Screening And Preliminary Identification Of Genes Related To Walnut Flower Bud Sexual Differentiation Based On Transcriptomic Analysis

Juglans regia L is a woody plant of Juglans in walnut family,which has excellent economic and nutritional value.However,in the process of flower bud sexual differentiation,the number of male and female flower bud differentiation is uneven,and the number of female flower is far less than that of male flower,the process of male flower development consumed a lot of energy,which is important for development of female flower,leading to low fruit setting rate and seriously affecting its economic benefits.Therefore,in order to solve this problem,regulating the flower bud differentiation process has practical application value.This study mainly focus on the molecular mechanism of walnut flower development,so as to regulate the direction of flower bud differentiation,and reduce the imbalance number of male and female flowers.AG is one of the key genes to regulate the development of stamen and carpel,FT regulates the flowering time,they play a very important role in the process of flower development.Therefore,it is of great significance to study the expression pattern and function of AG and FT in walnut to understand the flower bud differentiation and flowering regulation of walnut.The main results are as follows:1.Transcriptome sequencing of male and female flower buds at different stages of walnut,and get 177.4G of raw datas,each sample can get more than 93% of the effective data after filtering and screening.In this experiment,2167 differentially expressed genes between male and female flower buds of walnut were screened,among which 1005 upregulated genes and 1162 down regulated genes.1321 differentially expressed genes were screened in female bud for different stages,including 671 up-regulated genes and 650 down-regulated genes.1443 differentially expressed genes were screened in male bud for different stages,including 662 up-regulated genes and 781 down-regulated genes.Through the analysis of GO function and KEGG metabolic pathway,it is found that the differential genes are mainly enriched in the pathways of plant hormone signal transduction,organic matter synthesis,photosynthesis,sugar metabolism,etc.According to the results of differential gene expression analysis,GO function annotation and KEGG enrichment analysis,18 genes that may regulate flower bud differentiation were screened out.The quantitative expression of genes was verified by q RT-PCR,and the results were consistent with the transcriptome sequencing results.2.In this experiment,two AG genes and one FT gene in walnut were cloned,named Jr AG1,Jr AG2 and Jr FT,respectively.The total length of Jr AG1 coding region is 768 bp,the predicted amino acid length is 255 amino acids,p I value is 9.14,the protein instability coefficient is 56.77,the total average hydrophilicity is-0.577,the predicted hydrophilic protein,the phylogenetic tree analysis shows that the relationship with Betula luminifera is the closest.The total length of Jr AG2 coding region is 747 bp,the predicted amino acid length is 248 amino acids,the p I value is 8.98,the protein instability coefficient is 59.71,it is presumed to be an unstable protein,the total average hydrophilicity is-0.712,it is predicted to be a hydrophilic protein,and the phylogenetic tree analysis shows that it has the closest relationship with Ziziphus jujuba.Both Jr AG1 and Jr AG2 have the MIKC domain of MADS box and the unique AG I and AG II motifs.The total length of Jr FT coding region is 525 bp,and the predicted amino acid length is 174 amino acids,including a PEBP domain.The p I value is 6.73,and the protein instability coefficient is 46.28,which is supposed to be an unstable protein.The total average hydrophilicity is-0.391,which is predicted to be a hydrophilic protein.The phylogenetic tree analysis shows that the genetic relationship with Hydrangea is the closest.3.In this experiment,we constructed the plant overexpression vectors of Kpn I / Bam HI double enzyme 2301s:: Jr AG1,Kpn I single enzyme 2301s:: Jr AG 2 and Kpn I / Bam HI double enzyme 2301s:: Jr FT,and transfer the vetors into Arabidopsis thaliana to vetify the function of the gene.Preliminary identification at the molecular level confirmed that the gene was successfully transferred into Arabidopsis.Phenotype observations found that,the transgenic plants of Jr AG1 have abnormal development of stamens,leading to inability to bear fruit,the transgenic plants of Jr FT have early flowering traits.These results indicate that these genes are related to flower development.