Screening And Functional Study Of Receptor-like Kinase Genes In Potatoes In Response To Stress Signals

Receptor like Kinase(RLK),a transmembrane protein,which is located on the cell membrane,and its molecular structure and function is similar to that of receptor protein kinases in animal.It plays an important regulatory role in most of life activities,such as growth and development,protein activity regulation and adversity adaptation.However,the identification and analysis of RLKs subfamily in potatoes(Solanum tuberosum),such as cysteinerich receptor-like kinases(CRK)and lectin receptor-like kinases(Lec RLKs),as well as the screening of resistances related RLKs in potatoes have not been reported.In this study,the potato Favorita,Longshu No.7 and Atlantic were used as experimental material.By using bioinformatics,transcriptome sequencing techniques(RNA-Seq),quantitative Real-time PCR(q RT-PCR)and agrobacterium infiltration techniques,the RLKs especially the resistance related RLKs were identified and verification when the leaves and tubers of potato inoculated with Phytophthora infestans(Pi)and Fusarium sulphureum(Fs),or illuminated with constant white fluorescent light.In the control group,the samples were treated with distilled water or in darkness.The main results are as follows:1.Based on the bioinformatics analysis,RLKs in potatoes were identified and analyzed by extracting the global genome of potato and the transcriptome data from the published articles.Results showed that 479 RLKs were identified in potato genome,which can be divided into 47 subfamilies.Among them,a large number of members of the subfamilies PERK-1,WAK LRK10L-1,Cr RLK1L-1,LRR-XI-1and LRR-XII-1 have partied in partial duplication,and the duplicated characteristics of the subfamilies Cr RLK1L-1,LRR-XI-1 and LRR-XII-1 are unique to potatoes.Members of LRR-XII-1,SD-2b and WAK LRK10L-1 in the RLK subfamilies have partied in tandem duplication,among which the duplicated characteristics of SD-2b and WAK LRK10L-1 may be unique to potatoes.Based on the transcriptome data in the published articles,we found that the number of genes in Clades L-LEC,LRK10L-2,SD-2b,WAK and WAK LRK10L-1 involved in biological stress were more and larger proportion than the other clades.When the potato inoculated with Candidatus Liberibacter solanacearum(Cls)and Phytophthora infestans,(Pi),the differential expression genes were found in some of the subfamilies,indicating that these genes may play an important regulatory role in the process of disease resistance and can be used as candidate genes for further research.2.CRK members were identified and the physical and chemical characteristics,evolutionary characteristics,subcellular and chromosome location,and the expression patterns were analyzed.The expression patterns of CRK were detected through the q RTPCR technology when responded to salicylic acid(SA),jasmonic acid(JA)and pathogen infection.Results showed that eight CRKs were identified,and there were many hormone regulation and stress response elements in the promoter region.Among the eight CRKs,the expression of CRK4(PGSC0003DMG400018101)and CRK8(PGSC0003DMG400015171)increased with the extension of inoculated time.Therefore,we concluded that these genes may respond to multiple fungal signals,and played an important role in potato's broad-spectrum resistance and can be used as the candidate genes for further disease resistance and functional analysis.3.113 Lec RLKs were identified in the potato genome,including 85 G-Lec RLKs,26 L-Lec RLKs and 2 C-Lec RLKs.The evolutionary tree of Lec RLKs were constructed by multiple sequence alignment,and the chromosome positions,gene duplication and expression patterns in response to pathogen infection were analyzed.The results showed that the potato Lec RLKs could be divided into 7 clades,which randomly distributed on 12 chromosomes,and 47 members participated in tandem duplication and 9 members participated in partial duplication,indicating that the amplification of Lec RLKs family was mainly caused by tandem duplication.There were many differentially expressed genes which were responded to plant pathogenic fungi and bacteria infection.Among them,PGSC0003DMG400031861 and PGSC0003DMG400044943 were strongly induced by Fs and Pi.Therefore,these genes can be used as candidate genes for functional analysis and resistance breeding in the further.4.The global gene expression profiles of potato variety Favorita was investigated when potato tubers were placed in light and darkness.Compared to the control group,1 288,1 592,1 737,and 1 870 differentially expressed genes were detected at different time point,and in the sample of the four time points the expression of 393 differentially expressed genes were up-regulated,and four steroidal glycoalkaloids(SGA)related genes(HMG1,PGSC0003DMG400013663;SGT1,PGSC0003DMG400011749;SGT2,PGSC0003DMG400017508;SGT3,PGSC0003DMG400011740),were included.In addition,Mapman showed the enriched Bins was related to PS light reaction,protein degradation,the biological stress and abiotic stress.Remarkably,disease-resistant genes,such as IMPA1(PGSC0003DMG400014989),SPK1B(PGSC0003DMG400006184)and WDR5B(PGSC0003DMG400019361)were coexpressed with multiple SGA biosynthetic genes(HMG1,SGT1,SGT2 and SGT3).Therefore,we speculated that SGA biosynthetic genes had a regulatory role in the process of plant disease resistance.Moreover,28 RLKs were differentially expressed during the process of light induction,which demonstrated the regulatory role of RLKs in environmental adaptation.5.SD-2b-1(PGSC0003DMG400031861),a member of SD-2b subfamily was cloned and the overexpression vector was successfully constructed.Functional identification was performed by agrobacterium infiltration method.It was found that when potato leaves infiltrated with the recombinant plasmid SD-2b-1-p FGC5941,the spot diameter was significantly smaller than that of the control group when infected with Pi,indicating this gene had the function of resisting Pi infection.Moreover,the recombinant plasmids of SD-2b-1 and YL322-d1-GFP were constructed and the expression pattern in the protoplast of Arabidopsis thaliana were detected.Results showed that SD-2b-1 was located on cytomembrane,which was consistent with the predicted results of bioinformatics.