Preliminary Validation Of Disease And Stress Resistance Function Of Hevea’s Hb-novel-miR0523-3p Gene

Rubber trees(Hevea brasiliensis)are an economically important tropical tree species in China.Its main product is natural rubber,which is an essential industrial material.Hainan and Yunnan Province are the main planting areas in China.This tropical perennial tree species is sensitive to biotic and abiotic stresses.The main diseases of rubber trees are powdery mildew and anthracnose.The disease process is that fungi invade the leaves of the whole rubber tree growth period.When it is serious,it will cause a large area of fallen leaves in rubber forest,which has a great impact on the growth of rubber trees and seriously affects the yield of natural rubber.At present,chemical prevention and control is the common method,but chemical prevention and control pollutes the environment,and the economic benefit is not high.Therefore,the demand for disease resistant,stress resistant and high-yield varieties is more and more urgent,but the conventional cross breeding of rubber tree has a long breeding cycle and slow effect;Molecular breeding technology is not mature,there is no successful report of transgenic rubber tree operation,at the same time,there is a lack of available resources for stress resistance and disease resistance.A new miRNA(hb-novelmiR0523)was found after powdery mildew infection in our previous experiments.In order to further verify the function of hb-novel-miR0523,we constructed Arabidopsis overexpression plants to study the function of hb-novel-miR0523.The main results are as follows:(1)hb-novel-miR0523 gene was cloned.Three classes of vectors were constructed:p GEM-T-hb-novel-miR0523 as sequencing vector,p UC19-hb-novel-miR0523 as protoplast expression vector,and p CAMBIA1301-hb-novel-miR0523 as plant expression vector.Homozygous transgenic Arabidopsis thaliana has been obtained,Which were constructed by Agrobacterium-mediated genetic transformation system.(2)In Arabidopsis protoplasts,the transient expression of hb-novel-miR0523 significantly activated the expression of FRK1,indicating that the miRNA can activate the immune response system in plants.(3)hb-novel-miR0523 can respond to SA/JA stimulation,which indicates that hbnovel-miR0523 may participate in the resistance process of Arabidopsis through the SA/JA pathway.(4)Overexpressed of hb-novel-miR0523 significantly enhanced salt tolerance,drought resistance and disease resistance of Arabidopsis.Further study showed that FIB-7b may be the target gene of this miRNA.In summary,we identified a novel miRNA,hb-novel-miR0523,that responds to the stimulation of powdery mildew in Hevea brasiliensis;Overexpression of hb-novelmiR0523 in Arabidopsis significantly enhanced salt tolerance,drought resistance and disease resistance of Arabidopsis,and it was found that FIB-7b might be its potential target gene.These results provide a reference for further studies on miRNA function in Hevea brasiliensis and provide genetic resources for stress and disease resistance breeding of Hevea brasiliensis in the future.