Analysis Of Calcium Stress Transcriptome And MiRNA Of Mulberry And Functional Identification Of Related Genes

Calcium（Ca）is an essential element in plant growth and development.Not only as an essential element,providing and storing nutrients for plants;At the same time,as the"second messenger",it regulates the physiological and metabolic activities in the body to ensure the normal growth of plants.The great difference of calcium content in soil and the degree of absorption of calcium by plants have a great influence on the growth and development of plants and their physiological activities.In this experiment,the results of transcriptome sequencing and smallRNA sequencing under different concentrations of calcium stress were analyzed by using mulberry as the research object.The differentially expressed genes were cloned,microRNA and target genes were identified and analyzed.The results of this study are helpful to elucidate the efficient utilization of calcium in plants,and provide a certain molecular mechanism and theoretical basis for plants to resist calcium stress.The results are as follows:1.Physiological and biochemical indexes detection,sequencing data analysis and differential gene expression identification of calcium stress in mulberryMulberry seedlings were treated with 4 concentrations of CaCl₂·2H₂O（MS liquid medium containing 0 mg/L,220 mg/L,440 mg/L and 660 mg/L CaCl₂·2H₂O,440 mg/L CaCl₂·2H₂O as the control group and the other three groups as the experimental group）,and each group had three replications.The physiological and biochemical indexes were determined as follows:soluble protein（Cpr）,superoxide dismutase（SOD）,peroxidase（POD）,malondialdehyde（MDA）and proline（PRO）.The results showed that the five indexes all had different variation trends,which proved that calcium had an effect on the growth and development of mulberry.Four mulberry leaf samples under calcium stress were selected for transcriptome sequencing and smallRNA sequencing.In the transcriptome sequencing results,the available clean reads obtained after a series of treatments such as filtration and GC content distribution detection were 94.89 G,in which the number of Q20and Q30 bases in each sample accounted for more than 92.44%.Twelve genes with great differences were selected from the differentially expressed genes of the transcriptome for q RT-PCR to verify the reliability of transcriptome data.By GO annotation,it was found that the differential genes were enriched in biological process（BP）,molecular function（MF）and cellular component（CC）.In KEGG pathway,the three groups of different gene comparison combinations were mainly concentrated in the ribosome,photosynthesis,proteasome and other metabolic pathways.In the analysis of smallRNA sequencing,a total of 8.208 G of original data were obtained from the three comparison combinations.53 known miRNAs and 61 precursor hairpin structures were identified.In addition,the library predicted 42 novel miRNAs and 51 novel hairpin structures.A total of 36 differential miRNAs were identified in the three comparison combinations.The 7 differentially expressed miRNAs were selected for q RT-PCR verification,and all the 7 miRNAs were significantly expressed,which was consistent with the sequencing results.GO annotation and KEGG pathway analysis were performed on candidate target genes.In GO annotation,the target genes of 0vs440and 660vs440 were enriched in molecular function（MF）.The candidate target genes of 220vs440were more concentrated in biological process（BP）and cellular component（CC）.In KEGG pathway,many target genes were enriched in starch and sucrose metabolic pathways,α-linolenic acid metabolic pathways and glycerolipid metabolic pathways.2.Cloning and expression of transcription factor MYB30 geneMmMYB30 gene was cloned from mulberry（Gen Bank:MZ343300）.The length of cds region was 867 bp,encoding 288 amino acids,the predicted protein weight was 32.53 k D and the isoelectric point was 6.47,belonging to the MYB transcription factor family.The tertiary structure of the species was predicted and the homology and relationship between the species and other plant species were analyzed by bioinformatics method.Quantitative analysis showed that the expression of MmMYB30gene was increased under calcium stress.MmMYB30 protein was successfully expressed in Escherichia coli using SDS-PAGE technique.3.Cloning and functional verification of calmodulin CML37 geneThe cds region of MmCML37 gene（Gen Bank:MZ343301）was 435 bp and encoded 144 amino acids.The molecular weight of MmCML37 protein was predicted to be 16.36 k D and its isoelectric point was 4.53.It belonged to the Calmodulin-like Family.Analysis of its homology and kinship with other species.Quantitative results showed that the expression of MmCML37 gene was up-regulated under calcium stress.Silencing MmCML37 gene in mulberry by VIGS technique.The MDA content,PRO content,POD activity,SOD activity and soluble protein（Cpr）content in mulberry leaves changed in different degrees under calcium stress.The results showed that the silencing of MmCML37 gene could cause changes in the activities of enzymes related to calcium stress in mulberry seedlings,which could affect the response of mulberry seedlings to external calcium stress.It further indicated that MmCML37 gene had a positive effect on the stress of mulberry seedlings.4.Transient overexpression of miR156g in mulberry was used to identify its calcium stress responseAfter the precursor sequence of miR156g was obtained,the secondary stem loop structure was predicted and the mature sequence was found to be located on the 5’arm.The transient transformation system of miR156g was constructed.After the transient overexpression of miR156g in mulberry seedlings,the expression levels of miR156g and its corresponding target genes were opposited,suggesting that miR156g plays an important role in mulberry seedlings’response to calcium stress.