| Environment safety is an important factor limiting the commercialization of these transgenic rice lines.It must be determined whether the escaped exogenous genes can survive in the local natural ecosystem or radiate beyond it.These are the main criteria for the assessment and monitoring of the environmental safety of transgenic rice.The fitness and differential gene expression of insect-resistance transgenic rice in farmland ecosystem have been reported extensively.However,to date,there have been no reports on the fitness performance,differential gene expression and protein interaction of insect-resistance transgenic rice derived from the seeds that escaped from the tillage system and those that dispersed into natural ecosystem.In the present study,the cry1Ab/c transgenic rice Huahui-1(HH1),cry1C*/bar transgenic rice TIC-19 and non-transgenic rice Minghui-63(MH63)are taken as materials to:(1)evaluate the fitness of these rice lines under simulating natural ecosystem including saline-alkaline soil,wasteland soil and weed competition;(2)evaluate the differential gene expression of T1C-19 by RNA-seq and proteomics under the weed competition and wasteland soil;and(3)explore the protein interaction between Cry1Ab/c and rice endogenous proteins in Huahui-1,the cellular localization of Cry1Ab/c protein and protein interaction between Cry1Ab/c and rice endogenous membrane proteins under farmland soil,and interaction between Cry1Ab/c protein and rice flowering protein Hd3a under saline-alkali soil condition.These results can provide scientific evidence for environment safety in the commercial cultivation of HHland T1C-19 in the future.The results were as follows:1.The fitness of transgenic rice Huahui-1 and T1C-19 under stimulating different natural conditionsThe two kinds of transgenic rice Huahuil and T1C-19 were grown on simulating natural conditions.The vegetative and reproductive fitness of the rice and the expression of exogenous Cry1Ab/c protein were assessed:The expression of two exogenous Bt proteins in the two kinds of transgenic rice grown on saline-alkaline soil was lower than that in the strain grown on farmland soil.For HH1 rice,the vegetative growth abilities,evaluated by tiller number and biomass,and reproductive growth abilities,measured by filled grain number and filled grain weight per plant,showed a significantly higher fitness cost than that for the parental rice Minghui63 grown under the same soil conditions,and the magnitude of this fitness cost was relatively smaller than that of the rice grown on farmland soil.For T1C-19 rice,the vegetative growth,evaluated via plant height,tiller number,and biomass,and the reproductive growth indices,evaluated via the filled grain number and filled grain weight per plant,showed a significantly higher fitness cost than that for the parental rice Minghui63 grown under the farmland soil.Except for tiller number and biomass of T1C-19,which were significantly lower than those of MH63,there were no significant differences in other vegetative growth indices between T1C-19 and MH63 in alkaline-saline soil.The reproductive growth indices of T1C-19 were significantly higher than those of Minghui63.Under farmland soil and alkaline-saline soil conditions,heading time of HH1 was delayed by 12d and 25d than Minghui63.However,the heading time of T1C-19 was similar to Minghui63.The expression of two exogenous Bt proteins in the two kinds of transgenic rice grown on wasteland soil without weed completion,farmland soil with weed completion,and wasteland soil with weed completion were lower than that in the strain grown on farmland soil without weed completion.For HH1 grown on farmland soil and wasteland soil without weed completion,except for plant height,the vegetative growth abilities,evaluated by tiller number and biomass,and the reproductive growth abilities,measured by filled grain number and filled grain weight per plant,showed a significantly higher fitness cost than that for the parental rice Minghui63 grown under the same soil conditions.However,for HH1 grown on farmland soil and wasteland soil with weed completion,the vegetative growth abilities,evaluated by tiller number and biomass,and the reproductive growth abilities,measured by filled grain number and filled grain weight per plant,didn't show a significant difference with the parental rice Minghui63 grown under the same soil conditions.For TIC-19 grown on farmland soil and wasteland soil without weed completion,the vegetative growth abilities,evaluated by tiller number and biomass,and the reproductive growth abilities,measured by filled grain number and filled grain weight per plant,showed a significantly higher fitness cost than that for the parental rice Minghui63 grown on the same soil conditions.However,for T1C-19 grown on farmland soil and wasteland soil with weed completion,the vegetative growth abilities,evaluated by tiller number and biomass,and the reproductive growth abilities,measured by filled grain number and filled grain weight per plant,didn't show a significant difference with the parental rice Minghui63 grown under the same soil conditions.2.The differential expression of gene in T1C-19 under weed competition and wasteland soil conditionsThe two kinds of transgenic rice T1C-19 and MH63 were grown on simulating farmland soil and wasteland soil without weed completion,farmland soil and wasteland soil with weed completion.The differential expression gene and differential expression protein were analyzed by RNA-seq and TMT-based quantitive proteomics,respectively:The results of RNA-seq showed that,compared with MH63,a total of 3,786 different expression genes were found in T1C-19,including 150 shared different expression genes representing 0.4%of the rice genome under farmland soil and wasteland soil without weed competition,under farmland soil and wasteland soil without weed competition.The analysis of 150 shared different expression genes could be associated to exogenous cry1C*/bar genes.The KEGG pathway analysis showed that 150 shared different expression genes were significantly related to four pathways,including the signal pathway of photosynthesis,rhythm changing of light reactions,plant cell endocytosis and cell apoptosis.The result of qRT-PCR showed the expression level of these seven shared different expression genes,including Lhcbl,Lhcb2,Lhcb3,Lhcb5(related to photosynthesis),FKF1,PRR(related to rhythm changing of light reactions),and HSP70(related to plant cell endocytosis and cell apoptosis)related to these four pathways was down regulated in T1C-19 than that for the parental rice MH63 under four planting conditions.The aforementioned findings corroborated the results of RNA-seq.The exogenous Cry1C*protein could interact with six shared different proteins,such as photosynthetic antenna protein Lhcbl and so on,related to these four pathways mentioned above by bimolecular fluorescence complementation.Then for T1C-19 and MH63,compared with farmland soil without weed competition,the number of different expression genes was 1,084 and 1,234 under wasteland soil without weed competition,963 and 1,226 under farmland soil with weed competition,1,053 and 1,505 under wasteland soil with weed competition,respectively.A total of 2,743 and 2,200 different expression genes represented 8.50%and 6.86%of the rice genome under three stressed conditons.Therefore,the results showed that TIC-19 underwent extensive changes in gene expression under different planting conditions similar to parental MH63.Additionally,although there appeared to be a large number of stress-induced changes to different expression genes that were specific to T1C-19 or to MH63 rice under all the three stresses,the differences were not reflected in the over-all pattern of distribution of the genes among the different functional classes.By this criterion,it is speculated that the TIC-19 and MH63 were significantly indistinguishable,and exogenous cry1C*/bar gene insertion may not significantly change the adaptability for these stresses.The results of proteomics showed that,compared with MH63,a total of 2,177 different expression proteins were found in T1C-19,including 121 shared different expression proteins representing 1.7%of the rice proteome under farmland soil and wasteland soil without weed competition.The analysis of 121 shared different expression proteins could be associated to exogenous cry1C*/bar genes.The KEGG pathway analysis showed that 121 shared different expression proteins were significantly related to ribosome involved signaling pathways.Then for T1C-19 and MH63,compared with farmland soil without weed competition,the number of different expression proteins was 411 and 300 under wasteland soil without weed competition,662 and 430 under farmland soil with weed competition,309 and 375 under wasteland soil with weed competition,respectively.A total of 839 and 1,028 different expression proteins represented 14.52%and 11.87%of the rice genome under wasteland soil without weed competition and under wasteland soil with weed competition,respectively.Therefore,the results showed that T1C-19 and MH63 underwent extensive changes in protein expression under different planting conditions.Then,Additionally,although there appeared to be a large number of stress-induced changes to different expression proteins that were specific to TIC-19 or to MH63 rice under all the three stresses,the differences were not reflected in the over-all pattern of distribution of the genes among the different functional classes.By this criterion,it is speculated that the TIC-19 and MH63 were significantly indistinguishable,and exogenous cry1C*/bar gene insertion may not significantly change the adaptability for these stresses.3.The interaction of exogenous Cry1Ab/c protein and endogenous protein in insect-resistant transgenic rice Huahui-1Firstly,the study used exogenous Cry1Ab/c protein as bait to screen rice cDNA library through the yeast two-hybrid screening system.And finally,60 rice endogenous proteins interacting with exogenous Cry1Ab/c protein were obtained,including defense responses,redox reaction(mainly in photosynthesis),substrate metabolism,and some unknown functional proteins.Six putative proteins which involved in photosynthesis and nine putative proteins which involved in defense responses were further confirmed that they could interact with exogenous Cry1Ab/c protein by subcellular co-localization,bimolecular fluorescence complementation,and co-immunoprecipitation assay in tobacco cells.In addition,by using rice protoplasts and tobacco leaves as materials,exogenous Cry1Ab/c protein could locate at cell membrane,nucleus,and cytoplasmic membrane by situ immunohistochemistry,situ immunofluorescence,western blot(detecting cell membrane proteins and cytoplasmic proteins separately),and subcellular localization.Exogenous Cry1Ab/c protein could interact with two membrane proteins V-H+-ATPase and Ca2+-ATPase.The interaction between Cry1Ab/c protein and five predominant rice proteins in the heading stage was tested by yeast-two-hybrid technique,subcellular co-localization,BiFC and CO-IP.The result showed that Cry1Ab/c only could interact with node protein in the heading stage and was coded by gene Hd3a,and the level of mRNA Hd3a was significantly down regulated in HH1.It is speculated that all of them may play a key role in delaying heading date of HH1rice. |
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