The Transcriptome Analysis Of Malus Hupehensis Under Drought Stress And Functional Verification Of MdWRKY40 Gene

The cultivation area and yield of apple are first in the world.Dwarf rootstocks M9,M26 is currently widely used in apple anvil dense planting cultivation patterns,have good dwarf and early flowering effect,but the root shallow,not drought-tolerant,in poor soil conditions,lack of water area is weak,low yield.This restricts the popularization of Apple Dwarf rootstock dense planting with higher benefit.It is an urgent problem for cultivate drought-resistant rootstock suitable for arid areas in apple production.In this study,transcriptome sequencing was performed on Malus hupehensis under drought treatment,and multiple families of WRKY transcription factors were found to be differentially expressed.Members of the WRKY transcription factor family were identified by apple genome,and the expression analysis was conducted.The higher expression multiple of MdWRKY40 was functionally verified to study its possible role in drought resistance.The main results are as follows:1、In this study,rootstock Malus hupenensis were treated with control and drought treatment.After the fifth day,young leaves were collected and sequenced by transcriptome.The results showed that 4200 genes were differentially expressed,of which 1449 genes were up-regulated and 2751 genes were down-regulated.The differential gene KEGG analysis showed that the genes related to metabolism were significantly enriched,and the metabolic pathways such as photosynthesis carbon metabolism,amino acid synthesis,starch and sucrose metabolism,and purine metabolism were significantly enriched.There were 259 transcription factors differentially expressed,and multiple members of the MYB,NAC,ERF,HSF and WRKY families related to stress resistance were differentially expressed.2、According to the apple genome database,113 members of WRKY transcription factor family were identified,they can be divided into three subfamilies:Group Ⅰ(9),Ⅱ(87)and Ⅲ(17).Multiple WRKY genes were highly expressed in apple flowers,fruits,leaves,roots,stem tips and stems.However,MdWRKY29b was only expressed in the root system,and MdWRKY74b was only expressed in the stem.Analysis of WRKY expression under drought and salt stress showed that 11 WRKY genes were up-regulated under salt induction,and 16 WRKY genes were up-regulated under drought induction,among which MdWRKY40 was highly expressed under drought stress.3、MdWRKY40a and MdWRKY40b have no transcriptional activation activity in yeast.MdWRKY40a and MdWRKY40b transcription factors were able to bind to MdNCED3b and MdNCED5b promoters by yeast one-hybrid assay.MdWRKY40 may participate in drought response by regulating ABA synthesis.4、The over-expression vector of MdWRKY40b gene was constructed,and obtain transgenic Arabidopsis seeds by agrobacterium mediated inflorescence infection.It provided materials for functional verification of MdWRKY40b gene in the next step.