Developing Novel Transgenic Herbicide-tolerant Rice

Weed pest is one of the major factors that restrict rice production.Controlling weed effectively could improve rice production and promote the development of novel rice production techniques.Glyphosate and glufosinate are the most widely used broad-spectrum herbicide,but they can not be used in rice production directly.Developing transgenic glyphosate-tolerant rice and transgenic glufosinate-tolerant rice could improve the efficiency of weed cotrolling,reduce rice production cost and promote the development of direct-seeded rice.However in China,developing transgenic herbicide-tolerant rice is restricted by the lack of herbicide tolerant genes with agricultural practice value and independent intellectual property rights.Hence,it is of great meaning to cloning novel herbicide-tolerant genes with high efficiency.In this research,a novel glufosinate-tolerant gene(RePAT)and two novel glyphosate-tolerant genes(aroAJ.sp and I.variabilis-EPSPS)cloned by Professor Ziduo Liu's team were transferred into japonica rice variety Zhonghua11 and indica rice variety Minghui86.The agricultural practice value of these novel herbicide-tolerant genes was verified and novel transgenic herbicide tolerant rice varieties with commercial potential were bred,which could serve as good resources for the development of transgenic rice in China.To develop transgenic glufosinate-tolerant rice,a novel glufosinate-tolerant gene RePAT was codon optimized according to codon bias in monocots(designated as RePAT*),synthesized,constructed into the transformation vector and transferred into japonica rice variety Zhonghua11 by Agrobacterium-mediated.transformation.130 positive transgenic rice plants were obtained,80%of which grew normally under the application of 1000 mg/L glufosinate.2 transgenic lines(PAT7 and PAT11)with a single copy of RePAT*integrated into the intergenic region and normal agronomic performances were selected by molecular detection and evaluation of agronomic performances at T0 and T2 generation.At T4 generation,stable inheritation and expression of RePAT*in PAT7 and PAT11 homozygous were confirmed,but the transcript of RePAT*was terminated abnormally in PAT7,by Southern blot,Northern blot and 3' RACE analysis.Glufosinate tolerance assay was also performed in the field,in which major agronomic performances of PAT7 and PAT11 were not affected by 5000 g/ha glufosinate(equivalent to 10 times of the dosage for agricultural practice).PAT11 was finally selected as the candidate transgenic line according to molecular characteristic of transgene and glufosinate tolerance assay.At T6 generation,major agronomic performances of PAT11 were not affected by 5000 g/ha glufosinate in field assay,suggesting that RePAT*is valuable gene for developing transgenic glufosinate-tolerant crop and PAT11 has commercial potential.To develop transgenic glyphosate-tolerant rice,novel glyphosate-tolerant gene aroAj.sp and I.variabilis-EPSPS were codon optimized according to codon bias in monocots,synthesized,constructed into the transformation vector and transferred into japonica rice variety Zhonghua11 by Agrobacterium-mediated transformation.In 3 independent replicated transformation experiments,aroAj.sp*and I.variabilis-EPSPS*were used as the selectable marker,and the results of these experiments suggested that the resistant calli formation efficiency was about 50%and the positive ratio of transgenic plants was about 100%,which indicating that aroAJ.sp*and I.variabilis-EPSPS*could be used as the selectable marker gene in rice transformation.At Ti generation,glyphosate tolerance of transgenic Zhonghua11 with a single copy of aroAj.sp*or I.variabilis-EPSPS*was analyzed,the results of which suggested that transgenic rice with I.variabilis-EPSPS*was more tolerant to glyphosate than those with aroAJ.sp*,therefore I.variabilis-EPSPS*was more suitable for breeding glyphosate-tolerant rice.I.variabilis-EPSPS*was used to develop glyphosate-tolerant rice.115 transgenic lines were obtained by transferring I.variabilis-EPSPS*into the japonica rice variety Zhonghua11.3 candidate transgenic lines(ZY21,ZY25 and ZY29)with a single copy of I.variabilis-EPSPS*integrated into the intergenic region were selected according to Southern blot and inverse PCR.At T3 generation,expression of I.variabilis-EPSPS*in ZY21,ZY25 and ZY29 was confirmed by Northern blot and Western blot,and major agronomic performances of ZY21,ZY25 and ZY29 treated with different dosages of glyphosate were confirmed to be not significantly different from that of the control even though the highest glyphosate dosage was as high as 8400 g/ha(equivalent to 10 times of the dosage for agricultural practice).According to the molecular characteristic of transgene,agronomic performances and glyphosate tolerance assay,ZY21 was finally selected as the candidate transgenic line.At T5 generation,agronomic performances of ZY21 were not significantly affected by 8400 g/ha glyphosate,suggesting that ZY21 has commercial potential.60 transgenic Minghui86 lines were obtained by transferring I.variabilis-EPSPS*into the elite indica rice variety Minghui86.3 candidate transgenic lines(MY28,MY50 and MY51)with a single copy of Lvariabilis-EPSPS*integrated into the intergenic region were selected according to Southern blot and inverse PCR.The transcript of Lvariabilis-EPSPS*was confirmed to be intact and there was translation of Lvariabilis-EPSPS*in leaf and root of MY28,MY50 and MY51 by Northern blot,3'RACE and Western blot.Major agronomic performances of MY28,MY50 and MY51 were also not affected by 8400 g/ha glyphosate in the field assay.MY28,MY50,MY51 were hybridized with non-transgenic sterile line ?-32A.Agronomic performances of the corresponding hybrids were not affected by 8400 g/ha glyphosate in the field assay,suggesting that MY28,MY50 and MY51 can be used to develop hybrid rice.Based on agronomic performances,molecular characteristics and glyphosate tolerance,MY28 was selected as the candidate transgenic line used for developing commercial transgenic rice.This research proved that the glufosinate-tolerant gene RePAT*and the glyphosate-tolerant gene I.variabilis-EPSPS*are valuable gene for developing herbicide-tolerant crops.Novel herbicide-tolerant rice PAT11,ZY21 and MY28 have commercial potential and may promote the development of transgenic rice in China.