Cloning And Functional Identification Of MYB4a Transcription Factor In Tobacco

Tobacco(Nicotiana tabacum)is one of the important economic crops in China.The production of high-quality tobacco is of great significance to improve the national economy.The types and contents of phenols are closely related to the quality of tobacco leaves.The production of phenols mainly comes from phenylpropane metabolism pathway,which is an important secondary metabolism pathway in plants.It also involves the production of secondary metabolites such as flavonoids,lignin and stilbenes.These secondary metabolites play an important role in improving the quality of plants and resisting external stress.MYB transcription factors play an important role in the secondary metabolism of phenylpropane,response to stress and other physiological activities.Therefore,it is of great significance to clone MYB transcription factors which related to phenylpropane secondary metabolism,and to study its regulatory mechanism for improving tobacco quality and stress resistance.In this study,the EST sequence of MYB transcription factor differentially expressed in a high-quality tobacco hybrid and its parents was used as the motif,and the full length of MYB c DNA was cloned by RACE technology,and its sequence structure and subcellular location were analyzed;the overexpression vector and CRISPR/Cas9 vector were constructed,and the transgenic plants were obtained through genetic transformation to identify the MYB transcription factor in phenylpropanoid on the regulatory functions of alkanes synthesis and response to abiotic stress,providing basis and gene resources for in-depth study of the molecular regulatory mechanism of the gene and the cultivation of high-quality and multi-resistant tobacco varieties.The main research results are as follows:1.Based on the EST sequence of a MYB transcription factor differentially expressed in high-quality tobacco hybrids and their parents,the MYB gene was cloned from tobacco leaves by RACE technology and named Nt MYB4a(Gen bank accession number: MN178131).The total length of the c DNA of the gene is 816 bp,encoding 271 amino acids.It has two typical MYB domains and 35 phosphorylation sites.The subcellular is located in the nucleus,cell membrane and cytoplasm.The amino acid sequence of Nt MYB4 a has the highest homology with the MYB4 protein predicted by Nicotiana sylvestris(XP?009798928.1),which is 99.64%,and the homology with the MYB4 protein determined by Nicotiana attenuata(OIT38711.1),which is 77.69%.Phylogenetic tree analysis showed that Nt MYB4 a was closely related to wild tobacco MYB4,and was clustered into a class of transcription activating factors such as Eriobotrya japonica Ej MYB1 and Arabidopsis thaliana At MYB14.2.In order to identify the function of Nt MYB4 a,we constructed Nt MYB4 a overexpression vector and CRISPR/Cas9 vector.The results showed that compared with wild-type tobacco,Nt MYB4 a was over expressed in over expressed plants,and the expression of PAL,C4 H,4CL,DFR and ANS genes related to anthocyanin synthesis was up-regulated,the anthocyanin content increased by 65%,and the color deepened;Nt MYB4 a was down regulated in knockout mutant,the anthocyanin related enzyme gene was down regulated,the anthocyanin content decreased by 55%,and the color became lighter.The content of phenolics in over expressed plants was higher than that in wild type,and the content of chlorogenic acid in stems,leaves and flowers was 1.24,2.71 and 2.15 times higher than that in wild type,respectively.It was found that the expression of PAL,C4 H,4CL and HCT genes involved in chlorogenic acid synthesis pathway was up-regulated in the over expressed plant stems,leaves and flowers,among which HCT gene was up-regulated the most,while it was down regulated in the knocked out plant stems,leaves and flowers,indicating that Nt MYB4 a may promote the synthesis of chlorogenic acid by regulating the expression of chlorogenic acid synthesis related enzymes.3.The results showed that Nt MYB4 a was expressed in different tissues of tobacco at different development stages.Before flowering stage,Nt MYB4 a was mainly expressed in roots,while in mature stage,it was mainly expressed in middle leaves.It was found that the content of chlorogenic acid was the highest in seedling stage and higher in root stage.With the development of growth stage,the content of chlorogenic acid began to decline,while in mature stage,the content of chlorogenic acid was higher in middle leaf and less in root.The correlation analysis showed that there was a positive correlation between Nt MYB4 a and chlorogenic acid content,indicating that Nt MYB4 a may have a positive regulatory effect on the synthesis of chlorogenic acid in tobacco.4.The expression of Nt MYB4 a was induced by low temperature(4 ?),PEG,Me JA,Na Cl,ABA and dark treatment,but the expression pattern was different due to different stress types.Theexpression of Nt MYB4 a was significantly up-regulated after the induction of low temperature,Na Cl,PEG,Me JA and dark,among which the induction of low temperature and Me JA was the most obvious;however,the expression of Nt MYB4 a did not change significantly under ABA stress,suggesting that the transcription regulation of Nt MYB4 a may not depend on the ABA signaling pathway.Under the stress of low temperature,PEG and Me JA,the expression of Nt MYB4 a in overexpressed plants was less affected,the proline content was higher,the malondialdehyde content was lower,but the expression of Nt MYB4 a in knockout plants fluctuated greatly,sometimes significantly higher,sometimes lower than that in wild type,the proline content was lower,the malondialdehyde content was higher.These results indicate that Nt MYB4 a is a response gene to abiotic stresses such as peg,low temperature and Me JA.