Identification Of StLOR Gene Family And Study On Function Of StLOR7 In Potato(Solanum Tuberosum L.)

LURP1?Late up-regulated in response to Hyaloperonospora parasitica?belongs to the LOR gene family,playing a key role in resistance to Oomycetes pathogens in plants.To date,studies on the function of the LURP1 gene have not been completed and are mainly limited to Arabidopsis thaliana.Its research in potato?Solanum tuberosum L.?has not been reported yet.Furthermore,no systematic identification of the LOR gene family has been reported in any species.A new gene with unknown function but can interact with dehydration responsive element?DRE?was found in our previous study.Aligned by the Basic local alignment search tool?BLAST?,this gene was identified as StLURP1.In this study,the potato StLOR gene family was identified at the genomic level by bioinformatics.The research was mainly focused on the gene StLOR7 interacting with DRE.In order to elucidate the biological function of the potato StLOR7 gene,the overexpression vector pBI121-StLOR7 was constructed and transformed into the potato tetraploid cultivar"Atlantic".To clarify the role of StLOR7 in the protein interaction network,the yeast?Saccharomyces cerevisiae?two-hybrid system bait vector pGBKT7-StLOR7was constructed by directional cloning of homologous recombination,and the potato cDNA library was screened.The main results are as follows:1.Sixteen members of the potato StLOR gene family containing the complete open reading frame and LOR domain were identified and renamed as StLOR1-16 based on their chromosomal distribution.The physical and chemical properties,gene structure,protein motif of StLORs and the intra/inter-species phylogenetics were clarified.A variety of stress-and plant hormone-related cis-acting regulatory elements were found in the upstream non-coding region of StLORs,suggesting that the family can respond to multiple stresses and phytohormone treatments.Based on public transcriptome data,the distribution of the StLORs in different tissues of potato and the expression pattern under different stresses and hormone treatments were visualized.It revealed that the distribution of the StLORs was tissue specific.Besides,two StLOR members were able to respond to heat stress?3,9?while three members could respond to GA₃treatment?1,2,12?;StLOR11,12 And StLOR1,2 were found to respond to BAP and ABA treatment,respectively;the expression of StLOR14,15 increased significantly under BABA treatment while StLOR4,8,16 went down;StLOR5 could respond to mechanical damage specifically.However,the expression of StLORs does not appear to be induced by salt and osmotic stress.2.The expression patterns of 16 StLOR members under 4 stresses?osmosis,drought,cold and heat stress?and 4 plant hormones?GA₃,ABA,SA and MeJA?were examined by quantitative real-time polymerase chain reaction?qRT-PCR?.All family members except StLOR13 and 14were able to respond to salt stress;StLOR2 and StLOR13 were specifically induced by salt stress and cold stress,respectively;StLOR4,6 and 7 could respond to abscisic acid;StLOR1,3,5,15,16 could respond to a variety of different hormone treatments;StLOR8 could be induced by heat stress.This study also examined the relative expression of StLOR7 over time under the above eight treatments.It was found that StLOR7 can only respond weakly to heat stress,drought stress and SA treatment.Yet the gene was strongly induced by GA₃,MeJA and salt stress—its expression level was extremely up-regulated at 24 h,reaching a maximum at 48 h.3.The overexpression vector pBI121-StLOR7 driven by the CaMV 35S promoter was constructed and transformed into the tetraploid cultivar"Atlantic"by potato chip transformation method.After PCR detection of the NPT II?neomycin phosphotransferase gene?and qRT-PCR test for the expression of StLOR7,the gene has been successfully inserted into the genome of transgenic plants.4.In this study,the CDS region of StLOR7 gene was cloned,and the yeast two-hybrid bait vector pGBKT7-StLOR7 was constructed by homologous recombination.Using the zygosity of yeast,the bait protein hybridized with the cDNA library and plated on high stringency screening medium.126 candidate positive clones were screened,and 88 of them could hydrolyze the chromogenic substrate X-?-Gal in the staining screen.The positive rate is close to 70%.Twelve kinds of StLOR7-interacting proteins with correct ORF were obtained,including DnaJ family protein,glycerophosphodiesterase,polyphenol oxidase,proline-rich protein,actin depolymerization factor,heavy metal transporter.Annotation analysis of Gene Ontology?GO?of interactors suggests that StLOR7 may be involved in important biological processes such as plant pathogen defense,stress response,heavy metal detoxification,microfilament movement,lipid metabolism,protein folding and phosphorylation.