Analysis Of Molecular Mechanism Of Cold Tolerance And The Interaction Network Between MKK2,MAPK5 And ICE1 In Dajiao

Banana is a popular tropical fruit and a staple food for more than 500 million people in Africa.Low temperature is one of the main environmental factors that limit its growth and development,yield and quality and ecological distribution.In the production practice,it was found that the Dajiao,which belongs to the Musaceae Musa genus,like Cavendish banana,has strong cold resistance,and which is an ideal material for mining the coldresistant gene resources of banana for improving cold-resistant traits of bananas and cultivating new cold-resistant germplasm.The previous study of the research group showed that the MAPK cascade-ICE1 pathway,especially MKK2,MAPK5 and ICE1,played a critical role in Dajiao cold resistance,however,their interaction regulation mechanism is unclear.In order to further explore the molecular mechanism of cold tolerance difference of Dajiao,cross-linked mass spectrometry was used to identify the proteins interacting with Dajiao MKK2,MAPK5 and ICE1 in heterologous overexpressed Mp MKK2,Mp MAPK5 and Mp ICE1 rice lines under low temperature treatment,and combined with metabolomics and transcriptome methods to further analyze the molecular mechanism of cold tolerance difference between cold-sensitive Cavendish banana and cold-tolerant Dajiao at the cellular level.The main results are as follows:1.Using the rice genetic transformation system,rice lines with heterologous overexpression of GFP/m Cherry tag-Mp MKK2,Mp MAPK5 and Mp ICE1 were obtained for cross-linking mass spectrometry study.At the same time,the embryogenic cell suspension(ECS)of a new banana variety ‘Zhongjiao 1’(Musa spp.Cavendish;AAA Group cv.‘Zhongjiao 1’)was induced for histological study and creation of new banana germplasm.2.The total proteins of wild type and transgenic rice were extracted before and after low temperature treatment,and six differential proteins interacting with Dajiao ICE1-were identified by Co-IP cross-linking mass spectrometry,including ATP synthase subunit beta,Serine hydroxymethyl transferase,ATP-dependent zinc metalloprotease FTSH 2,ATP synthase subunit alpha,Photosystem II CP47 reaction center protein and Chlorophyll a-b binding protein 2;Six differential proteins interacting with Dajiao MAPK5-,including ATP synthase subunit beta,Serine hydroxymethyl transferase,ATPdependent zinc metalloprotease FTSH 2,Photosystem II CP47 reaction center protein,Chlorophyll a-b binding protein 2 and Transketolase;And one differential protein interacting with Dajiao MKK2-,Histone H4.This shows that ATP energy metabolism,photosystem and photorespiration play an important role in the cold tolerance of Dajiao.3.Through metabonomic analysis,it was found that there was a great difference in the response mode of lipids to low temperature between cold-sensitive Cavendish banana and cold-tolerant Dajiao.It was identified that a group of core metabolites were specifically up-regulated after 24 hours of cold stress.KEGG pathway enrichment analysis showed that these metabolites were mainly enriched in linoleic acid metabolism,α linoleic acid metabolism and unsaturated fatty acid biosynthesis pathway.In addition,it was found that there was no significant difference in ABA content and ABA-GE content between the two banana varieties under cold stress and 24 hours of cold treatment,and their content did not increase with the increase of cold stress time.This indicates that Dajiao does not participate in cold response through ABA-dependent signal transduction pathway.4.Through transcriptome analysis and real-time RT-q PCR verification of 18 cold response genes,including MKK2,MAPK5 and ICE1,the results showed that these 18 genes showed significant differences under cold stress.Under cold stress,the induced expression of MAPK signal pathway genes MEKK1,MKK2,MAPK5 and ICE1 and plantpathogen interaction(PP)pathway gene WRKY2/19/71 combined with the high expression of other cold response related genes may be one of the main reasons why Dajiao has better cold tolerance than Cavendish banana at the transcriptional level.To sum up,this study analyzed the molecular mechanism of the difference in cold tolerance of Cavendish(Dajiao)banana from three aspects: protein interaction,metabolic level and transcriptional level,and clarified that Dajiao responded to cold stress mainly through lipid metabolism pathway and ABA-independent MAPK cascade-ICE1 signal transduction pathway.The results will provide theoretical basis and improvement goal for banana cold tolerance molecular breeding.