Cloning And Functional Analysis Of MaWRKY24 And MaWRKY80 Transcription Factors

As a monocotyledonous plant,banana is distributed in tropical and subtropical regions and one of the most popular fresh fruits in the world.However,banana industry in China is seriously damaged by Fusarium wilt.Meanwhile,the roots of banana are shallow,the leaves are evergreen,the water demand is large,and it is easy to suffer from drought stress,which seriously affects the yield and quality of fruit.Therefore,excavation of stress-resistance genes cultivates new varieties of banana through molecular breeding,so as to enhance the adaptability to the adverse environment and promote the healthy development of banana industry.As an important transcription factor, WRKY acts as the vital role in plant growth and response to various stresses.This study analyzed the expression levels of MaWRKY24 and MaWRKY80 in Fusarium wilt and drought stress,and heterologous expression of MaWRKY24 and MaWRKY80 in Arabidopsis to explore their biological functions in response to stress.The main results are as follows:1 By qRT-PCR analysis,the expression level of MaWRKY24 is regulated by Fusarium wilt of banana,which is significantly up-regulated at 3 h and 51 h,significantly down-regulated at 27 h.With the intensification of drought stress,the expression level of MaWRKY80 is significantly up-regulated.2 MaWRKY24 and MaWRKY80 were cloned to the pEGAD vector,which was transiently expressed in tobacco leaves by Agrobacterium-mediated method,and its subcellular localization was observed.It was found that these two genes were localized only in the nucleus.Using tobacco leaf protoplasts for the dual LUC activity,it was revealed that W-box can affect the transcription activation activities of MaWRKY24 and MaWRKY80.3 In banana protoplasts transient expression of MaWRKY24,the levels expression of MaATG8f and MaATG8g are significantly higher than 35::GFP.The double LUC luciferase assay shows that MaWRKY24 can activate MaATG8f and MaATG8g promoter activities.Bioinformatics analysis shows that there are two W-boxes in MaATG8f promoter and one W-box in MaATG8g.It is found through ChIP-PCR and EMSA that MaWRKY24 directly binds the W-box of MaATG8f and MaATG8g promoter and acted as a transcriptional activator of MaATG8f and MaATG8g.4 To further study the response of MaWRKY24 and MaATG8f/g to Foc 4 infection,we obtain the MaWRKY24 and MaATG8f/g overxepressing in Arabidopsis.Under the infection of Foc 4,compared with wild-type Arabidopsis,overexpression of MaWRKY24 and MaATG8f/g is not conducive to the growth of Arabidopsis seedlings,decrease the relative chlorophyll content,increase the relative content of H₂O₂and O₂^-.It can be seen that the overexpression of MaWRKY24 and MaATG8f/g negatively regulates Foc 4 infection in Arabidopsis.5 Under drought stress,MaWRKY80 overexpressing lines still maintain good growth and no wilting.In addition,the survival rate of MaWRKY80 overexpressing lines is significantly higher than that of wild type,and the survival rates of wild type,OE-3,and OE-6 are 26.3%,92.2%,and 90%,respectively.At the same time,the water loss rate of MaWRKY80 overexpressing lines is significantly lower than that of wild type.The MDA content of OE-3 and OE-6 is also lower than that of wild type Arabidopsis.6 Under drought stress,the MaWRKY80 overexpressing increases antioxidant enzyme activities and antioxidant substance content,thereby making the ROS levels in leaves significantly lower than that of the wild type.In addition,MaWRKY80 regulates stomatal movement and leaf water holding capacity,reduces transpiration rate,improves water use efficiency and assimilation rate by regulating the expression levels of NCEDs and ABA biosynthesis.7 ChIP-PCR and EMSA confirm that MaWRKY80 can directly and specifically interact with W-box in the At NCEDs and MaNCEDs promoter,thereby regulating ABA levels and playing a positive role in regulating drought resistance.In summary,this study identifies two MaWRKYs.We identified potential target genes and analyzed their functions in the stress of banana,and finally analyzed the new mechanism of MaWRKYs participating in the plant's adversity response,providing candidates for the cultivation of new banana varieties gene.