Cloning And Functional Characterization Of Ndw Gene Regulating Necrosis And Dwarf In Tomato Under Low Temperature

Tomato(Solanum lycopersicum)is one of the extensively grown vegetable crops around the world.However,tomato is subject to various biotic and abiotic stresses,thereby limiting its productivity and quality.As a thermophilic plant,cultivated tomato suffers damage resulting in slow growth,leaf wilting,even flowers and fruits falling,and fruit deformity,when exposed to temperatures below 13°C.Thus,low temperature is one of the main limiting abiotic factors affecting tomato distribution,growth,development,and production.In addition,tomato production is strongly challenged by biotic factors such as tomato grey mold(Botrytis cinerea).Therefore,exploration of the key genes responding to cold tolerance,and Botrytis cinerea disease resistance,analyzing the mechanism of gene function have important value in tomato.In this study,we used S.pennellii introgression line IL6-2 as material,map-based cloning was used to locate the regulatory genes of the semi-dwarf and necrotic phenotype in IL6-2 under low temperature,and the function of the candidate genes was verified.The main research results are summarized as follows:1.The phenotypes of 50 introgression lines(M82 as the background material)were observed.It was found that IL6-2 showed dwarfism,stunting,leaf curl,and even progressive necrosis compared with M82 plants when plants grew at 12°C,while this phenotype did not appear in other introgression lines.Physiological index measurement showed that the relative electrolyte leakage,MDA content and H₂O₂ were remarkably higher in IL6-2 than those in M82 at 12?.By contrast,the proline content was significantly lower in IL6-2 than that in M82.The results suggest that IL6-2 is more sensitive to low temperatures compared to M82.2.The leaves of IL6-2 and M82 seedlings were treated with low temperature for 0h,24h and 48h,respectively,and RNA-seq analysis was performed.A total of 3,459 genes were identified as significantly differentially expressed between M82 and IL6-2,and there were1,439,2,170 and 888 differentially expressed genes(DEGs)at 0h,24h and 48h,respectively.Functional cluster analysis of DEGs showed that the differentially expressed genes between IL6-2 and M82 were mainly in transmembrane receptor protein kinase activity,transmembrane receptor activity,transferase activity,protein kinase activity,which were mainly involved in biosynthesis of secondary metabolites pathway,plant hormone signal transduction pathway and plant-pathogen interaction pathway.3.For mapping the gene that regulate the dwarfing necrosis of IL6-2 at low temperature,an F₂ mapping population was created by self-pollination of a cross of IL6-2×M82.Through the genetic analysis of characters indicating that ndw mutant IL6-2 is controlled by a single recessive gene.Based on the mapping population,insertion/deletion(In Del)markers were developed,NDW was delimited to an interval between 37.95 Mb-39.14 Mb on chromosome 6,a fragment of approximately 640 kb.Further expanding the genetic isolation population,developing new In Del markers for fine mapping,and finally NDW gene was mapped to an approximately 77.4 kb fragment between 38.68 Mb-38.76 Mb.4.Seven putative ORFs were predicted by sequencing and analyzing the target?77.4 kb sequence in M82 using gene prediction software GENESCAN and FGENESH.According to genomic annotation information,the best hits of these ORFs included the protein arginine N-methyl transferase(ORF1),receptor-like protein kinases(ORF2–ORF4),glycosyltransferase(ORF5),peroxisomal biogenesis factor(ORF6),and zinc finger protein(ORF7).Sequence alignments showed that each candidate gene had nucleotide differences in the promoter,exon,or intron regions.RNA-seq analysis of differentially expressed genes,combined with q RT-PCR analysis of the relative expression of seven candidate ORFs,indicated that ORF2(Solyc06g060680.1.1)and ORF3(Solyc06g060690.2.1)were extremely lower in IL6-2 than in M82,while ORF5(Solyc06g060710.2.1)was significantly up-regulated in IL6-2.ORF2,ORF3 and ORF5 showed significant difference between IL6-2and M82.Therefore,we speculated that these three ORFs are the candidate genes which regulate the semi-dwarf and necrotic phenotype in IL6-2.5.Over-expression vectors and RNA interference expression vectors were constructed for the three candidate genes,respectively,and the transgenic plants were obtained by genetic transformation of tomato.The transgenic plants were tested positive and the candidate gene relative expression level was measured.The expression level of candidate gene was evidently up-regulated in overexpression transgenic lines and down-regulated in RNAi transgenic lines were selected for phenotype identification.The results showed that overexpression of ORF3(Solyc06g060690)restored necrotic phenotype in IL6-2 under low temperature,the plant heights of transgenic plants OE-2,OE-7,and OE-9 were significantly higher than that of IL6-2,and were almost the same as that of M82.The relative electrolyte leakage and MDA content were remarkably lower in OE plants and M82 compared with IL6-2.In contrast,the proline content was significantly higher in OE plants and M82 compared with IL6-2.There was no significant difference between the phenotype of ORF2(Solyc06g060680.1.1)and ORF5(Solyc06g060710.2.1)transgenic lines and the non-transgenic control materials at12?.Taken together,these results indicated that Solyc06g060690 was NDW,and played an important role in plant growth and cold tolerance in tomato.6.In the disease assays,we measured the average disease lesion area per leaflet and analyzed the planta fungal growth in transgenic plants.These results indicated that overexpression of NDW reduced susceptibility to Botrytis cinerea compared to IL6-2,and knockdown NDW in M82 plants resulted in increased sensitivity to Botrytis cinerea compared to M82.These results indicated that NDW played a role in regulating the sensitivity to Botrytis cinerea in tomato.7.Sequence alignment analysis showed that NDW contains an ORF of 1122 bps in length,and contains two exons and an intron,encoding a 342 amino acid protein.The annotation information is a receptor-like protein kinase in the SGN database,and its amino acid sequence contains a conserved domain of the PKC-like super family.The kinase domain shared high sequence homology with that of other RLK genes in the Solanaceae species,and there are many conserved domains with potato,pepper and tobacco.Phylogenetic tree analysis shows that NDW shares the highest homology with Solanum tuberosum,and exhibited approximately 79.12%identity with XP?015161010.1(S.tuberosum),and it has low homology with other crops.8.It was confirmed that NDW gene was constitutively expressed in all tissues,including roots,stems,leaves,flower buds,flowers,and fruits,most highly in the root and stem of M82plants.The results of subcellular distribution showed that NDW protein was localized to the plasma membrane and cytosol.It shows that it plays the function of receptor-like protein kinase,perceiving extracellular signals and conducting them into the cell.9.Analysis the expression levels of some genes related to ABA synthesis,decomposition and signal transduction pathway in NDW transgenic lines.The results showed that NDW up-regulated the expression level of some ABA receptor genes,ABA synthesis related genes,and ABA signal transduction positive regulatory genes.On the contrary,the expression levels of some ABA signal transduction negative regulatory genes and ABA decomposition pathway genes were significantly decreased.These results indicated that NDW gene is related to ABA accumulation,and may regulate plant growth and stress response through ABA-dependent pathway in tomato.