Identification And Functional Verification Of LEA3 Protein From O.ochrocephala

The rapid spread of Oxytropis ochrocephala Bunge maximus seriously destroys the ecological diversity of the grasslands and further affects the ecological balance of the grassland.Therefore,it is particularly urgent to explore its spreading mechanism.The rapid spread of O.ochrocephala sinensis is closely related to its strong resistance.However,the current research mainly focuses on distribution and disaster investigation,allelopathy,poisoning mechanism,and medicinal value research.There is no research on the mechanism of stress resistance.Late embryogenesis abundant protein(LEA)is an important protein that plants respond to stress,and has the function of protecting plant tissues from water stress injury.Our previous study also showed that the LEA gene was significantly up-regulated under stress treatment.Therefore,this study intends to use LEA3 gene of O.ochrocephala as the research target,and conduct research on gene cloning,bioinformatics analysis,transcription level expression analysis,and functional analysis to clarify the Oo LEA3 gene’s response to adversity stress of O.ochrocephala intermedia.The mechanism of action provides a basis for the analysis of the intrinsic mechanism of O.ochrocephala spread,and also provides new ideas and methods for the study of plant drought resistance breeding.The main research contents and results are as follows:1.Obtain related sequences from the existing transcriptome data of this research group and use Primer software to design specific primers to successfully clone the gene and named Oo LEA3.The total length of Oo LEA3 gene is 570 bp,with an open reading frame of 522 bp and encoding 173 amino acids.Bioinformatics analysis showed that Oo LEA3 protein is a hydrophilic protein with no signal peptide structure and splice sites,no transmembrane domain,multiple phosphorylation and glycosylation sites.Compared with known LEA3 family proteins,Oo LEA3 protein contains four highly conserved repeat motifs(TAQAAKDKTQQ),which is a typical LEA3 family protein.Trifolium subterraneum analysis showed that the genetic relationship of the Clover of Oo LEA3 protein was closest,followed by the kinship of Cicer arietinum and Medicago truncatula.2.The q RT-PCR method was used to analyze the expression characteristics of Oo LEA3 under drought stress,high salinity and low temperature stress,and under the treatment of abscisic acid,ethylene and gibberellin.The results showed that: drought,high salt Oo LEA3 gene expression was significantly induced by low-temperature abiotic stress.Under hormone treatment,Oo LEA3 gene was highly induced by ABA,whereas ethylene and gibberellin treatment had no significant effect on the expression of Oo LEA3 gene.This result indicates that Oo LEA3 may be involved in the ABA-dependent signaling pathway and respond to the abiotic stress-resistance response of O.ochrocephala through this pathway.3.The prokaryotic expression recombinant vector p ET32a-Oo LEA3 of Oo LEA3 gene was constructed and transformed into E.coli BL21 star(DE3),which was induced by IPTG.The sorbitol osmotic stress treatment was performed on Escherichia coli containing the p ET32a-Oo LEA3 recombinant plasmid and the control bacteria(containing p ET32 a empty vector)to observe the plaque morphology and growth curve of E.coli under stress conditions.The results showed that the recombinant strains expressing Oo LEA3 protein had significantly larger plaques than the control strains under stress conditions and the growth status was better than that of the control strains,indicating that the LEA3 protein of O.ochrocephala was involved in the anti-permeability reaction of E.coli.4.The effects of Oo LEA3 protein on the activity and aggregation rate of lactate dehydrogenase(LDH)under repeated freezing and thawing and dehydration stress were studied in vitro.The results showed that the activity of LDH enzyme added with Oo LEA3 protein was significantly higher than that of the control.When the ratio of the protein to LDH to be measured was 20:1,the activity was retained to the maximum and the retention rate was over 80%.The LDH aggregation rate was significantly decreased in the presence of Oo LEA3 protein.It showed that Oo LEA3 protein could reduce the aggregation rate of LDH and protect LDH enzyme activity under freeze-thaw and dehydration stress.5.The eukaryotic expression recombinant vector p REP3X-Oo LEA3 of Oo LEA3 gene was constructed and transferred into yeast Schizosaccharomyces pombe(S.pombe).The recombinant S.pombe(p REP3X-Oo LEA3)and the control S.pombe(p REP3X)were subjected to sorbitol osmotic stress treatment to observe the changes in plaque morphology and the growth curve of S.pombe.The results showed that when S.pombe(p REP3X-Oo LEA3)was subjected to osmotic stress,its plaque was significantly larger than that of the control S.pombe(p REP3X),and its growth tendency was also better than that of the control strain.The results showed that Oo LEA3 protein could increase the penetration resistance of S.pombe.6.The Arabidopsis thaliana(Col-0)was transformed by Agrobacterium tumefaciens-mediated floriculture.The transgenic plants were screened by 1/2 MS medium containing hygromycin and identified by DNA level,transcription level and protein level to obtain the T3 generation.Positive transgenic plants,and determine their resistance indicators.