Cloning And Functional Analysis Of NAC Gene Responding To Drought Stress In Maize

In the process of growth and development,plants are often affected by biotic or abiotic stresses,such as disease and insect pests,high temperature,drought,salinity and hormones,they seriously affect the growth,yield and quality of crops.Meanwhile,in order to adapt to and resist all kinds of adversity,a series of effective protection mechanisms have been formed by sensing the stress signals and regulating the expression of genes involved in stress response in the long term evolution of plants.NAC transcription factor,as a unique regulatory factor,plays an important role in plant morphogenesis,metabolism and response to stress.In this study,8 NAC candidate genes related to drought resistance in Maize were cloned from maize inbred line B73 and carried on the bioinformatics analysis of them;The expression patterns of them under drought stress were analyzed by real-time quantitative PCR.In addition,up-regulated genes were used to analyze the nucleotide polymorphism and deduced amino acid sequence of maize inbred lines with different drought resistance;at the same time,The overexpression and abiotic stress tolerance of Arabidopsis thaliana were also identified.Here are the main results:1.Bioinformatics analysis of 8 ZmNAC genes(ZmNAC6,ZmNAC29,ZmNAC54,ZmNAC55,ZmNAC71,ZmNAC87,ZmNAC99,and ZmNAC102)were cloned,The results showed that the full-length of genes were 1847 bp,1105bp,2227 bp,1988bp,2170 bp,1135bp,1907 bp and 2152 bp respectively,consistent with the theoretical size,In addition to ZmNAC54 containing 4 introns and 5 exons,the other 7 candidate ZmNAC genes all contained 2 introns and 3 exons.Protein domain analysis revealed that the N-terminal of the drought resistance of 8 maize candidate ZmNAC genes had a conserved NAM domain;Phylogenetic analysis showed that the 8 maize ZmNAC genes were divided into two categories;The cis-element revealed that in addition to containing core promoter elements such as CAAT-box and TATA-box,abiotic stress and hormone responsive elements are also present in the promoters of the 8 ZmNAC genes,such as MBS,HSE and ABRE,etc.2.The expression of ZmNAC gene under different drought stress was analyzed by qRT-PCR technique.The results showed that ZmNAC6 gene was up-regulated in moderate and severe drought conditions;However,ZmNAC99 and ZmNAC102 genes were up-regulated only under the condition of moderate drought and severe drought.On the contrary,ZmNAC55 was down regulated under moderate drought and severe drought.3.The deduced amino acid sequences of ZmNAC99 and ZmNAC102 from different drought resistant maize inbred lines were compared by ClustalW.The results showed that ZmNAC99 and ZmNAC102 had different amino acid differences in different drought resistant inbred lines,and the difference between the inbred lines with similar drought resistance was significantly less than that of drought resistant inbred lines,and we speculated that the difference of genetic structure may have an effect on the drought resistance of maize.4.The plant expression vectors of ZmNAC54,ZmNAC55,ZmNAC87,ZmNAC99 and ZmNAC102 were successfully constructed,and transformed into Arabidopsis thaliana by dipping flower method to obtain T1 generation seeds,At the same time the T1 transgenic Arabidopsis seed of ZmNAC99 and ZmNAC102 were screened by 1/2MS medium containing 100 ?g/mL Carbenicillin and 30 ?g/mL Kanamycin and PCR detected,until the T3 generation transgenic Arabidopsis seed were harvested.5.Under the drought stress simulated by water control and 300mmol/L mannitol,Compared with wild type,the roots of ZmNAC99 transgenic Arabidopsis plants were relatively longer and the relative water content of leaves was higher while the rate of water loss was low in adult plant stage.This suggests that ZmNAC99 may play a role in improving drought resistance in Maize.45?high temperature stress experiment showed that ZmNAC102 transgenic plants showed better tolerance than controls under high temperature stress.However,under the high salinity stress of 150mmo/L NaCl and the low temperature stress of 2 ?,there was no obvious phenotypic difference between two transgenic Arabidopsis plants and wild-type Arabidopsis,the results showed that ZmNAC99 and ZmNAC102 could not improve the ability of salt tolerance and cold tolerance.