Molecular Mechanism Of Melon Golden2-Like Transcription Factor Regulating Seed Germination By ABA

Seed dormancy and germination are two unique physiological processes in plants,which are crucial for plant growth and crop production.Cucnmis melo L.is one of the most important horticultural crops in China,and its seed germination involves numerous complex physiological and biochemical reactions.Plant abscisic acid(ABA),as a key hormone that can regulate seed germination,has good potential for agricultural applications.However,the molecular mechanism by which ABA regulates melon seed germination is currently unclear.Our research group previously cloned the key gene CmGLK that regulates the chloroplast development of melon using a yellowing mutant material M166,and constructed a Cmglk near isogenic line under the background of annual thin skin melon HB42.On the basis of this research,we used the HB42 and Cmglk near isogenic lines glk-NIL as experimental materials to statistically analyze the differences in seed germination rates between the two types of melon;Using exogenous ABA and ABA synthesis inhibitor Fluridone to treat HB42 and g/k-NIL seeds,analyze the effect of exogenous ABA on the germination rate of melon seeds;Based on transcriptome results,CmABAR genes related to ABA signaling pathway and ABA receptor were screened out,and the function of CmABAR gene was verified by homologous transformation of melon,and the relationship between CmGLK and the ABA receptor gene CnrABAR was analyzed by Y1H,LUC,EMSA and other experiments.The main research findings are as followings:1.We compared and analyzed the germination rate of HB42 and glk-NIL seeds,and the results showed that after 2 days of sowing in the culture medium,the germination rate of HB42 seeds reached 95%,while the germination rate of glk-NIL seeds only reached 56%,which is significantly lower than the germination rate of HB42 seeds,indicating that the CmGLK gene can participate in regulating the process of seed germination.2.We selected the period when the difference in seed germination rates between HB42 and glk-NIL was the most significant to measure the endogenous ABA content.We found that the endogenous ABA content of glK-NIL seeds was almost twice that of the wild type material HB42 in melon,indicating that the difference in ABA content was the reason why the germination rate of glk-NIL was significantly lower than that of the wild type material HB42.The germination rate of HB42 seeds treated with ABA was significantly reduced compared to the control(HB42 seeds without ABA treatment);The germination rate of g/k-NIL seeds treated with the ABA synthesis inhibitor Fluridone was significantly increased compared to the control(glk-NIL seeds without Fluridone treatment).The above results indicate that the CmGLK gene regulates seed germination rate through the ABA pathway.3.transcriptome sequencing analysis was carried out at the time when the seed germination rate of HB42 and glK-NIL was most significantly different,and 1933 differentially expressed genes(DEGs)were screened.Among them,825 differentially expressed genes were upregulated and 1108 differentially expressed genes were downregulated.Among these DEGs,there are 31 genes related to the ABA signaling pathway,most of which are ABA receptors and are related to seed germination.Among them,the CmABAR gene related to ABA synthesis and ABA receptors is significantly downregulated in glk-NIL,and its homologous motif in Arabidopsis is consistent with the AtABAR mutant phenotype and the Arabidopsis double mutant glklglk2 phenotype,all exhibiting yellowing phenotype.4.We demonstrated that CmGLK-can bind to the CmABAR promoter using Y1H(Yeast one hybrid)and EMSA(Electrophoretic mobility shift assay)experiments;Dual Luciferase reporter assay(LUC)results show that CmGLK is a transcriptional activator,which can enhance the activity of CmABAR promoter.The 35S-CmABAR-GFP fusion protein expression vector was constructed using homologous recombination method and transformed into tobacco leaf epidermal cells.Subcellular localization results showed that the CmABAR gene was located in the chloroplast membrane.The results of GUS staining and fluorescence quantitative PCR experiments indicate that the CmABAR gene is involved in the regulation of melon seed germination.5.Construct an overexpression vector of CmABAR gene and transform melon glk-NIL using Agrobacterium mediated method;Simultaneously construct a CRISPR-CAS9 gene knockout vector for the CmABAR gene,and transform it into wild-type melon materials XZM and VED.Currently,a CRISPR-CAS9 hybrid editing plant with CmABAR gene has been obtained.In the future,we will observe the phenotypes of different transgenic plants,compare the differences in germination rate between overexpressed and CRISPR-CAS9 transgenic plant seeds and their respective control plants,and analyze the biological function of CmABAR gene in regulating seed germination.The above results indicate that CmGLK may by binding to the G-box element on the CmABAR gene promoter,thereby promoting the germination of melon seeds and increasing their germination rate.