Development Of Multiresistant Transgenic Rice And Evaluation Of Resistance To Different Diseases And Insect Pests After Polymerization Of Resistance Genes

Weeds,diseases and pests pose serious threats to rice production and cause significant economic losses.Cultivation of rice varieties with resistance to herbicides,diseases and pests is believed to be the most economical and environment-friendly method to deal with these problems.The resistance of rice to herbicides and diseases and insect pests can be improved by aggregating multiple resistance genes into one rice variety.However,no rice variety with resistance to herbicide,borer,Nilaparvata lugens,bacterial blight and rice blast has been bred,and there is no related research report on whether the resistance level of each gene is enhanced or weakened after the common expression of disease and insect resistance genes in rice.In this study,a highly efficient transgene stacking system was used to assembly the synthetic glyphosate-tolerance gene(I.variabilis-EPSPS~*),lepidopteran pest resistance gene(Cry1C~*),brown planthopper resistance genes(Bph14~* and OsLecRK1~*),bacterial blight resistance gene(Xa23~*)and rice blast resistance gene(Pi9~*)onto a TAC vector.The construct was transferred into ZH11(a widely used japonica rice cultivar Zhonghua 11)via Agrobacterium-mediated transformation,and "multi-resistance rice"(MRR)with desirable agronomic traits was obtained.In addition,we constructed corresponding single gene transformation materials(OE-Cry1C~*,OE-Bph14~*,OE-Pi9~* and ZZXa23~*)for control.Through the resistance identification and RNA-Seq analysis of multi gene transformation materials and single gene transformation materials,the molecular mechanism of synergistic effect between different pest genes was preliminarily explored.The main results are as follows:1.Based on the sequence of resistance genes from wild rice,Bph14~*,OsLecRK1~*,Xa23~* and Pi9~* were synthesized.2.A highly efficient transgene stacking system was used to assembly the synthetic genes I.variabilis-EPSPS~*,Cry1C~*,Bph14~*,OsLecRK1~*,Xa23~* and Pi9~* onto a TAC vector.The construct was transferred into ZH11(a widely used japonica rice cultivar Zhonghua 11)via Agrobacterium-mediated transformation,and "multi-resistance rice"(MRR)with desirable agronomic traits was obtained.3.The six genes transferred from the multi resistant transgenic rice materials could be inherited stably and expressed normally.4.The results showed that the resistance to glyphosate,borer,Nilaparvata lugens,bacterial blight and rice blast were significantly enhanced compared with wild type control zhonghua11.5.Under the condition of no occurrence of diseases and insect pests by spraying pesticides,the results of agronomic traits examination of MRR and zhonghua11 showed that there were some differences in some agronomic traits between MRR and control zhonghua11,but there was no significant difference in yield.6.Under the condition of serious occurrence of diseases and insect pests,the results of agronomic traits examination of MRR and zhonghua11 showed that the yield of MRR was significantly higher than that of zhonghua11,indicating that MRR could effectively resist the invasion of diseases and insect pests in the field,thus avoiding yield reduction.7.The results of resistance identification of multi gene transformation materials and single gene transformation materials showed that the resistance to diseases and insect pests of multi gene transformation materials was higher than that of corresponding single gene transformation materials,which indicated that there was synergistic effect among different resistance genes of diseases and insect pests,which enhanced the disease and insect resistance of multi gene transformation materials.8.The results of RNA-seq analysis of multi gene transformation materials and single gene transformation materials showed that compared with single gene transformation materials,the multiple gene transformation materials had higher up regulation times of rice immune response related genes induced by diseases and insect pests.We speculate that the simultaneous expression of different resistance genes to diseases and insect pests can make the rice produce stronger immune response and enhance the resistance.The above findings suggest that the strategy of using multiple gene conversion can enable the rapid cultivation of rice varieties that can resist multiple biotic stresses and thereby provide cost-effective control and cure of pests.The multi resistant transgenic rice we cultivated showed good and stable resistance to glyphosate,borer,brown planthopper,bacterial blight and rice blast.Under field conditions when pest and disease infestation is severe,MRR materials can withstand attack by pest and disease and thereby avoid yield loss,and are of great application.The molecular mechanism of resistance gene co expression interactions has been preliminarily explored using the means of transcriptome analysis,i.e.,when different pest and pest resistance genes are simultaneously expressed,there is a synergistic interaction between each other to enable rice to develop more robust resistance responses and thus enhance resistance.It provides guidance ideas for the better cultivation of multi resistant transgenic crops with rationally paired resistance gene combinations in other crops in the future.