Study On The Function And Drought-resistant Mechanism Of Mapip1;1 In Banana(Musa Ssp.)

Banana(Musa ssp.)is an important major tropical fruit,with characters of high yield,high economical efficiency and stable yield relatively,but it is sensitive to abiotic stresses such as drought,low temperature and high salt.Especially the water stress will be resulting in lower yield and undesirable fruit quality.As the banana planting in Guangxi,Yunnan and other non-traditional distribution area,drought often cause serious lost of production.Meanwhile,banana is a homologous triploid species,so conventional hybridization breeding is very difficult.It is of great industrial value to enhance stress resistant germplasm and banana varieties through genetic engineering technology.And it is of theoretical significance to deeply analyze the mechanism of drought-resistant domestication of banana.Aquaporins(AQPs)are membrane proteins with the function of regulating the transport of water and other small molecules,and plays a vital role in plant stress adaptation.At present,studies on the stress resistance of banana AQP gene and its regulatory mechanism are very limited.In this study,with the MaPIP1,member of the AQP family whom were identified response to water stress,we successfully obtained the over expression transgenic lines mediated by 35 S promoter,performed the identification of drought resistance,cold and salt tolerance under controlled environments.We obtained multiple transcription factors interacted with the promoter and regulators by c DNA library screening with yeast single hybrid,and clarify MaMADS3 transcription factor positively regulated MaPIP1;1.The concrete research results are as follows:1.Construction of an overexpression vector with MaPIP1;1 and 35 S promotor,was used for agrobacterium-mediated genetic transformation in banana.With the method depended on staminate inflorescence explants succeffully developed in our lab.,202hygromycin-resistant transgenic lines were obtained,throughout PCR detection,23 lines were positive,The production rate is 11.4%.Further RT-PCR and Southern blot identified8 transgenic lines.Two lines with higher expression of MaPIP1;1 were propagated and used for next step identification of third generation transgenic lines.2.The expression level of the double transgenic lines overexpressed MaPIP1;1 was up-regulated.The two MaPIP1;1 transgenic lines and wild-type control were treated with drought,low temperature and high salt respectively under potted conditions.The results showed that the drought-resistance of overexpressed lines was enhanced,and the abovemental and root recovery ability after rehydration by drought treatment was significantly better than that of wild-type.At the same time,the low temperature and salt tolerance of transgenic plants were also significantly improved.The physiological index test shown that under drought,high salinity,low temperature stress and recovery treatment,the content of chlorophyll,soluble sugar,abscisic acid(ABA)and proline in transgenic plants was higher than that in wild type,while the content of ion leakage and malondialdehyde was lower than that in wild type.The content of K+ and Na+ in the leaves of transgenic lines was lower in the high-salt and restoration treatment,while the ratio of K+ /Na + was higher in the restoration condition.These results indicated that MaPIP1;1 enhanced resistance to abiotic stresses in banana by reducing membrane damage,improving osmotic regulation and ABA levels,and increasing K+/Na+ ratio.3.Furthermore,it was found that under drought,salt and low temperature treatment,genes for ABA biosynthesis(MaNCED1,MaNCED2 and MaAO)and genes in ABA signal pathway(MaSn RK2-11 and Mab ZIP49)in transgenic lines were significantly higher expressed than in that of wild type.In addition,MaSDR2 of two transgenic lines increased transcriptional activity under drought and low temperature stresse,Mab ZIP10 expressed higher under drought stress only,and Mab ZIP101 just increased expression under low temperature stress compared with the wildtype.These results indicated that the overexpression of MaPIP1;1 could stimulate ABA biosynthesis and enhance the activity of response signaling pathway,which may be closely related to the improvement of banana’s resilience to stress.4.For further understand the transcriptional regulation of MaPIP1;1,the 1362 bp upstream promoter sequence of the MaPIP1;1 was cloned and fused with the GUS gene to construct 4 gradient deletion vectors,and the transformed Arabidopsis thaliana plants were obtained after qualified detection.MaPIP1 was determined by GUS activity assay.The promoter core region was-1274 bp,the regions from-1274 to-813 were highly activated,and the regions from-1362 to-1274 had inhibitory effects on transcriptional activity.5.MaPIP1;1 promoter sequence was used to construct bait vector.Yeast single hybridization was used to screen the mixed c DNA library of different banana tissues under drought stress,and 23 genes for candidated interaction proteins were obtained.Among them,the expression pattern of MaMADS3 transcription factor under drought stress was in accordance with MaPIP1.1.Rotation verification showed that MaMADS3 and MaPIP1;1 promoter regions interacted each other.The results of dual luciferase experiments indicated that MaMADS3 can bind to the MaPIP1;1 promoter and positively regulate the transcription of MaPIP1;1.