| Micro RNA(miRNA)is a single-stranded small molecule RNA encoded by an endogenous gene in a living body.The mature miRNA is 21 nt to 22 nt in length.It can bind to a target gene in vivo,and the target gene can be cleaved or translation can be suppressed.Functional regulation,and its widespread presence in eukaryotes,regulates a variety of important biological pathways,such as growth and development,hormone response,and virus induction.In recent years,abiotic stressful events have frequently occurred in China and even in the world,which has brought great pressure on the production of crops.Non-biological adverse conditions include high-temperature cold damage,drought,and salt stress.With the deepening of research,it has been found that miRNAs also play an important role in plant abiotic stress.Mi RNAs regulate their tolerance by binding to target genes involved in plant abiotic stresses to inhibit their translation or cleavage of target genes.Expression,for the purpose of analyzing and verifying the function of small RNAs,can be achieved by constructing small RNA vectors for genetic transformation.In addition,the HSP70 protein family can help plants to deal with adverse environmental impacts.When exposed to unfavorable environmental stimuli,HSP70 induces large amounts of protein and helps plants respond to environmental stimuli.The main research object of this experiment was to construct miR311 genetic transformation system through the high-throughput sequencing technology in the novel miRNA-miR311 found in the stem tip tissues of Brassica napus.Through the phenotype identification of transgenic plants,the biological function was analyzed:1.Through analysis of the minimum folding free energy and secondary structure,it was found that it can form a stable stem-loop structure,which conforms to the structural characteristics of miRNA and confirms its existence reliability.2.Sequence comparison analysis of 10 novel miR311 target genes identified by sequencing of the degradation group revealed that five target genes belong to the structural heat shock protein HCP family and three target genes are the inducible heat shock protein HSP family.Two target genes belong to the transduction protein gene.The5’RACE technique was used to verify that two of the target genes(Bna Cnng03470 D and Bna A10g27060D)were indeed able to be cleaved in vivo.The interaction of the target gene with the miRNA further confirmed that novel miR311 was actually present in this study.To further clarify whether novel miR311 can affect plant response to abiotic stresses through the cleavage of the target gene HSP70-1,this study constructed a novel overexpression vector of novel miR311 to verify overexpression and to Positive Arabidopsis plants were subjected to high temperature,salt and other environmental treatments respectively.The results showed that novel miR311 was mainly expressed in roots and leaves and was relatively less expressed in flowers,stems and pods.Under high temperature stress,the appearance of positive and wild-type plants was not significantly different,but the target genes were The quantitative results showed that after high temperature treatment,the wild-type HSP70-1 levels increased significantly,while the positive strain HSP70-1 did not change much,and then the in vitro water loss rate experiment was done.The results showed that the high-temperature wild type plants have strong water retention.In positive strains,under salt stress,the growth of wild-type and positive-type plants was relatively weak,with little difference.The target gene quantitative results showed that the expression level in wild-type was higher than that in positive strains,but the wild-type plants did not show a ratio.Positive strains are more tolerant to stress. |
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