Mulberry Transcriptome,miRNA Analysis Under Cadmium Stress And Gene Function Identification

Nowadays,soil heavy metal pollution has become a serious soil pollution problem.If the soil contains excessive heavy metal cadmium,it will affect the normal growth of plants.Mulberry is tolerant to bad soil and hash environmental conditions,and the use of mulberry to improve the environment can reduce the cost of environmental governance.This paper analyzed the physiological and biochemical indexes,transcriptome and miRNA differences of mulberry under the stress of heavy metal cadmium.In this paper,the physiological and biochemical indexes,transcriptome,and miRNA differences of mulberry trees under heavy metal cadmium stress were analyzed,and genes related to cadmium stress were cloned and functionally identified.The research results are helpful to further elucidate the molecular mechanism of high-efficiency cadmium resistance in plants and provide a theoretical basis for breeding new varieties with high cadmium tolerance.The main research contents are as follows: 1.The physiological and biochemical indexes of young leaves and root of mulberry under cadmium stress were determined.The content of BCA,SOD,POD,Pro and MDA in mulberry changed under cadmium stress,which suggested that cadmium stress affected the normal physiological growth of mulberry.The differentially expressed genes(DEGs)in the young leaves and roots of mulberry under normal growth and cadmium stress were determined by transcriptomic analysis.Among them,1291 differentially expressed genes were found in mulberry leaves,712 of which were up-regulated and 579 were down regulated.A total of 3411 differentially expressed genes were found in mulberry roots,2735 up-regulated and 676 down regulated.GO annotation,KEGG enrichment and other bioinformatics analysis were carried out for DEGs in the young leaves and roots of mulberry and the expression levels of some genes in the leaves and roots of mulberry were detected to verify the transcriptome results.After transcriptome analysis,it was found that the genes related to the ribosome were most regulated.In addition,it was found that the expression levels of calcium-dependent protein kinase(CDPK26)gene and cyclic nucleotide gated ion channel(CNGC14)gene involved in mulberry signal transduction were up-regulated.2.Analysis of miRNA sequencing results of mulberryThrough the analysis of miRNA sequencing,18 miRNAs with different expression levels were found in mulberry leaves,among which 5 miRNAs were up regulated under cadmium stress,and 13 miRNAs were down regulated under cadmium stress.27 miRNAs were found to be differentially expressed in mulberry roots,of which 16 were up regulated by cadmium stress and 11 were down regulated.Based on this results,we analyzed the expression level of differential miRNA,annotated the target genes related to differential miRNA,enriched the function and other bioinformatics analysis,The expression level of differential miRNA in mulberry leaves and mulberry roots was validated and analyzed.Under cadmium stress,the expression of miRNA in mulberry trees regulating metabolic pathways is quite different.3.Cloning,expression analysis and functional identification of calcium dependent protein kinase(Ma CDPK26)gene form mulberryThe Ma CDPK26 gene was cloned from mulberry.The ORF length of the gene is 1826 bp.The gene encodes 607 amino acids.It's molecular weight and isoelectric point are 68.107 KD and 5.41 respectively(Gen Bank NO:MT431589).The function of Ma CDPK26 gene was predicted by three dimensional structure prediction analysis.Multiple sequence alignment of amino acids encoded by Ma CDPK26 and other homologous species was performed and subsequently phylogenetic tree was constructed to know its evolutionary relationship.The quantitative analysis showed that the expression of Ma CDPK26 gene increased under cadmium stress.Ma CDPK26 gene of mulberry can be expressed in E.coli.Using VIGS system,Ma CDPK26 gene in mulberry was silenced.The results showed that the expression level of Ma CDPK26 gene decreased significantly in the transgenic plants than the control group,this suggested that the gene was successfully silenced.The results showed that the physiological and biochemical indexes of mulberry samples in the experimental group and the control group were significantly different,while the data of the two groups were basically the same.Our findings showed that the expression of Ma CDPK26 gene could improve mulberry tolerance to cadmium stress.4.Cloning,expression analysis and functional identification of cyclic nucleotide gated ion channel(Ma CNGC14)gene form mulberryThe Ma CNGC14 gene was cloned from mulberry.The ORF length of the gene is 2235 bp.It encodes 744 amino acids.Its molecular weight is 85.422 kd and its isoelectric point is 8.99(Gen Bank NO:MT431588).Through homology comparison,it was found that the difference of Ma CNGC14 gene among different species was small and its conservation was high.The expression of Ma CNGC14 gene increased under cadmium stress.Using the VIGS system,we silenced as the Ma CNGC14 gene in mulberry.The results showed that the expression level of Ma CNGC14 gene in the transgenic plants which successfully silenced the gene decreased significantly.Our finding showed that the physiological and biochemical indexes of mulberry samples in the experimental group and the control group were significantly different,while the data of the two groups were basically the same.The results showed that the expression of Ma CNGC14 gene could improve the cadmium resistance of mulberry.