Analysis Of Transcriptome And MiRNA And Function Identification Of Related Genes After Colchicine Induction In Mulberry

Colchicine has a good induction effect and is the most widely used inducer for artificial chemical induction of polyploid plants.This article analyzed the results of physiological and biochemical indicators,transcriptome sequencing and small RNA sequencing after colchicine induction.Prokaryotic expression and subcellular localization of differentially expressed genes were studied,and miRNA and its target genes were identified and analyzed.It provides a solid theoretical basis and application basis for the excavation of excellent genetic resources of mulberry and molecular breeding of mulberry mutagenesis.1.Physiological and biochemical transcriptome and miRNA analysis after colchicine induction in mulberryAfter colchicine induction,malondialdehyde(MDA),superoxide dismutase(SOD),proline(PRO)and other physiological and biochemical indexes of mulberry showed that the content of MDA after induction was lower than that of the control group,while the content of SOD and PRO were increased.The above results indicated that the antioxidant activity of mulberry after induction was stronger than that of the control group.Transcriptome sequencing was used to analyze and compare the differentially expressed genes between colchicin-induced leaf samples and control leaf samples.In the first group,there were a total of 1130 differentially expressed genes,including 418up-regulated genes and 712 down-regulated genes.In the second group,there were 1,901 differentially expressed genes,including 799 up-regulated genes and 1102 down-regulated genes Bioinformatics analysis including GO annotation and KEGG enrichment were performed on the differentially expressed genes in the above mentioned transcriptome,and12 genes were selected to detect gene expression levels in leaf samples to verify the transcriptome results.In the analysis of small RNA sequencing,a total of 45 known miRNAs and 62 predicted mature new miRNAs were identified by RNA-seq and screening,with 37differential(DIFF)miRNAs,there were 19 up-regulated miRNA and 18 down-regulated miRNA,respectively.Then,PCR was performed Real-time fluorescence quantitative PCR(q RT-PCR)was used to verify the expression patterns of 8 miRNAs.The results laid a data basis for the functional identification of miRNAs in mulberry and the study of molecular biology,and further conducted a preliminary exploration for the molecular mechanism of colchicine induction and regulation in mulberry.2.Cloning and prokaryotic expression of MmKIN-14 N gene from mulberryThe MmKIN-14 N gene was cloned from Morus multicaulis Yu71-1.The length is 2286 bp,including a complete ORF of 2286 bp,which can encode a protein of 761 amino acids.The molecular weight of the protein is 86.41 k Da and the isoelectric point is 5.86.Gen Bank NO: MZ343300.The amino acid sequence conservative domain prediction on NCBI shows that the amino acid series encoded by the MmKIN-14 N gene belongs to the Motor＿superfamily superfamily.After optimization,MmKIN-14 N gene of mulberry was expressed in prokaryotic cells.The protein was detected by SDS-PAGE and Western blot test.The experimental results showed that MmKIN-14 N gene of mulberry was successfully expressed by IPTG induction,which provided a theoretical basis for studying the protein function of MmKIN-14 N gene of mulberry.3.Cloning and subcellular colocalization of Morus alba homologous protein-leucine zipper protein ATHB-40 geneThe MmKIN-14 N gene was cloned from Morus multicaulis Yu71-1,with a length of630 bp,including a complete ORF630 bp,which can encode a protein of 209 amino acids.The protein has a molecular weight of 24.09 k Da and an isoelectric point of 7.57.Gen Bank NO: MZ343299.The conservative domain prediction of amino acid sequence on NCBI showed that the amino acid series encoded by the MmKIN-14 N gene belong to the homeodomain superfamily and COG5576 superfamily superfamily.The MmKIN-14 N gene was subjected to a subcellular localization experiment in Arabidopsis,and the expression intensity of the target gene-GFP was compared with the GFP control.It is speculated that its luminescence is located in the nucleus.4.Transient transformation and overexpression of mulberry miRNA novel＿77 to identify its response function induced by colchicineThe potential novel precursor sequence pre-novel＿77 of the new miRNA novel＿77 was obtained,and its secondary structure was obtained using MFOLD software to construct the transient transformation system of miRNA novel＿77.After the transient overexpression of miRNA novel＿77 in mulberaceae,miRNA was obtained.The expression levels of miRNA novel＿77 and its corresponding target genes show an opposite trend,suggesting that miRNA novel＿77 is negatively regulated with the expression levels of target genes.