Development Of An Endogenous Reference Gene In Wheat And Transcriptome Analyses Of Transgenic Wheat Resistant To Fusarium Head Blight

With the rapid development of transgenic technology and industrialization of genetically modified crops,especially the commercial cultivation of transgenic staple crops,the security issues about the genetically modified food are also increasing.To protect consumers’ rights to obtain relevant information,labeling regulations for GM contents of food crops have been established in more than 50 countries and regions.Techniques based on q PCR have been widely applied for the detection and quantification of GM contents.With amplification of both a specific GM target sequence and a taxon-specific target sequence,researchers can estimate the relative ratio of GM materials in a given matrix.To develop such an assay,a prerequisite is to develop an endogenous reference gene system.Five endogenous reference genes have been reported for GM wheat detection and quantification;however,all this genes are not very ideal.Moreover,none of these endogenous reference genes was verified for detecting GM wheat contents with GM wheat materials.The Fusarium head blight(FHB)resistance transgenic wheat is one of the main research contents of transgenic wheat.FHB is a destructive disease of grain crops,with worldwide economic and health impacts.With the development of genetic engineering technology,greatly expands the scope of the use of wheat scab resistance sources,and provides a new solution for the creation of resistant sources and developing new resistance varieties.The author’s laboratory transformed an antibody fusion protein(Ech42CWP2)with glume specific promoter Lem2 to wheat variety Zhengmai9023,through inoculation identification,the level of FHB resistance of transgenic lines increased significantly.Although the resistance of the transgenic wheat had been improved,the molecular mechanisms,including how many and what genes changed before and after inoculation are remained unclear.In this study,we developed an ideal endogenous reference gene RPL21,which can be used in for GM wheat detection and quantification.And we first used wheat microarray to compare the transcripts difference between transgenic and non-transgenic genotypes before and after FG5035 inoculation and screened the different expression genes.Through bioinformatics,the metabolism pathways and resistance mechanisms these genes involved in were discussed.The main results are as follows:1.To choose a suitable reference gene in common wheat,large amounts of gene information were collected from Gen Bank and several candidates were evaluated.RPL21 gene in the common wheat genome was found to have low homology with the sequences from other non-wheat species,and the special primer pair RPL21-1F/1R was designed.18 different species and 16 different wheat varieties from different ecological regions were detected through conventional PCRs,demonstrating that the RPL21 fragment is wheat-specifict has no allelic variation among the wheat varieties.By Southern blot analysis and sequencing analysis,showed that the RPL21 gene may have low copy in the common wheat genome and annealing sites of the primer pair RPL21-1F/1R were only present in the A genome.So the RPL21 system was suitable for detection and analysis of Triticum cultivars and the RPL21 gene has the nessential condition of an endogenous reference gene.The LOD of conventional PCR was 2 copies of wheat haploid genome.The LOD and LOQ of real-time PCR were 2 and 8 copies of wheat haploid genome,respectively.All the data was lower than other reported wheat endogenous reference genes,or at the same level.These mean that the RPL21 has a higher sensitivity of detection.To establish a method to estimate the contents of transgenic wheat,two standard curves(one for endogenous gene RPL21 and the other for transgenic gene NPR1)through five 5-fold serial dilutions were performed in transgenic wheat line R4 and seven mixed samples with known content of NPR1 GM wheat were relatively accurately quantified using the established real-time PCR method.All of these results demonstrated that the RPL21 gene is a suitable endogenous reference gene for GM wheat detection,and the developed RPL21 system will facilitate the enforcement of GMO labeling for wheat in the near future.2.In this study,we used wheat microarray to compare the transcriptome difference between transgenic and non-transgenic genotypes before and after inoculation(0 h,12 h,24 h,48 h,and 72 h).80 probesets were constitutively different expressed in spikelets before inoculation,including 55 up-regulated genes and 25 down-regulated genes.Some pathogenesis-related genes had already started to increase expression in the transgenic wheat before the pathogen inoculation,which might result in the elevation of basal defense status in the plant.Compared to its parent wheat,total 2361 up-regulated and 2883 down-regulated expressed probesets in the transgenic wheat were identified;And the biggest differences of transcriptome between resistant and susceptible wheat genotypes occurred at 48 hai,and after that this the transcriptome expression model began to reverse.So,48 hai may be the critical moment to study the molecular mechanism of wheat FHB resistance.SAR related genes were up-regulated in both transgenic and non-transgenic wheat,but increased less in the transgenic wheat.The JA pathway,less content of ROS,better ability maintenance cellular redox homeostasis and chromatin-related proteins may participate in the transgenic wheat FHB resistance.After Fg inoculation,detoxifying-related proteins and transcription factors were lower in the transgenic wheat,which might be associated with the lighter disease and the less toxin production in the transgenic wheat;And cytoskeleton protein content was higher expression in transgenic wheat,reflecting the extent of cell damage was relatively small in transgenic wheat after inoculation,and from another side proved the level of FHB resistance in transgenic wheat was improved.