Functional Analysis Of MaCDPK4 In Bananas Infected With Fusarium Oxysporum F.sp.cubense

Banana is a herbaceous plant which lives in tropical areas and semitropical are as.However,many factors constrain its growing even causing a critical decreasing in yield such as banana leaf spot disease and banana panama disease.Thus,it is very i mportant to study the molecular mechanism of banana threatened by biotic stress.A nd it also contributes to future breeding for resistance to pathogens.Calcium depend ent protein kinases play a important role inplant resistance to pathogens.According t o data from banana AAA genome and what researchers had did before,this research took use of ORF of MaCDPK4 to build plant overexpressing plasmid.Then the pla smid was transformed to tobacco to study its functions.With yeast two-hybrid meth od,proteins which interact with MaCDPK4 were found.Results are as below:(1)To study proteins which interact with MaCDPK4,I built yeast two-hybrid D NA binding plasmid pGBKT-7-MaCDPK4.Then I transformed this plasmid to yeast AH 109 for testing Transcriptional Activation and Toxicity.In SD/-Trp/X-a-Gal,SD/-A de/-His/-Trp/X-a-Gal medium with agar,blue colonies didn’t be found.This results sh owed that it didn’t be activate by itself.In SD/-Trp medium with agar,colonies wi th pGBKT-7-MaCDPK4 or pGBKT-7 growed both the same,which showed no toxi city.So,it is appropriate to carryout next step.(2)A cDNA library built from banana infected by FOC4 and then I cotransfor med it to yeast AH 109 with DNA binding plasmid.I sent positive colonies to seque nce and BLAST sequences.Thus,I found SAMS,coatomer protein and other unknown proteins as candidate proteins which were interact with MaCDPK4.So,I built plas mid pGADT7-SAMS to retest the interaction in yeast.In SD/-Leu/-His/-Ade/-Trp/X-a-Gal medium with agar,there were blue colonies to ensure the possible interaction.(3)To study MacCDPK4’role in banana responsing to pathogens,I built plant o verexpression plasmid pVKH-MaCDPK4.I took use of agrobacterium-mediated transf ormation transforming plasmid to tobacco by infecting callus of tobacco.I got 5 tran sgenic tobacco strains through making seeds germinate in the screen medium with hygromycin(30ug/ml).I chose two of those strains to inoculate with FOC4.(4)An appropriate spore concentration(1×108)was used to infect tobacco which growed 10 days atter transplant from medium to pot.5 days after infection,growing of transgenic tobacco and wild type both were constrained.10 days after infection,le aves of wild type became yellow,however,leaves of transgenic tobacco were less yel low than wild type.Then,quantitive real-time PCR analysis showed that pathogens-rel ated significantly more increased in transgenic lines than in wild type.And in transg enic lines,relative gene expression of SA and JA pathway were obviously increased.The expression of NTSAMS in wild type decreased.In transgenic lines,the expression of SAMS first increased and then decreased.Based on the results above,MaCDPK4 interacted with SAMS.SAMS might be i nvolved in tobaccos resistant to FOC4.SAMS mightIt is possible that MaCDPK 4 gets involved in banana resistance to FOC4 by SA or JA pathway.