Cloning And Functional Studies Of ZmbHLH91 Gene In Maize

Maize(Zea mays L.)is an important food crop,mainly used as feed and industrial raw material.Abiotic stress seriously affects the growth,development and yield of maize,so it is of great significance to explore the response mechanism of maize under abiotic stress.bHLH(basic helix-loop-helix)transcription factor is one of the largest transcription factor families in plants and plays an important role in regulating plant growth and development,biosynthesis,stress response and signal transduction.The sixth leaf stage(V6),the twelfth leaf stage(V12),the tasseling stage(VT),and the milk stage(R3)are key periods of maize growth and development.In the early work,we carried out root phenotype identification and transcriptome sequencing analysis in the four key periods of maize growth and development.There was significantly differentially expressed in ZmbHLH91 during the adjacent periods of the sixth leaf stage(V6),the twelfth leaf stage(V12),and the tasseling stage(VT).The relative expression levels of ZmbHLH91 were higher in V6,V12,and VT,which were the active periods of root growth and development.We speculated that ZmbHLH91 may have important regulatory effect on the growth and development of maize root system.The growth and development of maize root system plays an important role in absorbing nutrition and water by plants,and it is also an important organization for sensing and responding to stress(such as drought,salinity,etc.),so we suspect that this gene may participate in various stress responses.In this study,the Maize Backbone Inbred Lines B73 and HZ178 were used as the experimental materials,and real-time fluorescence quantitative PCR(q RT-PCR)technology,yeast two hybrid technology,phenotypic identification technology of transgenic lines and so on were used to explore the molecular mechanism of transcription factor ZmbHLH91 in stress response and signal transduction.To provide a theoretical basis for cultivating new maize germplasm materials of stressresistant maize.The main results are as follows:(1)Bioinformatics analysis showed that the full-length ORF of ZmbHLH91 gene was 2112 bp,encoding 703 amino acids.The gene belongs to the bHLH family of transcription factors,the protein molecular weight is 75.3k D,and the theoretical isoelectric point(PI)is 6.01.There are many cis-acting elements related to stress and growth and development in the 2kb region upstream of the gene ATG.(2)The expression pattern of ZmbHLH91 gene was analyzed by real-time fluorescence quantitative PCR.The results of tissue differential expression showed that the expression of ZmbHLH91 in roots was higher,and the expression of ZmbHLH91 in young roots in trefoil stage was higher than that in mature roots in tasseling stage,and the expression of ZmbHLH91 was higher in the active stages of root growth and development,such as V6,V12 and VT.Stress expression analysis showed that the expression of ZmbHLH91 in roots could be induced by mannitol,salt and dehydration,and the expression of ZmbHLH91 in shoots could be induced by mannitol,dehydration and cold treatment.Hormone-treated expression analysis showed that ZmbHLH91 could be induced by JA,ABA and GA.It is confirmed that ZmbHLH91 gene plays an important role in maize root growth and development and stress response.(3)The root length of transgenic Arabidopsis thaliana was longer than that of wild-type on 1/2 MS medium containing salt,mannitol,JA and ABA.Under drought and high salt treatments,transgenic Arabidopsis thaliana grew better than wild-type.In addition,transgenic Arabidopsis thaliana had higher green leaf rate and POD activity after stress treatment,indicating that ZmbHLH91 is a positive stress response factor in high salt,drought and osmotic stress,which can improve stress resistance by increasing peroxidase activity and eliminating peroxides in plants.Further analysis of the changes in the expression of stress response marker genes RD29 A,RD29B,RAB18 and RD22 in transgenic Arabidopsis thaliana showed that the expression of stress response marker genes in transgenic Arabidopsis thaliana was up-regulated after stress treatment,and the expression level was higher than that of wild type,and the difference was significant.It is further proved that ZmbHLH91 plays an important role in resistance to stress,and it can deal with abiotic stress by removing peroxides and increasing the expression of stress-related genes.(4)The over-expression vector and RNAi vector of ZmbHLH91 gene were transformed into maize HZ178 inbred lines using the pollen magnetofection by magnetic nanoparticles-mediated.After bar(bialaphos resistance)and PCR detection,4 strains of T2 generation overexpressed maize and 2 strains of T2 generation RNAi transgenic maize were obtained.Then the phenotypes of transgenic lines were identified and analyzed in normal culture medium growth,drought treatment with 10% PEG addition and salt treatment with 150 mmol L-1 Na Cl.The results showed that under normal culture medium conditions,the root length and plant height of over-expressed lines were significantly higher than those of wild-type,and the root length and plant height of RNAi lines were lower than wild-type,and the difference was significant.Under the condition of salt treatment,the root length and plant height of over-expressed lines were higher than those of HZ178.Preliminary analysis showed that ZmbHLH91 was involved in the regulation of maize growth and development and the response to salt stress.(5)Yeast two-hybrid technique was used to explore the interacting protein of ZmbHLH91,and it was found that ZmbHLH91 interacted with MYC2 in JA pathway,suggesting that ZmbHLH91 might be involved in JA signaling pathway to response abiotic stress.