Genome-wide Identification Of GLP Family And Functional Study Of GLP1-5-1 In Dimocarpus Longan Lour.

Longan（Dimocarpus longan Lour.）,as an important economic fruit tree in the subtropical zone of Dimocarpus,belongs to Sapindaceae.Its embryonic development has an important impact on fruit quality and yield.Studying the molecular mechanism of longan embryos can provide a theoretical basis for improving the quality and yield of longan fruits.The establishment of the longan somatic embryogenesis system provides an excellent model system for studying plant embryogenesis.Germin like protein（GLP）is widely expressed in different organs of plants and plays an important role in various growth and development stages.However,there is limited research on the GLP gene during the embryonic development stage of longan,and the specific mechanism of action is still unclear.Previous laboratory studies have shown that GLP is a key factor in longan somatic embryogenesis and differentially expressed in the early stages of somatic embryogenesis.Therefore,this study focuses on the callus and root system of longan;Genome wide identification of the longan GLP family to explore the role of GLP in early somatic embryogenesis;Through subcellular localization,genetic transformation,Transcriptome and metabolic analysis,the function of DlGLP1-5-1 in early somatic embryogenesis was deeply studied;Further validate the mechanism of action of DlGLP1-5-1 in longan roots using the longan hairy root transformation system.The main research findings are as follows:1.Genome-wide identification,expression pattern and subcellular localization analysis of the GLP gene family in longanThe whole genome of longan GLP Gene family was identified based on the genome of the third generation of longan,and 35 DlGLP genes were identified.According to phylogenetic analysis,the GLP gene is divided into 8 subfamilies.By analyzing the physicochemical properties of its protein,gene structure,phylogenetic tree and collinearity,we found that the GLP gene is generally conservative,different family members may perform different functions,and family expansion may be mainly affected by tandem repeat.Based on the transcriptome data,it was found that some GLP genes were highly expressed in the early somatic embryogenesis and root system of longan,and responded significantly to high temperature,2,4-D and unicorn lactone treatment.Based on Transcriptome data,8 members differentially expressed in the early somatic embryogenesis of longan were selected for q RT-PCR validation.The results showed that 8 genes were highly expressed in the globular embryo（GE）stage.It was speculated that these genes might play an important role in the morphogenesis of globular embryos.The results of ABA and Me JA treatments showed that four DlGLP genes were significantly downregulated under ABA treatment;All 8 DlGLP genes were upregulated under Me JA treatment,suggesting that these GLP family members may participate in the early growth and development of longan somatic embryogenesis by responding to the regulation of Me JA and ABA.Based on the early somatic embryogenesis transcriptome database and q RT-PCR validation results of longan,the differentially expressed genes DlGLP1-5-1 and DlGLP5-8-2 were screened out for further functional verification.Subcellular localization prediction indicates that DlGLP1-5-1 is localized in chloroplasts and extracellular matrix;DlGLP5-8-2 is located in the cytoplasm.The corresponding overexpression vector was constructed and transformed into tobacco leaves through Agrobacterium tumefaciens.The results showed that DlGLP5-8-2 was located in the cytoplasm,and DlGLP1-5-1 was located in the chloroplast and extracellular matrix,suggesting that DlGLP5-8-2 played an important role in the growth and metabolism process,and DlGLP1-5-1 might participate in photosynthesis and multiple metabolic activities as a Signaling molecule.2.Validation of Longan GLP1-5-1/5-8-2 and WRKY11 regulatory networks and determination of physiological indicatorsBased on previous research in the laboratory,it was found that WRKY11 was overexpressed under the MAPK regulatory pathway,while GLP gene has been shown by previous studies to have Serpin activity.Through transcription factor target gene prediction,WRKY11 may regulate DlGLP1-5-1 and DlGLP5-8-2.Instantaneous transformation of longan protoplasts and longan callus and q RT-PCR validation revealed that overexpression of WRKY11 upregulated the expression of DlGLP1-5-1 and DlGLP5-8-2.The results of the dual fluorescence reporter enzyme assay showed that WRKY11 can activate the expression of DlGLP1-5-1 and DlGLP5-8-2,indicating that WRKY11 activates its transcription by binding to the promoter regions of DlGLP1-5-1 and DlGLP5-8-2.It is speculated that WRKY11 may regulate the growth and development of longan GLP1-5-1 and GLP5-8-2 in the early stage of somatic embryogenesis through the MAPK metabolic pathway.Previous studies have shown that the GLP gene and WRKY transcription factor may be involved in lignin synthesis.To further explore the relationship between WRKY11 and GLP,DlGLP1-5-1 with multiple WRKY11 binding sites and more pronounced activation was selected for research.Instantaneous transformation of longan EC revealed an increase in lignin content in longan EC after the instantaneous transformation of DlGLP1-5-1;The lignin content in mutant Dlglp1-5-1longan EC decreased;After instantaneous conversion of WRKY11,the lignin content in longan EC increased,indicating that WRKY11 and DlGLP1-5-1 are involved in the lignin biosynthesis pathway.3.Functional validation of DlGLP1-5-1 in the early stage of somatic embryogenesis of longanInstantaneous transformation of longan callus revealed that DlGLP1-5-1 may play an important role in the lignin synthesis pathway,and laboratory studies have found that lignin plays an important role in the early stage of longan somatic embryogenesis.Therefore,in order to further investigate the function of DlGLP1-5-1 in the early stage of longan somatic embryogenesis,overexpression of DlGLP1-5-1 stably transformed longan callus tissue,and it was found that DlGLP1-5-1 can promote longan somatic embryogenesis differentiation.The determination of lignin content and reactive oxygen species levels revealed that overexpression of DlGLP1-5-1 resulted in higher lignin content and stronger SOD and POD activities in longan spherical embryos.This suggests that DlGLP1-5-1 can promote lignin accumulation and differentiation in the somatic embryo stage by regulating ROS levels.Preliminary laboratory research found that longan callus tissue cannot undergo normal somatic embryogenesis under high temperature treatment（35℃）.High temperature treatment was performed on DlGLP1-5-1 transgenic cell lines,and it was found that overexpressing DlGLP1-5-1 transgenic cell lines can induce normal somatic embryogenesis in longan callus tissue under high temperature treatment,but their growth and development process are delayed,suggesting that DlGLP1-5-1 may enhance its high-temperature tolerance by increasing the lignin content of somatic embryos.Overexpression of DlGLP1-5-1 can promote lignin synthesis after stable transformation of longan spherical embryos.Therefore,in order to further study the regulatory network of DlGLP1-5-1 in early somatic embryogenesis,GE cell line and WT cells overexpressing DlGLP1-5-1were selected for Transcriptome and wide target metabolome sequencing.Through the transcriptome analysis of DlGLP1-5-1 overexpression cell lines,a total of 3016 differentially expressed genes were identified in DlGLP1-5-1 overexpression cell lines and WT overexpression cell lines.Through KEGG enrichment analysis,it was found that a total of 1019differential genes were enriched in 131 pathways.Among them,the most abundant differential genes are in plant MAPK signaling pathway,Plant hormone signaling pathway and phenylpropane biosynthesis pathway.It is speculated that DlGLP1-5-1 plays an important role in the early somatic embryogenesis of longan by regulating these three pathways.Metabolomic analysis revealed that a total of 519 differential metabolites were identified in overexpressing DlGLP1-5-1 and WT cell lines.It was found that differential metabolites were significantly enriched in the biosynthetic pathways of phenolic acids and flavonoids,with a total of 19 differential metabolites,of which only 4 metabolites were significantly downregulated.It is speculated that DlGLP1-5-1 plays an important role in the flavonoid and phenolic acid pathways.The analysis of phenylpropane KEGG enrichment pathway found that the content of coniferyl alcohol in DlGLP1-5-1 overexpression cell line was significantly increased,suggesting that DlGLP1-5-1 could increase the content of G-lignin.Conjoint analysis found that significantly different expressions of Cinnamoyl Co A reductase（CCR）,Cinnamyl alcohol dehydrogenase（CAD）,Ferulic acid 5-hydroxylase（F5H）and Peroxidase（POD）were found in DlGLP1-5-1 cell line overexpressing.The content of coniferyl aldehyde,coniferyl alcohol and syringa oblata in longan globular embryos overexpressing DlGLP1-5-1 increased significantly.The above results indicate that DlGLP1-5-1 may participate in the lignin synthesis pathway by regulating the expression of CCR,CAD,F5H,and POD.4.Genetic Transformation of DlGLP1-5-1 and WRKY11 in Longan Root SystemThrough transcriptome analysis of nine different tissue parts of longan,it was found that DlGLP1-5-1 had the highest expression in roots,suggesting that DlGLP1-5-1 played an important role in the growth and development of roots.Therefore,this experiment further verified the role of DlGLP1-5-1 in longan roots by means of overexpression of DlGLP1-5-1 and inhibition of expression of Dlglp1-5-1,transformation of longan roots,measurement of ROS level and lignin content in transgenic roots,and paraffin sectioning of adventitious root.The results showed that the strain overexpressing DlGLP1-5-1 had longer roots than the mutant strain Dlglp1-5-1,but more short adventitious root roots were found in the mutant strain Dlglp1-5-1.Then the lignin and H₂O₂content in adventitious root of different transgenic lines were measured.It was found that the lignin content in DlGLP1-5-1 overexpression lines was higher,and the H₂O₂content was lower;The mutant Dlglp1-5-1 strain has lower lignin content and higher H₂O₂content.It is speculated that DlGLP1-5-1 can affect the growth and development of plant roots by regulating lignin and H₂O₂content.Further validate the role of DlGLP1-5-1 in longan roots by overexpressing WRKY11 and transforming it into longan roots.It was found that overexpression of WRKY11 upregulated the expression of DlGLP1-5-1;The lignin content in the root system increases while the hydrogen peroxide content decreases,consistent with the trend after overexpression of DlGLP1-5-1.This indicates that DlGLP1-5-1 is positively regulated by WRKY11 in the root system,further proving that DlGLP1-5-1 affects the growth and development of plant roots by regulating lignin and H₂O₂ content.