Production Of Cry Proteins In Transgenic Bt Rice Tissues And Their Degradation In The Residues

The development and application of insect-resistant genetically modified(IRGM)rice provide a new and more economic,safety,and useful approach than chemical control for the control of rice pests.Many researches have been reported since the first transgenic Bt rice was developed.In the area of IRGM rice,China leads the world,and has developed a large number of new IRGM rice lines.These lines including mfb-MH86 expressing Cry1 Ab generated recently by mark-free transgenic technology and T2A-1 expressing Cry2 A and T1C-19 expressing Cry1 C,and the latter two lines have been in the application of bio-safety certificate stage.All the three Bt rice lines have shown great commercial potential.As a main food crop,it is very important to assess their safty fully and compositively before commercialized.An important aspect of the environmental risk assessment of transgenic Bt rice is to assess the potential risks of their Cry protein on non-target organisms,such as natural enemies,soil organisms and aquatic organisms in farmland ecosystem.Therefore,making clear content,insecticidal activity and degradation of Cry protein in plant residues of Bt rice is the important basis for systematic evaluation of the environmental risk of exogenous Cry protein produced by Bt rice.So,mfb-MH86 and its corresponding nontransformed isoline Minghui86(MH86)were used for evaluating the seasonal expression of Cry1 Ab in the Bt rice line and its efficacy for control of the target pest,Chilo suppressalis.In addition,T2A-1,and T1C-19,and their corresponding non-transformed near isoline Minghui 63(MH63)were used to study the degradation of Cry protein in rice residues.We used litter bags to determine degradation patterns of Cry2 A and Cry1 C proteins in rice leaf residues,covered on soil surface or buried in horizontal position at a depth of 5 cm,from harvest to the next harvest.On the other hand,we monitored the dynamics of the decomposition of the two insecticidal proteins in Bt rice stubble under field conditions after harvest.The changes of Cry protein in Bt rice residues was monitored by the enzyme-linked immunosorbent assay(ELISA).1)Expression of Cry1 Ab protein in mfb-MH86 and its efficacy in controlling Chilo suppressalis:The expression of Cry1 Ab protein in mfb-MH86 leaves,stems and leaf sheaths(hereinafter referred to as stems),and roots was evaluated throughout the rice-growing season using an enzyme-linked immunosorbent assay.In addition,mfb-MH86 resistance to C.suppressalis,a major pest of rice,was evaluated in a laboratory bioassay with field-collected rice stems.Cry1 Ab protein levels of mfb-MH86 were highest in leaves(9.71±34.09g/g dry weight [DW]),intermediate in stems(7.66±18.51g/g DW),and lowest in roots(1.95±13.40 g/g DW).In all tissues,Cry1 Ab levels in mfb-MH86 were higher in seedling and tillering stages than in subsequent growth stages.In the laboratory bioassay,mortality of C.suppressalis after 6 d of feeding on mfb-MH86 stems was 100% throughout the rice-growing season;mortality of C.suppressalis when feeding on stems of the nontransformed isoline,MH86,ranged from 15.0 to 38.3%.The results indicate that Cry1 Ab protein levels in mfb-MH86 stems are sufficient to protect plants against C.suppressalis throughout the rice-growing season.2)The degradation of Cry protein in rice residues:(1)The residual contents of Cry proteins in stubble(including root and stalk)were significantly differently among different rice lines,different tissue types or different sampling dates.The initial amount of Bt protein in T2A-1 stubble was significantly higher than that in T1C-19 stubble.And the Bt protein contents in transgenic rice stalk residues are usually higher than those in the root remnants.During the whole sampling period,Cry2 A protein in T2A-1 stubble was gradually degrade after a period of the Cry2 A protein content increase,and the degradation rate of Cry2 A protein in stalk was faster than that in root.8 months after the beginning of the experiment,Cry2 A protein concentration in stalks decreased rapidly to 0.06±0.01?g/g DW,0.79% of the initial amount,and then continued to be degraded smoothly;while Cry2 A protein content in roots was still 0.09±0.01?g/g DW,accounting for 19.95% of the initial content,and then came to a state of fluctuation.Cry2 A protein in stalk and root residues still can be detected when the experiment was terminated.The degradation dynamic pattern of Cry1 C protein content in T1C-19 stubble was different with the Cry2 A protein content in T2A-1 stubble,presenting a decline trend on the whole,during which there was a short-term increase;and after 8 months,Cry1 C protein could not be detected in T1C-19 stalk and root residues.(2)The residual amounts of Cry proteins in leaves were significantly different among different rice lines,different treatments and different sampling dates.In general,the Cry protein content in T2A-1 leaf residues was higher than that in T1C-19 leaf remnants.Cry protein contents in leaf residues covered on soil surface were not significantly different with those in leaf residues buried in soil during the whole experiment period.However,there was significant difference between the two treatments during the period from December 2012 to May 2013.Cry2 A protein content in the T2A-1 leaves covered on soil surface was rapidly degraded to 15.62% of the initial content in the first 3 months,and then after two stable months re-enters into the accelerated degradation,and decreased to 0.03% of initial content at the end of the experiment;but there was a different Cry2 A degradation pattern in the leaves buried in soil,with the increase in the first 3 months and then decreasing,and lastly,reaching about 0.09% of the initial content at the end.In contrast,Cry1 C protein content in T1C-19 leaves,whether it is covered on soil or buried in soil,having a decline trend on the whole,and after 7 months,reaching the almost undetectable level.Meanwhile,the treatment of leaves buried in soil is more beneficial to the degradation of Cry1 C protein in T1C-19 leaves.In field conditions,the degradation of Bt protein in rice residues is affected by combination of temperature,precipitation and many other factors,showing seasonal characteristics,degraded rapidly in the spring and summer to the the very low or undetectable level.In conclusion,Cry proteins contents in residual tissues of T1C-19 and T2A-1 rapidly declined during early stage and slowly and stablely at a later stage.In order to reduce the residual amount,we combined with the degradation patterns of Bt protein in different rice residues,suggested that T1C-19 rice residues should be buried in soil with the plowing after the rice harvest,and T2A-1 rice residues should be covered on soil surface after the rice harvest.