,DREB1A,P5CS,SOS1,HKT-2 decreased after BASS5 silencing and was up-regulated after salt stress.These results indicate that plants silenced by the GhBASS5 gene show more salt tolerance.In this study,we obtained the GhBASS5 gene from upland cotton,analyzed the structure,expression and characteristics of the gene and its role in salt stress.Furthermore,silencing GhBASS5 increased the content of soluble sugar and H₂O₂ and caused a significant regulation of several sodium transporter genes under normal growth condition.Taken together,our results suggested that GhBASS5 might serve as a xylem Na⁺entry to control Na⁺loading directly,and/or influence the plastid function to retrograde other sodium transporters,thereby restricting Na accumulation to the shoots.It provides theoretical guidance for the further study of GhBASS5 gene,provides an effective genetic method for improving the tolerance of cotton to salt stress,and provides ideas for the salt tolerance mechanism of plants.