Drought Tolerance Identification And Mechanism Of Maize Mutant M716

Maize is an important food crop in China,and its yield is closely related to the agricultural economy.Drought,high temperature and other stress can seriously affect the yield and quality of maize.With the rapid development of biotechnology,it has become a research hotspot of plant genetic improvement to improve the resistance of crops in arid environment and cultivate new varieties with strong stress resistance by means of genetic engineering.In this study,maize inbred line Chang 7-2 and its mutant m716 were used as materials to determine the water loss rate and relative water content of maize leaves at three-leaf stage.C7-2 and m716 were subjected to drought treatment,and stomatal,photosynthesis and drought resistance physiological indexes were measured.After dehydration treatment of C7-2and m716,RNA-seq analysis and q RT-PCR verification were performed to screen the candidate gene ZmTHX15.In order to preliminarily study the function of this gene,the Trihelix transcription factor family of maize was analyzed by bioinformatics methods,and then the overexpression vector of ZmTHX15 was constructed and transformed into wild-type Arabidopsis for preliminary exploration of gene function.The difference between overexpression plants and wild-type under drought conditions was tested and drought tolerance was determined.The main results of this experiment are as follows :(1)m716 has stronger drought tolerance than C7-2.The physiological indicators such as antioxidant enzymes,osmotic substances and MDA were measured on C7-2 and m716 plants under normal growth and drought treatment conditions.It was found that m716 had higher antioxidant enzyme activity and osmotic substances accumulation under drought conditions than C7-2.more,less MDA was produced;leaf water loss statistical analysis found that the water loss rate of m716 leaves was slower than that of C7-2;stomatal analysis found that the stomatal density of m716 was only about 0.6 times that of C7-2;observation and analysis of xylem vessels found that drought Under these conditions,the vessel density of m716 is smaller,the inner diameter is larger,embolism is more likely to occur,the rate of water loss is reduced,and it is more drought-tolerant;the results of photosynthetic index measurement show that the chlorophyll fluorescence index and net photosynthetic rate of m716 are higher than those of C7-2,the accumulation of intercellular CO2 was less,which indicated that under drought conditions,with the closure of stomata,m716 could make full use of intercellular CO2 and maintain the stability of photosynthesis.(2)RNA-seq analysis identified 1975 up-regulated genes in m716.Under dehydration treatment,1975 genes were up-regulated in m716.GO analysis and KEGG analysis showed that these differentially expressed genes were significantly enriched in response to abiotic stress,ABA-mediated signal transduction,protein phosphorylation and other pathways.Some differential genes were verified by q RT-PCR,and the gene ZmTHX15 that may respond to drought stress was selected as a candidate gene for further study.(3)Gene family analysis showed that ZmTHX15 responded to drought stress.The gene family of ZmTHX15 was identified and analyzed,and a total of 46 THX genes were obtained.Its members are divided into five subfamilies : GT-1,GT-2,GTγ,SH4 and SIP1.Through cis-element analysis of the family,it was found that many genes,including ZmTHX15,have cis-acting elements related to drought stress response,such as ABRE,DRE and MBS.Gene differential expression heat map showed that ZmTHX15 and other genes were strongly induced under drought conditions,and the expression level increased significantly,indicating that ZmTHX15 was involved in the response of plant drought stress.(4)Overexpression of ZmTHX15 transgenic Arabidopsis improved the tolerance to drought stress.In order to explore the function of ZmTHX15,we constructed transgenic Arabidopsis plants overexpressing ZmTHX15,treated them with drought and identified their drought tolerance.The results showed that under soil drought conditions,overexpression of ZmTHX15 transgenic Arabidopsis lines had stronger water retention capacity,higher antioxidant enzyme activity,more osmotic substance accumulation,less oxidative damage and stronger drought tolerance than wild-type plants.In conclusion,this experiment found that m716 is more drought-tolerant than C7-2 through experiments such as drought treatment,physiological index measurement,stomatal analysis and xylem vessel analysis.Through RNA-seq analysis and q RT-PCR verification,the candidate gene ZmTHX15 was screened out,which belongs to the Trihelix transcription factor family.The bioinformatics analysis of the family to which this gene belongs showed that many genes,such as ZmTHX15,contained drought stress-related response elements,and were strongly induced to express under drought treatment.The drought tolerance of ZmTHX15 transgenic Arabidopsis was determined,and it was found that ZmTHX15 could improve the drought tolerance of transgenic plants.In conclusion,this study not only provides theoretical support and genetic resources for maize drought tolerance genetic breeding,but also provides new clues for analyzing the drought tolerance regulatory network of maize.