Screening And Interaction Analysis Of Genes Related To Functional Male Sterile In Eggplant

Male sterility has significant economic and social benefits in production breeding.Applying male sterility lines to eggplant breeding can solve the problems existing in eggplant hybrid seed production,improve seed quality,and reduce costs.Functional male sterility is a special type of plant male sterility:it can't be scattered due to anther not cracking,which leads to infertility,but its pollen is normally fertile.The mechanism of anther not cracking of functional male sterility has not yet been determined.At present,through a large number of studies on mutant genes of Jasmonic acid?JA?biosynthetic pathway structural genes,signal transduction genes and transcription factors,it is generally believed that JA plays important roles in regulating anther indehiscence,filament elongation and pollen development.In the current study,we performed a transcriptome analysis to identify differentially expressed genes?DEG?in eggplant S12?sterile anthers?and F142?normal anther development?to reveal differences in anther dehiscence networks.DEG enrichment analysis and endogenous hormone measurements highlight the role of the JA signal transduction pathway in anther dehiscence.We analyzed the relationship between JA pathway genes through yeast two-hybrid and yeast one-hybrid analysis,explored the molecular mechanism of functional male sterility in eggplant,and provided a basis for revealing the mechanism of functional male sterility.The main research results are as follows:1.Preliminary screening of differentially expressed genesTranscriptome analysis was performed on anthers with normal cracking eggplant?F142?and anthers indehiscent eggplant?S12?,and a total of 2670 differentially expressed genes were screened.Among them,1928 differentially expressed genes were significantly up-regulated and 742 differentially expressed genes were significantly down-regulated.The enrichment analysis of differentially expressed genes showed that amino and nucleotide sugar metabolism pathways?Metabolic pathways?,plant hormone signal transduction,inositol phosphate metabolism pathway?Amoebiasis?,and flavonoid biosynthesis pathway?ECM-receptor interaction?,the highest degree of fatty acid biosynthesis.We further screened these differentially expressed genes to find differentially expressed genes related to anther dehiscence.These genes mainly included plant hormone transduction genes.We then analyzed the gene expression levels of key genes in the jasmonic acid,auxin,gibberellin,abscisic acid,cytokinin,ethylene and brassinolide signaling pathways.In the JA signaling pathway,five key genes were identified,including DAD1,LOX,COI1,JAZ1,and JAR1.Two of them increased significantly and three decreased.At the same time,we identified one key gene in the CTK signaling pathway and two genes in the abscisic acid signaling pathway.However,four key genes were identified in the IAA signaling pathway.In the ETH signaling pathway,four key genes were identified;which were significantly up-regulated.Four genes were significantly upregulated in the BR pathway.In addition,a total of 9 genes were enriched in other hormone signaling pathways,and 2 genes were significantly down-regulated.2.Cloning and expression analysis of JA pathway gene in eggplantSmLOX,SmDAD1,SmJAR1,SmCOI1,and SmJAZ1 were amplified by PCR.The total length was 2508 bp,744 bp,1191 bp,1653 bp and 756 bp respectively,encoding836,248,397,551 and 252 proteins respectively.The relative molecular weights were94.52,26.89,43.74,62.18 and 28.00 respectively,and the isoelectric points were 5.3,8.23,7.71,6.24 and 9.22 respectively.Quantitative analysis of SmLOX,SmDAD1,SmJAR1,SmCOI1 and SmJAZ1 expression levels in F142 and S12 revealed that JAZ1and JAR1 expression levels were significantly up-regulated in S12,while SmLOX,SmDAD1 and SmCOI1 expression levels were significantly down-regulated.3.Subcellular localization of eggplant Sm COI1 proteinAgrobacterium LBA4404-mediated transformation of the vector into tobacco leaf epidermal cells revealed that the green fluorescence in the tobacco leaf epidermal cells transformed with the pCAMBIA1300-GFP-Sm COI1 vector which the green fluorescence detected in the cells was only distributed in the nucleus,indicating that the eggplant SmCOI1 protein was localized in the nucleus.4.SmLOX,SmDAD1,Sm JAR1,SmCOI1 and SmJAZ1 protein interaction analysisThe yeast two-hybrid studies found:LOX?AD?+COI1?BK??LOX?AD?+JAZ1?BK??DAD1?AD?+JAR1?BK??DAD1?AD?+COI1?BK??DAD1?AD?+JAZ1?BK??JAR1?AD?+JAZ1?BK??LOX?BK?+COI1?AD??LOX?BK?+JAZ1?AD??DAD1?BK?+JAR1?AD??DAD1?BK?+COI1?AD??DAD1?BK?+JAZ1?AD??JAR1?BK?+JAZ1?AD?were not allowed in DDO?A?SD?-Leu?-Trp?AbA?,QDO?SD?-Ade?-His?-Leu?-Trp?colonies growed on the agar medium;but LOX?AD?+DAD1?BK??DAD1?AD?+LOX?BK??COI1?BK?+JAZ1?AD??JAZI?BK?+COI1?AD?could.The DDO?A?SD?-Leu?-Trp?AbA?and QDO?SD?-Ade?-His?-Leu?-Trp?protein combinations were used in DDO?A?SD?-Leu?-Trp AbA?,QDO?SD?-Ade?-His?-Leu?-Trp?agar medium grew colonies,indicating that only DAD1 protein interacted with LOX and COI1 protein interacted with JAZ1,but there was no interaction between other proteins.Furthermore,it was proved by prokaryotic expression that the test result was the same as that of yeast two-hybrid.5.Cloning and expression analysis of SmDAD1 promoter in eggplantThe SmDAD1 promoter obtained by the chromosome walking method was 746 bp long.It was found that the DAD1 promoter contains multiple TATA-Box and CAAT-Box key cis-acting elements,and contains multiple light-responsive elements A-Box,G-Box,Box III,GATA-motif,hormone-related elements ABRE,GARE-motif,meristem expression element CAT-box,MYB binding site,etc.GUS staining analysis revealed that SmDAD1 was not expressed in Arabidopsis stems,but was expressed in roots,leaves and anthers.Spraying transgenic embryos with different plant hormones found that MeJA,ABA,GA,SA,IAA,and ACC could all induce GUS expression to varying degrees.Among them,the expression of GUS induced by MeJA and ABA was extremely significant,and the expressions of GUS induced by GA,SA,IAA and ACC were significant.6.Analysis of the interaction between Sm LOX,SmJAR1,SmCOI1,SmJAZ1 proteins and SmDAD1 promoterYeast one-hybrid identification showed that Y1H?prDAD1+LOX?,Y1H?prDAD1+JAR1?,Y1H?prDAD1+COI1?,and Y1H?prDAD1+JAZ1?could not grow white plaques on SD?-Leu?AbA⁴⁰⁰,and the positive control could grow white plaques,and the negative control couldn't grow normal plaques,indicating that the LOX,JAR1,COI1,and JAZ1 proteins couldn't interact with the SmDAD1 promoter,so the downstream reporter gene couldn't be activated,and plaque couldn't grow.The results of the double luciferase system test were consistent with the results of the yeast one-hybrid test.