Identification,Expression And Regulation Of MaC3H Gene In Banana(Musa Acuminata L.AAA Group,cv.Cavendish) Under Infection Of Fusarium Oxysporum F.sp Cubense Tropical Race 4

Banana is one of the important food crops in the tropical and subtropical regions,and is also a famous tropical fruit.At present,due to various biological and abiotic stresses in production,the banana industry is severely constrained.Especially,fusarium wilt of banana has posed a great threat to banana production in recent decades.In fusarium wilt of banana,the disease caused by physiological race(Foe TR4)has not yet been effectively controlled.To study the pathogenesis of fusarium wilt induced by Foe TR4 in banana is the basis of an in-depth understanding of the pathogenesis of fusarium wilt and establishing effective preventive measures.In this study,we measured lignin content in roots from three banana varieties,Brazil plantain(Musa acuminate L.AAA group cv.Cavendish ’BaXiJiao ’),GCTCV 218(Musa acuminata L.AAA group cv.Cavendish ’Formosana ’)and GCTCV 119(Musa acuminate L.AAA group cv.Cavendish ’Formosana’),and determined the relationship between the lignin content and disease resistance of bananas.The full-length sequence of p-coumarate 3-Hydroxylase(C3H)was cloned and analyzed,which is a key enzyme gene in the lignin biosynthetic pathway.We also analyzed the expression of C3H at 0 day,2 days,4 days and 6 days after Foe TR4 inoculateion in resistance and susceptible cultivars using qRT-PCR method.The full-length promoter of C3H gene was cloned and analysis of its acting elements.The co-expression network and transcriptome data were used to screen the related transcription factors,and the yeast one-hybrid technique was used to verify the transcription factor interacting with the C3H gene.The main results are as follows:1.The content of lignin in disease-resistant and susceptible banana varieties are different from each other.Obviously,The lignin content of disease-resistant cultivars,GCTCV 218 and GCTCV 119,was higher than that of susceptible cultivar Brazil plantain,indicating that the disease resistance of banana may be related to the content of lignin.2.The banana A genome database combined with the transcriptome analysis showed that there were two MaC3H genes in the pathway of banana lignin synthesis,and gene MaC3H expression is observed but gene MaC3H1 didnot express.We establish C3H gene protein sequence alignment in banana,Arabidopsis thaliana and rice by using bioinformatics methods.Those results showed that the MaC3H gene,C3H gene in banana,contains a conserved cytochrome P450 domain,which confirmed the acquired gene is MaC3H gene.3.Through the qRT-polymerase chain reaction(PCR)analysis,the results show that C3H gene expression of Brazil plantain,GCTCV 218 and GCTCV 119,three different banana varieties,are improving after inoculated by Foe TR4;Among the three different banana varieties,the relative value of two disease-resistant varieties,GCTCV 218 and GCTCV 119,were obviously higher than that of susceptible cultivar Brazil plantain,after 2 days root inoculation of pathogenic bacteria,disease-resistant varieties GCTCV 218’s relative expression quantity equals twice that of Brazil plantain;And when the root inoculation of pathogenic bacteria reach 4 days,disease-resistant varieties GCTCV 218’s relative expression quantity equals three times that of Brazil plantain,and disease-resistant varieties GCTCV 219’s relative value equals two times that of susceptible cultivar Brazil plantain.These results come to a conclusion that Foc TR4 inoculation made C3H gene’s different amount of expression,induction expression value of disease-resistant varieties significantly higher than that of susceptible cultivars.4.Cloning MaC3H promoter sequences can see the promoter length is 1482 bp,and by cis-acting element analysis we can obtain TATA-box and GC-box structure core promoter sequences,also including photorresponse cis-acting element,involving in response to jasmine element response(MeJA)cis regulatory elements,ethylene response element,gibberellin response element,MYB binding sites,taking part in the defense and stress reaction cis element and auxin response element,etc.5.Using WGCNA(weighted gene co-expression network analysis)algorithm to establish MaC3H co-expressing network,data shows that there are 37 transcription factors may be linked to MaC3H gene promoter.At the same time,the lab builded 11 Brazilian Manila transcriptome data,including fruits development and postharvest fruits mature process;abiotic stress such as low temperature and salt stress,osmotic stress,and root inoculation of pathogenic bacteria TR4,etc.;In all these cases,MaC3H gene all reach the high level expression(>20);Among co-expressing with MaC3H’s transcription factors,there are 30(81%)genes reach a high level expression of these transcription factors after affected by Foe TR4 root inoculation.Which means that these transcription factors are all inducted by Foe TR4.6.Cloning the cDNA sequences of the three transcription factors(MaMYB1,MaMYB2,MaMYBS)that may co-express the MaC3H promoter in the network.Yeast one-hybrid system was used to perform the binding experiment with MaC3H.The results showed that MaMYB1,MaAYB2,MaMYB3 respectively interacted with MaC3H.It is speculated that MYB transcription factors can regulate the expression of MaC3H gene,and then influence the synthesis of lignin and further regulate the disease resistance of bananas.