Mining Of Differential Genes Related To Dormancy Release Of Pyrus Calleryana Decne Seeds And Preliminary Study On The Regulation Mechanism Of PcNAC30

Dormancy is the biological characteristic of seeds in order to adapt to climate change,ensure germination and survival or resist adverse environmental conditions.Pyrus calleryana Decne is the main rootstock variety of pear,and its seeds have dormancy phenomenon,which can only be broken by stratification;However,in agricultural and forestry production,seeds usually need to germinate quickly and form seedlings neatly in order to obtain high benefits.from this point of view,the characteristics of seed dormancy are not conducive to the development of rootstock production;and the current industry is lack of simple and effective methods to identify seed dormancy status and to quickly break seed dormancy.While the mechanism of seed dormancy release remains unclear,and restricts the development of related industry..In this study,the seeds of Pyrus calleryana Decne were used as materials to analyze the changes of hormones and sugars during the dormancy release progress,and transcriptome sequencing were applied to identify the differentially expressed structural genes and transcription factors regulating seed dormancy release.Furthermore,the functions of key NAC genes were preliminarily identified through gene expression analysis and yeast one hybrid system,which laid an important theoretical foundation for the establishment and development of related technological systems.The main results are as follows:1.The changes of hormones and sugars during seed dormancy release were measured and a comparative transcriptome sequencing analysis was applied to detect the differentially expressed genes involved in seed dormancy release were screened.In the process of dormancy release of pear seeds,ABA content decreased significantly,GA3 and IAA increased significantly,and ABA/GA3 also showed a downward trend,which was consistent with the theory that ABA dominated the previous study,and IAA and GA3 antagonized ABA to promote dormancy release.Starch and sucrose decreased gradually during dormancy release,while glucose and fructose increased,the results indicated that sugar transformation were necessary for seed dormancy release and provided energy available for seed dormancy release.Through transcriptome sequencing,a total of 8048 differentially expressed genes(DEGs)were identified with a series of genes involved in the biosynthesis,degradation and signal transduction of hormones and carbohydrates.At the same time,differentially expressed transcription factors(TFs)belong to 16 TF families were obtained by transcriptome sequencing,with the most TFs belong to ERF gene family,followed by bHLH,bZIP and NAC genes.Based on the DEGs screened by transcriptome sequencing,an effective method was developed to identify the dormancy status of pear seeds.Two highly expressed genes were selected from DEGsand the corresponding probes were designed.Based on the color changes in PCR-enzyme-linked immunosorbent reaction,the dormancy state of seeds can be identified quickly and efficiently.2.PcNAC30 regulated the promoter of PcHAB1 and involved in the dormancy release process of pear seeds.Through transcriptome sequencing,phylogenetic analysis and qPCR correlation analysis,two differentially expressed NAC genes(named PcNAC30 and PcNAC31)and four PP2C genes(named PcHAB1,PcHAB2,PcABI1 and PcABI2)were isolated.During dormancy release process,the expression of PcNAC30 and PcNAC31 genes were significantly decreased while that of PcHAB1,PcHAB2,PcABI1 and PcABI2 were increased.Furthermore,yeast one hybrid experiment indicated that PcNAC30 could directly bind to PcHAB1 promoter,regulate the expression of PcHAB1 gene,and then involved in formancy release of pear seeds.