Cloning And Functional Verification Of Cold Resistance IlMYB97 Gene From Iris Laevigata

In the Northeast of China,cold is the main factor affecting the promotion and application of most garden plants,and such low temperatures have important effects on the growth and development,trait performance and even survival of exotic plants,so research related to plant cold tolerance mechanisms and cold tolerance breeding is an important research content in the field of landscape plant applications.As one of the largest transcription factor families in plants,MYB transcription factors play an important role in plant growth and development,stress response and other aspects.As one of the northernmost hostas in China,Iris laevigata is a valuable plant material for studying the cold resistance of flowers.In prior work,we discovered a MYB transcription factor of the R2R3 type called IlMYB97（OP484854）,which may be connected to the cold response.In this study,we cloned this gene and,in combination,transformed it into model plants to confirm its functionality.We discovered that the IlMYB97gene has transcriptional activation activity,which might enhance plant cold tolerance and photosynthetic features.The findings of this study serve as a guide for the creation and use of IlMYB97 as a genetic resource for cold tolerance breeding and as a theoretical foundation for the investigation of cold tolerance in iris plants.These findings are summarized as follows:（1）The sequence of IlMYB97 gene was obtained from transcriptome,cloned and sequenced,and analyzed by online bioinformatics:the ORF region of the gene is 1251 bp long,encoding 416 amino acids,the molecular weight of the protein is 45.68 k Da,the theoretical isoelectric point is 6.23,which is an unstable protein;the lipolysis index is 20.38,the total average hydrophilic value is-0.688,which is a hydrophilic protein;there is no transmembrane structure and signal peptide.There is no transmembrane structure or signal peptide;α-helix and irregular coiling are the main components in the secondary structure of the protein;the protein is predominantly serine phosphorylated;subcellular localization predicts that the protein is localized in the nucleus.IlMYB97 protein is related to the MYB protein of the monocotyledonous plant Dioscorea cayenensis subsp.rotundata nearest;using Arabidopsis thaliana as a reference,protein interaction function prediction showed that this protein may be related to pollen tube development,plant immune response,flower organ development,and regulation of biotic and abiotic stresses.（2）The plant overexpression vector pCAMBIA1300-IlMYB97-GFP was successfully constructed and heterologously transformed into tobacco and arabidopsis by leaf disc method and inflorescence infestation method respectively,and three strains of highly expressed T₂transgenic Nicotiana tabacum and two strains of T₂ transgenic Arabidopsis thaliana were successfully identified by PCR,and the yeast expression vector pGBKT7-IlMYB97 was constructed,and the protein was found to have transcriptional activation activity by transcriptional activation assay,and the transcriptional activation domain was located at the C-terminus of the protein.（3）The IlMYB97 gene of I.laevigata can improve the cold resistance of plants.The germination and growth of transgenic tobacco seeds and control treated with-20℃ were better than wild type and no-load.The growth status of transgenic tobacco plants under 4℃ and-2℃ stress and transgenic arabidopsis plants under-5℃ treatment were stronger than the control.Under stress,trans-IlMYB97 gene tobacco had lower MDA,relative conductivity,and less membrane system injury than wild-type transgenic tobacco;having a higher content of osmoregulatory substances like proline and soluble sugar could better maintain the osmotic pressure inside and outside the cell,making transgenic tobacco have stronger osmoregulatory ability;and transgenic tobacco’s antioxidant enzymes SOD,POD,and CAT activities were higher.As a result,it can scavenge reactive oxygen species and minimize oxidative damage.The root conformation of transgenic tobacco was considerably different from that of the control after cold induction at 4℃,suggesting the gene may also be involved in root development.（4）The growth characteristics and photosynthetic physiological assays of the IlMYB97-transformed tobacco and the control showed that the plant height of the transgenic tobacco was slightly shorter than that of the control,the leaves tended to grow upward,and the pollen germination was not significantly different from that of the control.The trans-IlMYB97 gene tobacco had increased chlorophyll content,net photosynthetic rate,stomatal conductance,transpiration rate,and non-photochemical quenching coefficient NPQ than the control.The IlMYB97 gene may also promote transgenic tobacco adaptation to low temperature and other stress-resistant settings via plant photosynthesis and photoprotective response.