Genome-wide Identification Of HAM Family And Expression Regulation During Early Somatic Embryogenesis In Dimocarpus Longan Lour.

Dimocarpus longan Lour.,which belongs to Sapindaceae family,is widely distributed in southern China and is one of the characteristic economic fruit trees in subtropical tropics.The somatic embryogenesis（SE）system of longan is an excellent model system for woody plants.Auxin and cytokinin are important phytohormones in the longan SE,which play an important role in regulating the proliferation of embryonic heal tissues and maintaining embryonicity in longan.Studies show that auxin and cytokinin can regulate the WUSCHEL-CLAVATA3（WUS-CLV）signaling pathway.HAIRY MERISTEM（HAM）regulates the WUS-CLV signaling pathway in embryonic cells and plays an important role in plant cell differentiation.Currently,the mechanisms of auxin and cytokinin regulation during longan SE are unclear,and no studies on the Dl HAM gene in longan have been reported.Therefore,this study combined high-throughput RNA-seq,physiological and biochemical assays,cell biology studies and genetic transformations to investigate the regulatory roles of auxin and cytokinin,as well as the Dl HAM family in longan early SE.1 Transcriptomic analysis of longan embryogenic heal tissues under auxin and cytokinin treatmentThe embryogenic callus was maintained by auxin,auxin and cytokinin treatment,and cytokinin promoted differentiation of embryogenic callus.In this study,RNA-seq high-throughput sequencing was performed on longan embryogenic callus（EC）grown in MS as a control（named CK）,and on longan EC treated simultaneously with auxin（named D）,cytokinin（named KT）,and auxin and cytokinin（named D＿KT）as a test group.A total of 177.86 Mb of sequencing data was obtained,and the comparison rate with the reference genome ranged from81.73 to 85.46%.A total of 21,884,21,807,21,835,and 21,849 genes were annotated in the D,D＿KT,KT,and MS samples,respectively.There were 19 up-regulated differentially expressed genes（DEGs）and 17down-regulated DEGs in D vs D＿KT;518 up-regulated DEGs and 189down-regulated DEGs in MS vs D;492 up-regulated DEGs and 258down-regulated DEGs in KT vs D＿KT;MS vs D＿KT up 550 DEGs and down 245 DEGs;MS vs KT up 24 DEGs and down 14 DEGs.The results of q RT-PCR expression pattern validation were consistent with the FPKM values,indicating that the transcriptomic data were accurate and reliable.Comparative transcriptome analysis revealed that longan EC were predominantly responsive to auxin induction,and a total of 11auxin-related DEGs were obtained（four auxin response factors ARFs,three auxin response proteins IAA,three auxin response genes GH3,and one auxin export carrier protein gene PIN）.Based on the DEGs,GO functional pathways are primarily involved in biological processes,molecular functions,and cell components.KEGG pathways significantly enriched after auxin treatment are mainly metabolic pathways,phenylpropanoid biosynthesis,pyrimidine metabolism,and alpha-linolenic acid metabolism,whereas KEGG pathways significantly enriched after cytokinin treatment are mainly ether lipid metabolism,biotin metabolism,brassinosteroid biosynthesis and homologous recombination.The results assume that auxin can regulate the metabolism of alpha-Linolenic acid and taurine metabolism in longan EC.2 Effects of taurine and linolenic acid on the morphogenesis,physiology and biochemistry of longan somatic embryogenesisIn this study,longan EC was treated with different concentrations of taurine and linolenic acid.The results showed that high concentrations of taurine（0.5,5,and 10 m M）could promote the longan early SE,and taurine can promote the development of longan somatic embryo.Linolenic acid（5,50,and 100μM）could inhibit the longan early SE,but the ability of SE was restored after incubation.By measuring the activity of superoxide dismutase and peroxidase of longan EC treated with taurine and linolenic acid,,It is hypothesized that the taurine and hypotaurine metabolism pathways and the alpha-Linolenic acid metabolism pathway affect the ability of somatic embryos to resist oxygen to influence the process of SE.3 Genome-wide identification,bioinformatics and expression analysis of longan Dl HAM family membersThe longan Dl HAM family consists of four members,all of which are hydrophilic proteins without transmembrane structure and signaling peptides,with similar physicochemical properties,all of which are expected to be localized in the nucleus;all of which are randomly distributed on three chromosomes;all members have multiple cis-acting elements on their promoters related to hormones,stress and growth;the Dl HAM gene family is closely related to Lchinesis and Nephelium lappaceum.Dl HAM was not used in different embryonic cultures of longan;Dl HAM was tissue specific;Dl HAM also responded to different hormone treatments,with IAA,GA₃,ABA,and Me JA promoting its expression.High concentrations of taurine inhibited Dl HAM3 expression.50μM and 100μM of linolenic acid significantly promoted Dl HAM1expression and 5μM significantly inhibited Dl HAM4 expression.The Dl HAM family is hypothesized to be responsive to various treatments,including gibberellin and linolenic acid,and is involved in the regulation of somatic embryogenesis and the growth and development of various organs in longan.4 Subcellular localization and functional analysis of some genes of Dl HAM in longanThe results of the subcellular localization prediction showed that Dl HAM2 and Dl HAM4 proteins were localized in the nucleus.In addition,the Agrobacterium fusion expression vectors p CAMBIA1302:Dl HAM2:GFP and p CAMBIA1302:Dl HAM4:GFP were constructed by a seamless cloning technique.Then,fusion expression vectors for each of the two gene members were transformed by injection to infest the inner epidermis of onions.The results verified that the Dl HAM2 and Dl HAM4proteins were localized in the nucleus,which hypothesized that the two may be activation-regulated transcription factors with similar functions.The recombinant vectors p1301:Dl HAM3:GUS and p1301:Dl HAM4:GUS were transformed into longan EC using Agrobacterium tumefaciens in over expression of the transgenic cell lines.On this basis,the study investigated the interactions among Dl HAM family members and the regulatory patterns of WUS-CLV pathway-related genes in the over expressed cell lines.The results showed that Dl HAM3 significantly promoted the expression of Dl HAM1,Dl HAM4,Dl WUS,and Dl CLV1-1;Dl HAM4 significantly promoted the expression of Dl HAM2,Dl HAM3,Dl WUS,Dl CLV1-1,Dl CLV1-2 and Dl CLV2,and significantly inhibited the expression of Dl HAM1.Dl HAM3 and Dl HAM4 are closely related to Dl CLV1-1 and Dl WUS and involved in the WUS-CLV pathway.In addition,HAM is involved in the gibberellin signaling pathway,and endogenous GA₃was measured in transgenic longan EC.Over expression of p1301:Dl HAM3:GUS resulted in a significant decrease in endogenous gibberellin in longan EC.It is hypothesized that the Dl HAM3 gene significantly inhibits the production of endogenous GA₃in longan EC,thus affecting the process of somatic embryogenesis.