| Food safety of GM food crops has always been a hot topic of public controversy.It is also an excuse why GM food crops have not been widely commercialized in the world yet.From 2012-2016,our research group developed a system named "gene switch" by using rice green tissue-specific expression promoter rbcS,site-specific recombination system Cre/loxP,nuclear localization signal krp2 and terminator nosT.The exogenous genes controlled by this system could be expressed precisely only in green tissues which provided a new and feasible solution for food safety issues of GM food crops.In order to successfully push the "gene switch" system from basic research to agricultural production application,this paper focuses on the studies of the creation of parental germplsm and two-line insect and herbicide resistance-hybrid rice with null-expression in the endosperm.The main results and conclusions are as follows:1.The work status of gene switch system on Two-line hybrid Rice has been proved to be feasible:five different insect-resistant hybrid combinations 201S-K1/ZR6-LB2,201S-K4/ZR6-LB2,201S-K17/ZR6-LB2,201S-K17/551-LB6 and 604S-K5/ZR6-LB2 were prepared by using "gene switch" system,the content of Cry1Ab/1Ac protein both in the leaves and stems were detected at three stages(tillering stage,flowering stage and filling stage)respectively.Also,the content of CrylAb/lAc protein in brown rice and milled rice at maturity stage was detected.The results showed that the insect-resistant prote:in CrylAb/lAc could be effectively expressed in the leaves and stems of the above five hybrid combinations.Among them,the content of CrylAb/lAc in the leaves was between 270ng/g-1020ng/g,while that in the stems was between 160ng/g-410ng/g.Additionally,it showed no significant difference on the content of CrylAb/lAc in brown rice and milled rice between all hybrid combinations and wild type control.Resistance test results obtained under indoor artificial infestation and field natural outbreaks showed that the insect resistance of all the hybrid combinations were good.The lethal rate of the indoor leaf sample was 100%,which was significantly higher than that of the control 12.04%.The damage rate of the leaffolder in the field was only 0.72%,which was significantly lower than that of the control 62.81%.The results of agronomic traits investigation showed that there was no adverse change in all trait indexes compared with the wild type control.These results suggest that the "gene switch" system works sucessfully in these two-line hybrid combinations.Also,the results prove that it is feasible to breed insect-resistant two-line hybrid rice with null expression of insect-resistant protein in endosperm by using the "gene switch" system.2.The marker-free insect-resistant two-line hybrid rice was successfully created by application of "gene switch" system.A new round of genetic transformation and germplasm innovation was carried out.As a result.the number of new 201S-KEY(containing Cre gene)and 551-LB(containing CrylAb/IAc gene)independent transformants were obtained.The marker-free lines containing 201S-KEY9,KEY12 and 551-LB1,LB7,LB13 and LB16 were screened out.Five hybrid combinations 201S-K9/551-LB1,201S-K9/551-LB7,201S-K9/551-LB13,201S-K9/551-LB16,201S-K12/551-LB1 were then made.ELISA analysis showed that CrylAb/lAc protein was effectively expressed in all these five combinations,and the protein content was between 130ng/g-630ng/g.3.The marker-free herbicide-resistant two-line hybrid rice was also successfully created by application of "gene switch" system.RePAT gene which is a new glyphosate-resistant herbicide gene provided by Professor Liu Ziduo of Huazhong Agricultural University was transferred into the restorer line 112.Through PCR and Southern blot analysis,a set of marker-free and single-copy transgenic lines were obtained.After that,we chose some of those lines to cross with the previously obtained 201S-KEY lines.Five hybrid combinations were also obtained:such as 201S-K17/112-LR1,201S-K4/112-LR16,201S-K1/112-LR21,201S-K1/112-LR24 and 201S-K4/112-LR26.QRT-PCR analysis of the F1 hybrids showed that the RePAT gene could be expressed normally in all these five hybrid combinations at RNA level.Resistance identification results by smearing glyphosate on leaves in seedling and tillering stage showed that when glyphosate was applied at 250 mg/L,the transgenic leaves were completely unaffected.When glyphosate concentration reached 350 mg/L,the transgenic leaves began to be slightly affected.In contrast,when the concentration of glyphosate was 100 mg/L,it began to affect non-GM leaves.When the concentration of glyphosate reached 150 mg/L,the non-GM leaves showed severe symptoms of damage,leading to yellowing and wilting.These results indicated that the herbicide-resistant gene RePAT controlled by gene switch also works normally in the two-line hybrid rice. |
