CsMYB Transcription Factors Participate Jasmonic Acid Signalling Transduction In Response To Cold Stress In Tea Plant(Camellia Sinensis)

The freezing caused by low-temperature in spring not only affect the growth and development of the tea plants(Camellia sinensis),but also lead to a decline in yield production and quality,which seriously affects economic benefits,and has become a prominent problem in tea industries.Therefore,it is significant to deeply understand the cold resistance mechanism of tea plants to effectively alleviate the damage caused by low temperature.Transcription factors(TFs)plays a key role in a variety of physiological activities such as plant growth and stress response,and are a key link in regulating the expression of functional genes and the stress response system.The MYB transcription factor family is one of the largest transcription factor families in plants,and the main transcription factor of R2R3-MYB plays an important role in the process such as plant resisting low temperature and other stresses.Methyl Jasmonates(MeJA)is a kind of plant endogenous regulatory substance widely used in agricultural production,which could improve the tolerance of plants to cold stress by regulating a variety of physiological substances.The MYB transcription factors can also participate in a variety of jasmonic acid reaction processes,mainly by synthesizing anthocyanins and other substances to improve the cold resistance of plants.However,the study of MYB transcription factor in the transfer of jasmonic acid signal response at low temperature in tea plants has not been reported.Therefore,this study conducted single and combination treatments of low temperature stress and exogenous MeJA,and measured physiological indicators related to resistance,physiological indicators related to quality and endogenous hormone content.At the same time,three of the R2R3-MYB cold resistant genes were discoveries,and carried out functional identification to reveal the internal molecular mechanism between MYB transcription factor,MeJA and cold resistance.The main results are as follows:1.Using meta-analysis,the reported effects of overexpression of MYB genes among the 17 species on the response to low temperature stress,drought stress,and salt stress were quantitatively analyzed,and the variable parameters affecting the effect sizes of exogenous MYB gene were evaluated.The result showed that overexpression MYB transcription factor members reduced the REL(related electrolyte leakage)of plants in response to cold stress and the water loss of plants in response to drought sress.And increased the survival rate of plants in response to drought stress and physiological indicators such as germination rate,root length and fresh weight in response to salt stress.In addition,the analysis showed that the treatment medium,donor/recipient species,and donor type significantly influence the effects of MYB-overexpression on drought stress tolerance.And the donor/recipient species,donor type,and stress duration all significantly affected the extent of MYB-mediated salt stress tolerance.These findings help to study the role of MYB gene family in plant stress tolerance and the application of genetic engineering in plant stress tolerance breeding.2.Using ’Longjing 43’ as the material,sparying water under normal temperature conditions as a control,the contents of POD,CAT,SOD,PPO,C4 H,tea polyphenols(TPs)and endogenous jasmonic acid in the tea plants were significantly increased under the exogenous MeJA treatment.The cold stress treatment as a control,under the combination treatments of low temperature and exogenous MeJA,the content of anthocyanin,soluble sugar,soluble protein,REL,proline,POD,CAT,PAL and C4 H activities and endogenous jasmonic acid in tea plants was significantly increased,indicating that MeJA can effectively enhance the cold resistance of tea plants.3.Refer to the CsMYB gene expression datas in tea plant genome database under MeJA and cold stress treatments.Using ’Longjing 43’ as the material,the single and combination treatments of cold stress and exogenous MeJA were carried out respectively.The key CsMYB genes of CsMYB45,CsMYB46 and CsMYB105 that are responsive to MeJA and cold stress were screened by qRT-PCR analysis.4.Using Gateway reaction,the overexpression vectorsof 35S::CsMYB45-EGFP,35S::CsMYB46-EGFP and 35S::CsMYB105-EGFP were be constructed.Subcellular localization of tocacco epidermal cells showed that the three target genes were all located on the nucleus.The prokaryotic expression experiment found that exogenous MeJA treatment can improve the cold tolerance of E.coli overexpressing CsMYB45,CsMYB46 and CsMYB105.In addition,the CsMYB45,CsMYB46 and CsMYB105 genes were transfered into Arabidopsis thaliana by inflorescences disseminated method,and T2 generation plants were obtained after antibiotics screening.5.The yeast expression vectors of pGBKT7-CsMYB45,pGBKT7-CsMYB46 and pGBKT7-CsMYB105 were constructed.The results showed that only CsMYB45 protein has self-activation activity by yeast expression experiment.Through yeast two-hybrid test,it has been preliminarily determined that CsMYB46,CsMYB105 interact with the inhibitor JAZ protein in the jasmonic acid signaling pathway,CsJAZ3,CsJAZ10,and CsJAZ11,respectively.