Knockdown Of Gene CYP81A6and Insertion Of Exogenous Gene EPSPS Effect On Gene Expression In Rice

To study the effects of knockdown of rice detoxification gene CYP81A6by RNA interference and insertion of exogenous gene EPSPS on gene expression in transgenic rice, here we report the transcriptome profiling analysis of bentazon sensitive transgenic rice S11, glyphosate-tolerant transgenic rice S6and normal control rice S2. Using high-throughput Illumina RNA-seq, mRNA of transgenic rice and its parent (control) were deeply sequenced, sequences information obtained and functional genes comparison against the rice gene database were analyzed. Main findings of the study are as follows:1. Genes expressed differentially in these two transgenic rice. A total number of34,792unigenes were obtained. Comparative analysis revealed that3,529genes and5,795genes significantly differential expressed in S11and S6respectively. Among the differential expressed genes in S11,1,018genes were showed to be upregulated and2,511genes were indicated to be downregulated. Distribution of the differential expressed genes in two samples showed that2,533genes expressed in both S2and S11,363genes only expressed in S11,633genes expressed just in S2. Focusing on the genes significantly differential expressed in S6,2,994genes were showed to be upregulated and2,801genes were indicated to be downregulated. Distribution of the differential expressed genes in two samples showed that4,671genes expressed in both S2and S6,480genes only expressed in S6, while644genes expressed just in S2. Comparative analysis showed that a number of1,613genes differential expressed in both S11and S6.2. Minority of resistance genes in transgenic rice significantly differential expressed. Comparative analysis with296resistance genes in the rice database showed that64resistance genes and74resistance genes differential expressed in S11and S6respectively, among them30genes were differential expressed in both two transgenic rice. Stress-resistant gene OsRMC and OsIRO2have been studied. Knocking down the expression level of gene OsRMC led to insensitive seed germination, enhanced growth inhibition. The rice bHLH protein OsIRO2involved in Fe uptake in plants, downregulated of this gene led to growth inhibition and yellow leaves. These two genes were significantly downregulated in both S11and S6, indicated that the salt tolerance of transgenic plants may reduce. Disease-resistant gene OsRacB is a negative regulator of rice basal resistance, over-expressing OsRacB increased symptom development in response to rice blast pathogens, upregulated of this gene may reduce the resistance of S6to rice blast. Oryza sativa defense responsive gene OsDR8was significantly downregulated in S6, repressed expression of this gene showed reduced resistance to rice bacterial blight and blast pathogens in S6.3. Differential expressed genes were significantly enriched in several GO terms and pathways. Gene enrichment studies and further analysis showed that differential expressed genes were significantly enriched in38GO terms and13KO terms in S11,’heme binding’(GO:0020037) and ’photosynthesis’(ko00195) were enriched at the most dramatically. While30GO terms and12KO terms were significantly enriched in S6,’metabolic process’(GO:0008152) and ’plant-pathogen interaction’(ko04626) were enriched at the most dramatically. Comparison analysis showed that19GO terms and6KO terms were enriched in both S11and S6. The target gene CYP81A6was associated with4GO terms, these GO term were all significantly enriched in the top5in S11, indicated that knockdown of gene CYP81A6resulted in the differential expression of the other genes, especially cytochrome P450family genes. Comparison of the expression of the genes involved in the5KO terms associated with gene CYP81A6showed that a P450gene was involved in each KO term, indicated that this gene may have a certain relationship with the target gene CYP81A6. Plant-pathogen interaction pathway was associated with the immune system of plants, the change of this pathway may result in variable effects of S6plants.4. Minority of cytochrome P450family genes expressed differentially. A total number of290P450family genes were annotated.58and85P450genes with differential expression were observed in S11and S6respectively. Comparative analysis showed that the expression of10genes changed in the opposite direction and22genes consistently changed between S11and S6,26genes only expressed in S11while52genes only expressed in S6. Homology analysis of RNA interference target gene fragment and the P450family genes revealed that gene CYP75A2homology with target fragment was up to100%. CYP75is a class of P450subfamliy genes in Solanum melongena, encoding flavonoid3’,5’-hydroxylase. Currently the function of CYP75A2is unknown in rice, homology analysis indicated that this gene may have a certain relationship with the target gene CYP81A6, the function of this gene still needs further study.In this study, comparative transcriptome analysis of transgenic rice S11, S6and normal control rice S2were analyzed in order to clarify the changes of gene expression in transgenic rice, the effects on these transgenic rice plants were further analyzed. These observations may provide valuable information from gene ecology to address the safety of genetically modified crops, and it could be used as a new method applied to the safety evaluation of genetically modified organisms.