Development Of MiRNA-SSR Markers And Functional Study Of MiR397 In Brassica Juncea

Brassica juncea belongs to Brassica genus of the cruciferous family.It is widely cultivated all over the world as an oil or vegetable crop.It has the characteristics of enriching a variety of heavy metals.MicroRNA(miRNA)is a type of small non-coding RNA ubiquitous in eukaryotes,which plays an important regulatory role in plant growth and development and response to adversity stress.In this study,a miRNA-S SR marker was developed based on B.juncea miRNA sequence data,molecular evolution analysis,expression analysis,and target gene-wide analysis of miR397 were performed,and an overexpression vector of miR397 was constructed to transform Arabidopsis thaliana.The main findings are as follows:1.Development of miRNA-SSR markers in B.juncea.Using MISA program to search the mustard miRNA sequence and transcriptome sequence.279 and 11 SSR sites were found in the mustard pri-miRNA and pre-miRNA,respectively,and 32,987 SSR sites were found in 115228 unigenes.These SSRs are mainly single nucleotide repeats,and the length of the SSRs is between 12-19 bp.The Primer3.0 program was used to develop 30513 pairs of SSR primers,26 pairs of which were randomly selected for verification,of which 6 pairs were polymorphic in 11 mustard varieties.The above results indicate that the development of SSR molecular markers based on miRNA sequences is an effective way to develop SSR markers in mustard.2.Analysis of bioinformatics and expression characteristics of miR397 in B.juncea.A total of 143 miR397 sequences from 68 plants were downloaded from the miRBase database,and multiple sequence alignments were performed to construct a phylogenetic tree and draw a conservative Logo map of mature sequences.The results showed that B.juncea miR397 is evolutionarily closely related to Arabidopsis miR397,and most of the bases in the mature sequence are very conserved.The prediction results of psRNATarget software show that miR397 target genes are mainly laccase genes.The miR397 promoter region of B.juncea has abscisic acid,salicylic acid,methyl jasmonic acid,anaerobic induction,rhythm regulation and other elements.The results of qRT-PCR showed that the expression level of miR397 in the roots of B.juncea seedlings was higher than that in the leaves,and the expression level of miR397 increased significantly under the conditions of Cd treatment,indicating that miR397 was involved in the response of mustard to cadmium stress.3.Genome-wide identification of the miR397 target gene LAC gene in B.juncea.BlastP comparison of Arabidopsis LAC gene family and B.juncea transcript data,and combined with protein structure domain analysis identified B.juncea 57 LAC genes.The analysis of physical and chemical properties showed that most of them were hydrophilic and stable basic proteins with an average molecular weight of 66.94 kD.Phylogenetic tree analysis showed that these genes could be clustered into 7 subfamilies.B.juncea LAC protein is located on the plasma membrane and various organelle membranes.Using the MEME program to analyze the protein conserved motifs,it was found that the B.juncea LAC protein family contained 1-19 motifs;GSDS analysis showed that the number of introns in the B.juncea LAC gene ranged from 4 to 15.The B.juncea LAC promoter region contains multiple cis-acting elements such as ABRE,ARE,and ERE that are related to plant growth and development and stress response.4.Construction of overexpression vector of MIR397 gene in B.juncea and its transformation into Arabidopsis thaliana.The MIR397 gene was cloned from the B.juncea genome and ligated to pCAMBIA1300 to construct an overexpression vector,which was then transformed into A.thaliana using agrobacterium-mediated transformation.Transgenic plants were obtained through hygromycin resistance screening and PCR detection,and homozygous overexpression lines were obtained through self-passage,which laid the foundation for further research on the function of B.juncea miR397.The results showed that overexpression of miR397 improved the phenotype of Arabidopsis root length and the number of fruit pods;within a certain cadmium concentration range,overexpression of miR397 improved cadmium tolerance of A.thaliana.These findings provide a foundation for further disclosing the function of miR397 in B.juncea.