Clone And Transformation Of Cold Resistance-related Gene JMJ15in Maize

Maize is a kind of grain crop which is grown the most widely and have thegreatest yield.And it has a variety of purposes such as food, industrial raw materialsand feed. The northeastern of China is the largest cultivated area of maize.But becauseof the geographical reasons, maize often experience low temperature and has lead tomost areas reduce yield.Currently chilling stress has become one of the mostimportant abiotic stresses in this region of China.So it is very important to study thechilling response mechanisms in maize.In this study, we cloned the histone demethylation-related gene JMJ15fromchilling-resistant maize inbred linesW9816using RT-PCR.The sequencing resultsshowed that the nucleotide sequence contained a complete open reading frame,its fulllength was1803bp,It encoded a protein of601amino acid and then usingbioinformatics analysis of this Amino acid sequence.Besides, we cloned JMJ15gene’spromoter successfully and analyze the core region.It’s the basics for further analyzethe mechanism of JMJ15gene for cold resistance.The dynamical changes of the mRNA transcription levels of this gene wasfurther determined by real-time reverse transcription PCR during Maize response tolow temperature,the result shows that this gene is related to chilling response inmaize.For further study, this experiment based on pCAMBIA3301plasmid, weconstructed two plant vectors:the expression vector35S::JMJ15and the ihp-RNAiexpression vector UBI::JMJ15.We transfered the JMJ15gene successfully toArabidopsis thaliana by Agrobacterium dip method, and obtained T3generationtransgenic plants. And then low temperature treated WT and the mutant,the resultshows that the JMJ15mutant more cold resistance compared with WT.We usedstatistical methods to observe phenotype differences between Arabidopsis mutantsand wild-type. The difference between them are significant.This result show thatJMJ15may regulate maize growth and development by histone demethylation. JMJ15was transferred into immature embryo and somatic embryo induced bymaize inbred lines Y423by Agrobacterium tumefaciens-mediated.then selected on themedium containing the concentration of bialaphos for3mg L-1.The transgenic plantswere confirmed by PCR and we have obtained T0generation plants of desired gene.13T0transgenic plants of Bar were obtained through infect immature embryo.9T0transgenic plants of Bar with2have desired gene were obtained through infectsomatic embryo.JMJ15gene was transformed into maize inbred lines10Z27、B73、W9816、W9805、KB411through pollen tube pathway and T0transgenic plants’seedwere obtained.This help us further study of JMJ15’s effect in maize.