| Copper(Cu)is not only a micronutrient for plant growth and development,but also a heavy metal element that pollutes the environment.In plants,Cu can be used as a cofactor of enzymes to promote the activity of related enzymes,so it plays an important role in the normal physiological metabolism,growth and development of plants.Due to the continuous use of copper-containing pesticides,the copper concentration in the field,field and orchard soil increases.In order to adapt to the changing copper ion content in the soil,plants must strictly control the transport of copper ion at the overall level and cell level.This experiment by mulberry as the research object,combined with the related physiological and biochemical index determination,differences in gene expression level appraisal,mi RNA expression of target gene regulation,appraisal of copper processing related transfer genes and some important mi RNA and its target gene expression analysis,in order to further illustrates the molecular mechanism of plant efficient absorption use copper,copper cultivate efficient use of new varieties has a certain theoretical and practical significance.The main research contents are as follows:1.Detection of physiological and biochemical indexes,analysis of sequencing data and expression identification of differential genes in mulberry under copper stressUnder copper stress,the expression levels of MDA,SOD,POD,BCA and PRO in mulberry were measured.Raw and copper-treated mulberry samples were selected for transcriptome and small RNA sequencing analysis using sequencing technology provided by Beijing nuohe zhiyuan technology company.Two transcriptome libraries were constructed,and 56,701,537 and 53,546,796 Clean reads were obtained by filtering the original sequences,with the base percentages of Q20 and Q30 above 91%.Based on the comparison results,variable shear analysis,prediction of new genes,analysis of gene structure optimization,and analysis of gene expression volume were carried out to screen out 5486 different genes in mulberry under normal growth and copper stress,among which 3078 genes were up-regulated and 2408 genes were down-regulated.Fifteen different genes were selected for expression verification and analysis,and the results showed that the relative expression levels of genes such as 21402441,21395828 and 21405820 were significantly different,which was basically consistent with the sequencing results.Two small RNA libraries were constructed,and the number of Clean reads and its proportion in the number of raw reads were 14,483,482(94.11%)and 11,598,984(97.25%).Based on the comparison results,conservative mi RNA analysis,Novel mi RNA prediction,mi RNA expression and differential analysis and prediction of mi RNA target genes were performed,and a total of 65 known mi RNA and 78 predicted mature mi RNA were identified,of which 40 mi RNA were differentially expressed under copper stress,including 27up-regulated mi RNA expression genes and 13 down-regulated mi RNA expression genes.The expression of 14 differentially expressed genes in mulberry was verified under untreated and copper treatment,and 11 mi RNAs(Novel-2,Novel-18,Novel-23,Novel-24,mi R156 a,mi R166 n,mir168a-3p,mi R171 e,mi R396 a,mi R398 b,mi R399f)were identified as significant differentially expressed by high throughput sequencing and q RT-PCR.2.Cloning,expression analysis and functional verification of ZIP4 gene of mulberry copper transporterMaZIP4 gene was cloned from mulberry with a sequence length of 1405 bp,containing a complete open reading frame of 1152 bp,encoding 383 amino acid residues,and was predicted to have a protein molecular mass of 95.32 k D and an isoelectric point of 5.02,belonging to the ZIP(ZIP,irt-like proteins)transporter family.Bioinformatics was used to analyze its homology with different plant species and evolutionary tree.The results of qrt-pcr showed that compared with untreated MaZIP4 gene,the expression regulation level of MaZIP4 gene would change in different degrees under different copper concentration stress.The VIGS system was constructed,and the MaZIP4 gene of mulberry was silenced successfully by using VIGS technology.After silencing MaZIP4 gene in mulberry,malondialdehyde(MDA)content,proline(PRO)content,superoxide dismutase(SOD)activity,peroxidase(POD)activity and soluble protein(BCA)content in leaves all showed different changes and fluctuations with the concentration of copper ion stress.The results showed that the MaZIP4 gene silencing of mulberry could cause the change of enzyme activity related to copper stress in mulberry,thus affecting the regulation of copper ions in mulberry.It further indicated that MaZIP4 gene played an important role in the regulation of copper stress in mulberry.3.Cloning,expression analysis and functional verification of mulberry copper transporter HMA2 geneThe MaHMA2 gene was cloned from mulberry with a sequence length of 1342 bp,acomplete open reading frame of 1194 bp,encoding 397 amino acid residues,and a predicted protein molecular mass of 42.852 k D and an isoelectric point of 7.53,belonging to the p-type ATPase transporter family.Bioinformatics was used to analyze its homology with different plant species and evolutionary tree.The results of qrt-pcr showed that,compared with untreated MaHMA2 gene,the expression regulation level of MaHMA2 gene under the stress of different copper concentrations showed different variation trends.The VIGS system was constructed,and the gene of mulberry MaHMA2 was silenced successfully by using VIGS technology.After MaHMA2 gene was silenced in mulberry,MDA content,PRO content,SOD activity,POD activity and soluble protein content in leaves all showed corresponding changes with the concentration of copper ion stress.The results showed that the silencing of MaHMA2 gene could cause the change of enzyme activity related to copper stress in mulberry,thus affecting the regulation of copper ions in mulberry,which further indicated that MaHMA2 gene played an active role in the response of mulberry to copper stress.4.Transient transformation overexpression of mulberry mi R396 a gene was used to identify its copper stress responseThe precursor sequence of mulberry mi R396 a was obtained,and the prediction of its secondary structure revealed that the mature mi R396 a was on the 3 'arm of the precursor.The transient transformation system of mi R396 a was constructed.After the transient overexpression of mi R396 a in mulberry trees,the expression levels of mi R396 a and its target genes were significantly different from those of the control group,and different copper stress concentrations would lead to different change trends.The results showed that mi R396 a plays an important role in copper stress regulation of mulberry. |
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