Cloning And Primary Functional Analysis On Stress Resistance Of IlMYB306 Gene In Iris Laevigata

This study is the National Nature Science Fund(31670344)and Central University Innovation team Project(No.2572016EAJ6).As a kind of aquatic flower with strong cold resistance in the north,Iris laevigata has high ornamental and application value.At present,research on resistance molecules such as cold resistance of I.laevigata is rarely involved.The MYBs gene family not only participates in the secondary metabolic reactions of plants,but also plays a huge role in responding to external abiotic stresses.In this study,I.laevigata was taken as the main research object.Based on the existing transcriptome data,the MYBs gene family of I.laevigata was analyzed,and the resistance gene IIMYB306 was screened and cloned to detect the expression of IlMYB306 gene under abiotic stress.Transferred to the model plant tobacco and its expression characteristics were studied.The main results are as follows:1.A total of 53 MYB transcription factor sequences were obtained from I.laevigata transcriptome data.According to the domain type classification,6 belong to the 1R/4R subfamily,3 belong to the 3R subfamily,20 belong to the R2R3 subfamily,and 24 belong to the MYB related protein.The sequence of MYB gene of I.laevigata is highly conserved,and the consenved sequences of MYB gene in the same subfamily are consistent.Subcellular localization predictions showed that most of the I.laevigata MYB transcription factors act on the nucleus.C37880.graph_c0 is localized to the cell membrane and nucleus,c42108.graph_c2 is localized to the chloroplast and nucleus,and c45677.graph_c0 is localized to the cell membrane and chloroplast.The motif composition of the R2R3-MYB protein sequence was analyzed by MEME online software,and five motifs were identified.A systematic comparison of the R2R3-MYB transcription factors of I.laevigata and Arabidopsis thaliana showed that the R2R3-MYB transcription factor of I.laevigata not only participates in the response of plants to abiotic stress,but also participates in other secondary metabolic processes such as anthocyanin synthesis.The abiotic stress-related R2R3-MYB transcription factor was screened and named IlMYB306.2.The target gene IlMYB306 was obtained by PCR amplification using the cDNA of I.laevigata root and leaf as template.The gene is 1024 bp in length and has an ORF of 906 bp and encodes 301 amino acids.Bioinformatics analysis showed that IlMYB306 has a molecular weight of 33.54kDa and a theoretical isoelectric point of 6.51,which is an unstable hydrophilic protein.There are two conserved domains,subcellular localization is predicted in the nucleus;no transmembrane structure,no signal Peptide sequence;contains 5 glycosylation sites,3 potential serine phosphorylation sites,8 potential threonine phosphorylation sites,5 potential tyrosine phosphorylation sites;protein secondary structure It is shown that IlMYB306 consists of 37.54%?-helix,5.56%fold extension,3.32%?-turn and 53.49%random curl,and the protein tertiary structure is predicted to be consistent with the secondary structure.By homology analysis with the MYB306 protein of different species in the NCBI database,it was found to have high homology with the MYB306 protein of Asparagus officinalis.It is speculated that this gene has a transcriptional regulatory function in response to abiotics.Stress,involvement in cell differentiation,and involvement in abscisic acid and jasmonic acid signaling also play a similar role.3.The expression of IlMYB306 gene in roots and leaves of seedlings was detected by QRT-PCR when induced by 4? low temperature and different stress levels of Nacl and PEG The results showed that the expression of IlMYB306 gene in I.laevigata was changed compared with the control group under three stresses,which fully proved that the gene was involved in the stress response of I.laevigata.Under the stress,the expression level of IlMYB306 gene in roots was higher than that in leaves;the overall expression under cold stress was higher than that under drought stress and salt stress,and it was up-regulated under cold stress;the expression increased under the initial drought stress.However,with the increase of stress time,the expression of this gene was down-regulated.Under salt stress,the expression of this gene was down-regulated in leaves,and the expression of the root in the early stage of stress was lower and the downward trend was observed with the increase of stress.It is speculated that the gene has anti-cold function and does not have drought and salt resistance.4.The pBI121-IlMYB306-GFP plant expression vector was constructed and transferred into wild type tobacco by Agrobacterium-mediated transformation.The transgenic tobacco seedlings were obtained by Kana resistance screening and PCR identification.The expression of the target gene in transgenic tobacco was detected by real-time fluorescence quantification,which proved that the expression level of the foreign gene in transgenic tobacco was significantly higher than that in the wild type.The protoplasts of transgenic tobacco were extracted and observed by laser confocal microscopy.The IlMYB306 protern was found to act on the nucleus.