Identification Of MiRNA Synthesis Pathway Related Genes And Preliminary Study DlAGO4 Function In Dimocarpus Longan Lour

Longan（Dimocarpus longan Lour.）is an important economical woody fruit tree in the tropical and subtropical regions of China.It has extremely high edible and medicinal value,but there are many problems in the production of longan that restrict its industrial development.Studies have found that the yield and quality of longan fruit are closely related to the development of longan embryos,so the study of longan embryo development is of great significance.Micro RNA（mi RNA）is a class of small molecules with a length of 21 to 24 nucleic acids,which plays an important role in plant growth and development.The formation and mechanism of mi RNA require the participation of many genes,and the 24nt mi RNA is the most in longan somatic embryos.Studies have shown that a certain degree of DNA methylation is conducive to the normal development of plant embryos,especially 24nt lmi RNA is closely related to DNA methylation,and the function of Argonaute protein in somatic embryogenesis of longan is not yet clear.This study intends to use the longan somatic embryogenesis system to identify and analyze genes related to the mi RNA synthesis pathway,promoter analysis,expression patterns at different stages of somatic embryos and exogenous hormones and abiotic stress treatment,Dl AGO4 subcellular localization analysis,and The effect of silencing Dl AGO4 on the early morphogenesis of longan somatic embryogenesis.The main findings are as follows:1 Identification and bioinformatics analysis of genes related to longan mi RNA synthesis pathwayFifteen related genes were identified from the Longan genome and named Dl DDL1,Dl DDL2,Dl DDL3,Dl DCL1,Dl SE,Dl HYL1a,Dl HYL1b,Dl HYL1c,Dl CBP20,Dl CBP80,Dl HEN1,Dl HASTY,Dl DCL3,Dl AGO4.Bioinformatics analysis shows that Dl DDL1,Dl HYL1a,Dl HYL1c,Dl SE and Dl AGO4 are basic proteins,and the rest of the members are acidic proteins;Except that Dl HASTY is a hydrophobic protein,the rest of the members are hydrophilic proteins;All genes are unstable Protein;15genes are precisely located on 8 chromosomes;Dl CBP80 is a secreted protein,and the remaining members are not secreted proteins;Except for the three members of the Dl DDLs protein that do not contain a transmembrane structure,the remaining proteins have multiple transmembrane structures;The results of the protein conserved domain show that each member contains its typical domain;Protein interaction analysis shows that there is a strong interaction between genes related to the mi RNA synthesis pathway:different genes have different evolutionary distances with other species Relationship;The genes related to the mi RNA synthesis pathway exhibit different expression patterns during the early development of longan somatic cells,suggesting that different members have different functional divisions and characteristics in the early stage of somatic embryogenesis.2 Analysis of promoters of genes related to longan mi RNA synthesis pathwayA 2000bp sequence upstream of the ATG start codon of the above gene was extracted from the longan genome for promoter analysis.Analysis of cis-acting elements revealed that:genes related to the mi RNA synthesis pathway contain a large amount of TATA-box and CAAT-box,indicating that normal transcription can be performed,and also include various optical response elements and hormone response elements;some genes also contain cells Cycle,day and night control,drought,stress,endosperm,meristem,low temperature and other functional elements suggest that these genes may be involved in hormone response,stress response,light response,circadian clock,drought and cold resistance,seed growth,embryo development and other processes.3 Longan mi RNA synthesis pathway related gene expression pattern analysisThe expression trends of the 15 genes in the three early stages of somatic embryogenesis in longan are obviously different.Dl DDL1,Dl HYL1b are not expressed,Dl DDL2,Dl DDL3 expression is relatively stable,Dl SE,Dl HYL1c,Dl CBP80,Dl HEN1 showed a downward trend and then increased,Dl HYL1a,Dl CBP20,Dl HASTY showed a downward trend,Dl DCL1,Dl DCL3 showed a downward trend,and Dl AGO4 and Dl DRM2 showed a rising trend and then a downward trend.Among them,Dl AGO4 had the highest expression level in the three stages.It is speculated that Dl AGO4 played an important role in the early stage of longan somatic embryos.The expression levels of Dl HYL1c and Dl DCL3 were significantly up-regulated after 24h of exogenous hormone ABA treatment;The expression levels of Dl DDL2,Dl DDL3,Dl DCL1,Dl SE,Dl HYL1c and Dl CBP20 were up-regulated after Me JA treatment for 4h,and the expression levels of Dl CBP80 were significantly up-regulated after 12h treatment;the expression of Dl DCL3 was up-regulated throughout the treatment;The expression of Dl HYL1c increased after 24h of SA treatment,and the expression of Dl DDL3,Dl DCL1,Dl CBP80,Dl HASTY,and Dl DCL3 increased significantly after 48h of treatment;The expression of Dl DDL3,Dl DCL1,and Dl HYL1c increased after 4h of GA₃treatment The expression level of Dl DDL2 was up-regulated at 8h,and the expression levels of Dl CBP80 and Dl DCL3 were significantly up-regulated at 24h and 48h,respectively,indicating that genes related to longan mi RNA synthesis pathway have different response mechanisms under the treatment of exogenous hormones.After salt stress treatment,the expression levels of Dl DDL2 and Dl SE increased significantly at 50mmol/L and 100mmol/L concentrations,the expression level of Dl DDL3 increased at 50mmol/L concentrations,and the expression levels of Dl HYL1c at 100mmol/L and 200mmol/L concentrations Increased,the expression level of other treatments decreased showed negative regulation.Under the treatment of 10%PEG6000,the expression level of Dl DCL1 increased at 8h,the expression level of Dl DDL2 increased at 12h and 24h,the expression level of Dl DDL3,Dl SE,Dl HYL1a and Dl DCL3 increased at 24h,and the expression level of Dl HYL1c at 8h,12h and 24h.The expression level increased significantly at 48h,and the expression level decreased significantly under the other treatments as negative regulation.The above results indicate that genes related to longan mi RNA synthesis pathway are widely involved in different abiotic stresses,but different members have different expression patterns.4 Longan Dl AGO4 subcellular localization studyThe Wo LF PSORT online website was used to predict that Longan Dl AGO4 may be located in the nucleus.At the same time,a1302-Dl AGO4-GFP expression vector was constructed on the basis of the full-length sequence of Longan Dl AGO4 c DNA.Agrobacterium was used to infect the tobacco leaves,and the fluorescence in the tobacco leaves was observed under a laser confocal microscope.The results showed that Longan Dl AGO4 was located in the nucleus,consistent with the predicted results.This result laid the foundation for further exploration of the function of this gene.5 Effect of Dl AGO4 gene silencing on early morphogenesis of longan somatic embryogenesisAccording to the ORF sequence of Dl AGO4 gene,design and synthesize Si RNA for knocking out the target gene,and select the target477 with the best inhibitory effect to carry out longan callus transformation experiment results.Slightly ahead of time indicated that the inhibition of D1AGO4 accelerated the early differentiation of longan somatic embryogenesis.It is speculated that this may be related to the reduction of early methylation of longan somatic embryogenesis.