Functional Analysis Of AhLecRK9 And AhLEAs In Peanut Under Aluminum Stress

Peanuts grown in the acidic soils of southern China are affected by aluminum toxicity,which inhibits root growth and makes peanut yields in this region lower than the national average.Aluminum-induced programmed cell death(PCD)is an important mechanism of aluminum toxicity,but its associated molecular mechanisms are far from being clarified.In this study,we focus on the upstream signaling components and downstream functional components of the peanut aluminum stress response process.Studies have revealed that lectin-like receptor protein kinases(Lec RKs),components of the cellular adversity signaling response,are involved in the regulation of PCD in plants,and late embryonic development proteins(LEA)are important stress-associated proteins with enhanced stress tolerance in plant stress response.Here,we analyzed the function of Ah Lec RK9 and LEAs under aluminum stress to investigate the roles of these components in aluminum stress response in peanut,the result is as follows:1.A lectin-like receptor protein kinase gene,Ah Lec RK9,was obtained by homologous cloning in cultivar peanut.Bioinformatic analysis revealed that the gene belongs to L-type Lec RKs with a molecular weight of 81.75 k D,a theoretical isoelectric point of 6.28 and an instability index(II)of 39.15,which is a stable hydrophilic protein.The gene contains two transmembrane structural domains and no signal peptide.The phylogenetic tree shows that Ah Lec RK9 has the highest homology with Arabidopsis Lec RK-IX.1/2 and pear Pb LRK138.2.The q RT-PCR results revealed that there was no significant difference in the expression of Ah Lec RK9 under aluminum stress in ‘ZH2’(Al-sensitive variety),and the expression of Ah Lec RK9 increased significantly under aluminum stress for 4h and 8h in ‘99-1507’(Al-tolerant variety).Tissue specificity showed that Ah Lec RK9 gene expression was highest in flowers,followed by roots,stems and leaves in both ‘ZH2’ and ‘99-1507’ varieties.Subcellular localization studies showed that the Ah Lec RK9 gene is localized to the plasma membrane.3.Successful acquisition of Ah Lec RK9 overexpression transgenic Arabidopsis thaliana,Arabidopsis homozygous mutants and mutant restorer lines.The results showed that under normal conditions,Ah Lec RK9 transgenic Arabidopsis exhibited large rosette leaves,early shoots,high plants,large seeds and high 100-seed weight,whereas the mutant phenotype was the opposite.Under aluminum stress,the relative root elongation of the overexpression Ah Lec RK9 was longer and the mutant was shorter compared to wild type(WT),whereas the relative root elongation of the restorer lines was similar to WT.Under drought stress,the relative root elongation of Ah Lec RK9 transgenic Arabidopsis was longer than that of WT,and aboveground growth was also significantly inhibited.Rosette leaves are similar in size to WT.Under salt stress,germination of seeds from transgenic Ah Lec RK9 and restorer lines was severely inhibited,while mutant seeds were able to germinate normally.Compared with WT,the relative root elongation of seedlings of overexpressed Ah Lec RK9 and restorer lines was shorter and the aboveground parts were also more severely inhibited,while the relative root elongation of the mutant was similar to that of WT.4.Using the peanut genome database,126 Ah LEAs genes were identified in peanut and the 126 genes were classified into eight subfamilies,including LEA1,LEA2,LEA3,LEA4,LEA5,Pv LEA18,Dehydrin and SMP.Sequence analysis showed that most Ah LEAs(85.70%)had no or only one intron.LEA genes were randomly distributed on 20 chromosomes.Compared with tandem duplication,segmental duplication played a more critical role in Ah LEAs amplication.Synteny analysis showed that some Ah LEAs genes come from a common ancestor,and genome rearrangement and translocation occurred among these genomes.Peanut LEA is a close genetic relationship to soybean LEA than Arabidopsis.Almost all promoters of LEAs contain ABRE,MYB recognition sites,MYC recognition sites,and ERE cis-acting elements.5.Analysis of transcriptome data under different embryonic stages,tissue-specific,hormonal and abiotic stresses.The results showed that most of the LEAs were expressed in the late stages of peanut embryonic development.LEA3(AH16G06810.1,AH06G03960.1),and Dehydrin(AH07G18700.1,AH17G19710.1)were highly expressed in roots,stems,leaves and flowers.Moreover,100 Ah LEAs were involved in response to drought,low-temperature,or Al stresses.Some LEAs that were regulated by different abiotic stresses were also regulated by hormones including ABA,brassinolide,ethylene and salicylic acid,Ah LEAs that were up-regulated by ethylene and salicylic acid showed obvious subfamily preferences.6.Based on the analysis of expression patterns,the functions of genes responding to Al stress,drought and low temperature stress were verified in prokaryotic and eukaryotic systems.Overexpressing the Ah LEA1 gene enhanced cold and drought tolerance in bacteria but reduced the resistance of bacteria to high temperatures and salt.Overexpression of Ah LEA1 and Ah LEA3-3 enhanced cold and Al tolerance in yeast,and Ah LEA3-1 enhanced the drought tolerance in yeast.In addition,Ah LEA1,Ah LEA3-1 and Ah LEA3-3 are more sensitive to high temperature and salt.In this study,we conducted a preliminary investigation of the function of the peanut Ah Lec RK9 gene,as well as a systematic analysis of the peanut Ah LEAs gene family,and verified the function of genes related to aluminum stress,which is important for screening aluminum tolerance genes in peanut and exploring the mechanism of action of peanut in response to aluminum stress,and for improving peanut yield and screening excellent varieties.