Development Of Transgenic Rice With Resistance To Lepidoptera And Coleoptera Pests

Rice is one of the most important staple food crops in the world.Insect damage is one of main causes of rice yield loss.Rice leaf folder(Cnaphalocrocis medinalis),striped stem borer(Chilo suppressalis)and yellow stem borer(Tryporyza incertulas)are the major Lepidoptera pests in rice production.Amount of studies had been reported that transgenic Bt rice could effectively control lepidopteran pests.Most of the development transgenic rice carried the selectable marker genes(SMGs).However,the presence of SMGs in transgenic plants would cause a lot of adverse problems.In this study,cry1C*gene and cry2A*gene,respectively with hpt gene,were introduced into Minghui86(MH86)(Oryza sativa L.ssp.indica),an elite indica restorer line,by Agrobacterium tumefaciens-mediated'two T-DNAs in separate Agrobacterium strains' approach.Finally,the marker-free cry1C*and cry2A*insect-resistant rice had been developed by self-crossing segregation of transgenic plants,respectively.In addition,coleopteran rice water weevil(RWW)is another kind of pests in rice fields.It had been reported that Cry3 proteins had toxic to coleopteran pests.In this study,the cry3Aa*and cry3Bb*,which were synthesized with rice codon optimizing,were introduced into Zhonghua 11(Oryza sativa L.ssp,japonica),respectively,with the purpose of development transgenic rice which could control coleopteran pests.The mainly results in this study are as follows:1.Three marker-free cry1C*homozygous lines(named as 1CH1-2,1CH1-4 and 1CH1-9)were selected by PCR and Southern Blotting analysis in the T2 generation of pMF-1C*transformant,and three marker-free cry2A*transgenic homozygous lines(named as 7-61,8-30 and 8-62)were selected in the T2 generation of pMF-2A*transformant through the same methods.2.The protein contents of cry1C*and cry2A*homozygous lines and their corresponding hybrids were measured by enzyme-linked immune sorbent assay(ELISA).The results indicated that the Bt proteins of transgenic homozygous lines and their hybrids were efficiently expressed in leaves and stems at tillering stage,heading stage and filling stage.Besides,the Bt protein contents in the hybrids were lower than those in their corresponding homozygous lines.3.The result of the stem bioassay in the laboratory suggested that the cry1C*and cry2A*homozygous lines could efficiently kill the first-instar of yellow stem borer,and the larvae mortality was up to 100%;the insecticidal capacity of transgenic homozygous lines against striped stem borer was lower than that against yellow stem borer.The transgenic hybrid stems showed equivalent insecticidal activity with their corresponding homozygous lines.4.The insect-resistance assay in the field indicated that the three cry2A*homozygous lines were suffered a little damage from rice leaf folder,all of the transgenic homozygous lines were suffered very few damage from rice stem borers.The transgenic homozygous lines could effectively control rice stem borers and rice leaf folder in the field.5.Agronomic performance of cry1C*and cry2A*transgenic rice in the field were examined.Some agronomic traits of transgenic homozygous lines revealed differences compared with MH86,however,there were no significant difference in yield between transgenic homozygous lines and MH86.Most agronomic performance of transgenic hybrids showed no change with ? Youming86.All the agronomic performances were consistent between hybrid combination Hs-30 and ? Youming86.6.The flanking sequences of marker-free transgenic homozygous lines were isolated by Tail-PCR.The results showed that the T-DNA was inserted into chromosome 8 between Os08g19540 and Os08g19550 in line 1CH1-2,and the T-DNA was inserted into chromosome 2 between Os02g43680 and Os02g43690 in line 8-30.7.The constructs p3300-3Aa*and pDT3Aa were individually introduced into ZH11,and 2 single-copy families and 3 single-copy families with high expression level of cry3Aa*were obtained,respectively.The vectors p3300-3Bb*and pDT3Bb*were individually introduced into ZH11,4 single-copy families and 2 single-copy families with high expression level of cry3Bb*were gained,respectively.8.The result of feeding trace investigation assay showed that the total feeding trace length of rice water weevil adult fed on leaves of transgenic homozygous families were significantly shorter than that of feeding on ZH11 leaves,especially 3300-3Aa-9.It suggested that the cry3Aa*and cry3Bb*homozygous families could efficiently inhibit the feeding of rice water weevil adult.9.The results of transgenic plants feeding assays indicated that transgenic lines 3300-3Aa-9 and DT3Aa-20 could effectively kill the adults of rice water weevil in different stages of transgenic plant.