Genome-wide Identification And Functional Analysis Of RALF Gene Family During Early Somatic Embryogenesis In Dimocarpus Longan Lour

Longan(Dimocarpus longan Lour.)is an important subtropical economic fruit tree.The longan somatic embryogenesis system is an excellent model system for somatic embryogenesis in woody plants.The Rapid alkalinization factor(RALF)family is widely distributed in plants and fungi.RALF peptides are also considered to be a group of short peptide hormones with regulatory effects on plant cell expansion,root growth,sexual reproduction,and response to stress.Existing studies have demonstrated that RALF peptide has an important regulatory role in plant reproductive growth.The study of the expression pattern and function of the RALF gene family in the early somatic embryogenesis of Longan can provide a valuable reference for the molecular regulation mechanism during plant embryogenesis.Therefore,in this study,genome-wide identification,bioinformatics analysis,and expression pattern analysis of the Dl RALF gene family were performed based on the longan genome database.Validation of the sequences of candidate genes Dl RALF4,Dl RALF5,and Dl RALF9,and constructed the p CAMBIA1301 overexpression vector to transform into longan embryonic callus.Preliminary investigation of the biological functions of Dl RALF4,Dl RALF5,and Dl RALF9 at the early stage of longan somatic embryogenesis by genetic transformation experiments.And it was verified that the Dl RALF gene family antagonizes brassinolide to regulate early somatic embryogenesis in longan.The main results are as follows:1 Genome-wide identification and bioinformatics analysis of the Dl RALF familyThe Dl RALF gene family has 10 members.Four pairs of members with covariate relationships exist within this species.Strong covariance also exists with other plants in the Sapotaceae family.Subcellular localization is predicted to be all localized extracellularly.The promoters of Dl RALF family gene members contain a large number of lightresponsive elements,stress-responsive elements,tissue-specific growth elements,and hormone-responsive elements.The results of this study indicate that the Dl RALF gene family members are highly conserved,evolved in a similar manner to other Sapotaceae,and are functionally diverse.2 Analysis of the expression pattern of the Dl RALF gene familyAnalysis of the expression patterns of the Dl RALF gene family revealed that members of the Dl RALF gene family differed greatly in their expression patterns at different stages of somatic embryogenesis.Dl RALFs respond to a variety of treatments such as 5-azacytidine,temperature,drought,light quality,and Dl MIR408-OE.Based on the bioinformatics and expression pattern analysis of Dl RALF gene family members,we selected Dl RALF4,Dl RALF5,and Dl RALF9 as candidate genes,which have complete conserved motifs,respond to multiple treatments,and may be involved in the regulation of longan somatic embryogenesis.The candidate genes Dl RALF4,Dl RALF5,and Dl RALF9 responded to the regulation of growth hormone,abscisic acid,and methyl jasmonate in different patterns.It is hypothesized that the Dl RALF gene family may respond to a variety of treatments including,auxin and abscisic acid.And Dl RALFs are involved in regulating somatic embryogenesis and the growth and development of various organs in longan.3 Genetic transformation study of Dl RALFs in embryonic callus and effect of transcriptional levelTo further investigate the function of Dl RALFs in the early stage of somatic embryogenesis in Longan,the p CAMBIA1301 overexpression vector carrying key member sequences was stably genetically transformed into Longan embryonic callus to obtain Dl RALF4-OE,Dl RALF5-OE,Dl RALF9-OE,and Dl MIR408-OE Longan embryonic culture cell line materials and to explore the potential gene interplay regulatory network of Dl RALFs/Dl MIR408.The result shows that,Dl MIR408 upregulated the expression of Dl RALF4 and Dl RALF5,while significantly downregulating the expression of Dl RALF9.Dl RALF4 and Dl RALF9 down-regulate the expression of dlo-mi R408-3p.Dl RALF4,Dl RALF9,and Dl MIR408 regulate expression of Dl YUC family members Dl YUC4 and Dl YUC8,and also regulate expression of Dl GPI8,a member of the glycosylated phosphatidylinositol pathway,and Dl FER.However,there is variability in expression patterns.None of the effects of Dl RALF5 on the expression of the above members was significant.It is hypothesized that Dl RALF4 and Dl RALF9 together build a gene interplay regulatory network and participate in pathways such as growth hormone anabolism and glycosylated phosphatidylinositol pathway.Members of the Dl RALF gene family may be involved in growth and morphogenesis in Longan somatic embryogenesis through this pathway.4 Physiological and biochemical functions of Dl RALF4-OE,Dl RALF5-OE and Dl RALF9-OE cell lines in the early stage of somatic embryogenesis of LonganDetermination and comparative analysis of physiological and biochemical parameters in Dl RALF4-OE,Dl RALF5-OE,and Dl RALF9-OE longan embryonic cultures cell lines revealed that: the Dl RALF4-OE,Dl RALF5-OE,and Dl RALF9-OE cell lines all have the typical function of alkalinizing the extracellular medium.Compared with empty vector(EV),endogenous IAA content was increased in Dl RALF4-OE and Dl RALF9-OE cell lines,and endogenous ABA content was decreased in Dl RALF5-OE and Dl RALF9-OE cell lines,which all three genes have the effect of reducing endogenous BR content.Suggesting that the Dl RALF gene family has a role in regulating the content of the endogenous hormones IAA,ABA and BR in longan embryonic cultures.SOD and POD activities were significantly increased in Dl RALF4-OE,Dl RALF5-OE,and Dl RALF9-OE cell lines compared to EV.All of them are also effective in enhancing the flavonoid content.Dl RALF4 and Dl RALF5 significantly enhanced the hydroxyl radical scavenging rate,but Dl RALF9 decreased this index.All three candidate genes significantly increased the total antioxidant capacity of Longan somatic embryogenesis in the early stage.Therefore,it is hypothesized that the Dl RALF gene family enhances the antioxidant capacity of longan embryonic cultures by increasing the antioxidant content and antioxidant activity,thus ensuring the balance of redox environment in embryonic cells,and playing a role in maintaining the embryonicity of longan embryonic callus and resisting stress.5 Dl RALFs respond to exogenous brassinolide and are involved in regulating the early embryogenesis of Longan somatic embryosBrassinolide is antagonistic to RALF peptide in regulating root growth.And brassinolide has a role in regulating somatic embryogenesis in several species including cotton.To further investigate the interaction between the Dl RALF gene family and brassinolide and its function in the early stage of somatic embryogenesis by brassinolide treatment of Longan embryonic embryonic callus.The result shows that: Addition of exogenous brassinolide to global embryo(GE)induction medium or relay medium supplemented with high concentrations of the growth hormone analogue2,4-D both promoted early somatic embryogenesis in Longan,the most significant difference was found in the 0.5 mg/L concentration of brassinolide treatment.Exogenous brassinolide treatment inhibits the value proliferation of embryonic cultures of longan.Brassinolide inhibits both IAA and ABA contents,while brassinolide and 2,4-D may have antagonistic effects in regulating endogenous hormone contents.Dl RALF4,Dl RALF5,and Dl RALF9 were regulated by brassinolide and 2,4-D,but with different expression patterns in different concentrations,days,and combinations of brassinolide treatments.Dl RALF4,Dl RALF5,and Dl RALF9 all down-regulated the expression of the brassinolide synthesis gene Dl CYP92C6.In summary,key members of the Dl RALF gene family have antagonistic effects at brassinolide.Brassinolide promotes early somatic embryogenesis and inhibits the proliferation of Longan embryonic cultures.This may be regulated by the interactions of brassinolide with other hormones such as IAA,ABA,and RALF peptide hormones.In summary,Dl RALF gene family interacts with Dl MIR408 in the hormone crosstalk network and the glycosylated phosphatidylinositol pathway.And involved in the process of somatic embryogenesis in longan through interactions with plant hormones such as IAA,ABA,and BR.In addition,key members of the Dl RALF gene family play a role in regulating the oxidative environmental homeostasis and resistance to adversity stress in longan embryonic cultures by regulating the content and activity of antioxidants.