Development Of Glyphosate-resistance Rice With G2-aroA And The Assessment Of Reassembled G2-EPSPS After Splitted

Rice is an important grain crop in the world,occupying a main position in national economy.Paddy field weeding runs through the whole process of rice production,which increases the labor and economic input.The use of chemical herbicides has a very important significance to reduce the cost of rice production.Glyphosate is a broad spectrum,efficient,low toxicity,cheap,translocated herbicides,but glyphosate is rarely used in paddy field in a large area for its lethal effect on rice.By means of genetic engineering breeding of transgenic rice with resistance to glyphosate can save paddy weeding cost,reduce labor input,decrease the use of herbicides,and has good application prospect to increase farmers'income.But with the development of transgenic technology,transgenic safety problems have attracted widespread attention,especially the pollen mediated transgene flow problem has been the focus of public attention and part of the research community,however gene split technology can reduce transgenic drift frequency to a great extent.Therefore,this study transferred the gene G2-aroA which express G2-EPSPS protein in vivo into rice to develope glyphosate resistant transgenic rice,to explore glyphosate resistantance of transgenic rice with G2-aroA gene.And through the gene split technique study Intein mediated protein reassembly efficiency,which provided data support for the application to control the rice gene flow with gene split technology.The results are as follows:1.Development and Event-Specific Detection of Transgenic Glyphosate-Resistant Rice Expressing the G2-aroA Gene1)To investigate the agronomic performance of the two transgenic lines,seeds from ZH11,G2-6,and G2-7 were germinated in ultrapure water with 0,50,and 100 ppm glyphosate.The result showed that ZH11 germination was inhibited in 50 ppm,and germ soon rotted in 100 ppm,whereas G2-6 and G2-7 in 50 ppm and 100 ppm growth had no obvious difference from the control group,which indicated that the transgenic lines received glyphosate resistance from G2-aroA gene,and the glyphosate concentration of 50 ppm can be used as rice glyphosate fast screening at the bud stage.2)Spraying glyphosate at seedling stage,ZH11 was dead at 1000 ppm glyphosate concentration,while G2-6 and G2-7 at 20000 ppm still survived,which means the gene G2-aroA increases the glyphosate resistance of rice seedling by 20 times at least.By measuring height and statistical analysis after spraying,shoot heights of both transgenic lines were significantly decreased compared with untreated group.The height showed no significant difference when treated with 3000 ppm and 5000ppm,but lower than the control group significantly,and the height showed no significant difference between treatment under 8000 ppm-20000 ppm,but lower than that in 3000 ppm and 5000 ppm treatment sianificantly.3)By adding different concentrations of glyphosate in hydroponic solution,we measured the height of G2-6 and G2-7,the used a glyphosate dose-response curve to determine the relationship between plant height and glyphosate concentration.Calculateing theglyphosate concentration when the plant height is inhibited to 50%,we confirmed that the I₅₀0 of ZH11,G2-6 and G2-7 were 0.93,96.4 and114.07,respectively.It means that the glyphosate resistance of G2-6 and G2-7 were increased by 104and 123 times.4)By amplifying the T-DNA 5'flanking sequences of G2-6,certified the T-DNA insertion site was Chr 8:23685037.We download the up and dowstream sequences and designed specific primers G2-OsF and G2-OsR,designed the primer G2-TR according to the sequence near T-DNA left-border,established the specific PCR detection system and the detection PCR system with three primers method for G2-6 homozygous lines2.Application and efficiency of gene split technology in rice1)The flanking sequences of T-DNA insertion were amplified by modified Tail-PCR method from En transgenic lines?containing EPSPSn-In gene?and Ec transgenic lines?containing Ic-EPSPSc gene?,combined with Southern blot analysis exogenous gene copy number of transgenic rice gene,we confirmed that the T-DNA of En-1,En-12 and Ec-26 lines were inserted into the chromosome 2 with one copy,and Ec-22 line were inserted into the chromosome 6 with one copy.2)Taking En-1 and En-12 as the female parent,Ec-22 and Ec-26 as the male parent,we obtained the hybrid En-1/Ec-26,En-12/Ec-26 which EPSPSn-In and Ic-EPSPSc were inserted into homologous chromosome,and hybrid En-12/Ec-22 which EPSPSn-In and Ic-EPSPSc were inserted into nonhomologous chromosome through artificial hybridization.3)Through seedling glyphosate spraying experiment,we found that the hybrid material En-1/Ec-26 can survive after sprayed by 5000 ppm glyphosate,and the growth rate is significantly lower than those in control group.4)Different types of glyphosate resistant transgenic plants were analysises by spraying glyphosate,the result showed that the hybrid and G2-6 could tolerant 2000 ppm glyphosate,in contrast En-1,En-12,Ec-22,Ec-26,ZH11 died in ten days after spraying glyphosate.It indicated that both of the N-terminal and the C-terminal of G2-EPSPS protein can not perform glyphosate-tolerance after splitted.In the hybrid En-1/Ec-26,En-12/Ec-26 and En-12/Ec-22,the splitted protein fragments of G2-EPSPS were reconstructed by Ssp DnaE Intein,resulting in a functional EPSPS protein.5)By adding different concentrations of glyphosate in hydroponic solution,analysis the plant height of hybrid En-1/Ec-26,En-12/Ec-26,En-12/Ec-22,G2-6,G2-7 and ZH11 in the different treatment groups,calculate concentration of glyphosate when plant height was inhibited at 50%by the dose-responce curve,I₅₀ for hybrid En-1/Ec-26 is 23.78,for En-12/Ec-22 is 20.62,for En-12/Ec-26 is17.81,which was 25.6 times,22.1 times and 17.81 times higher than that of ZH11,and the tolerance of glyphosate was about 4-6.8 times lower than that of G2-6 and G2-7.6)The expression ratio of RNA in hybrid is lower than that in parent plants,about 50%of the total protein level by fluorescent quantitative PCR analysis in leaf.Total protein Western blot analysis revealed the presence of intact EPSPS protein in the hybrid.The results showed that Intein mediated protein reconstruction was completed in the hybrids,and the protein fragments were reassembled into complete functional protein.EPSPSn-In and Ic-EPSPSc protein fragments haven't been used were detected by anti-In-N and anti-Ec,the utilization efficiency of EPSPSn-In was 91%,67%,90%,and Ic-EPSPSc was 83%,81%and 87%respectively.