| Rice（Oryza sativa L）is the most important food crop in China and has a wide range of cultivation.Weeds can cause rice production declining dramatically or damaging the quality of rice.Controling weed with chemical herbicides is the most economical and effective method in agricultural production at present.Glyphosate is the most widely used broad-spectrum herbicide,but because it is also toxic to rice,it is difficult to be used in the process of rice production directly.It is of great significance to cultivate glyphosate-resistant transgenic rice by mining new glyphosate-resistant genes which can be applied to agricultural production.5-enolpyruvate shikimate-3-phosphate synthetase（EPSPS）is the key enzyme of shikimate pathway in plants and a target enzyme for the herbicide glyphosate.The glyphosate tolerance of plants can be effectively improved by transferring EPSPS genes insensitive to glyphosate or overexpression of EPSPS in transgenic plants.The EPSPS coding gene ELs-EPSPS from Liriope spicata（Thunb.）Lour is a novel gene that can be used to cultivate herbicide-resistant crops.Therefore,the target gene was transferred into japonica rice cultivars ZH11 by genetic engineering in order to verify the application potential of optimized ELs-EPSPS* and develop transgenic rice with glyphosate tolerance.The main results of this research are as following:1.The original glyphosate-resistant gene ELs-EPSPS was codon optimized according to codon bias in monocots,synthesized and constructed into the plant overexpression vector.2.ELs-EPSPS* was transferred into ZH11 by Agrobacterium-mediated transformation,and 91 transgenic rice plants were obtained.Through glyphosate resistance test,90% T0 transformed plants grew normally under the application 3600 mg/L glyphosate.3.At T0 generation,4 transgenic plants were obtained with high expression and single copy of ELs-EPSPS* integrated into the intergenic region were selected by Southern blot,inverse PCR and q RT-PCR detection.4.At T2 generation,the major agronomic performances of the candidate transgenic lines were not significantly different compared with the wild type ZH11.The field glyphosate resistance test showed that the transgenic line TG14 could tolerate the spraying of 9000 g/ha glyphosate at the seedling stage,the overexpression of ELs-EPSPS* resulted in higher tolerance to glyphosate（up to tenfold of the recommended dose）,and the transgenic line TG14 did not show drug damage and grew normally.The transgenic ZH11 line TG14 reached the requirement of glyphosate-resistant level in rice production and could be used as a donor for developing transgenic glyphosate-resistant rice.The transgenic with high glyphosate-tolerance was obtained,which proved that the novel glyphosate resistance gene ELs-EPSPS* is an available glyphosate-resistance gene resource,which has important value in the cultivation of glyphosate-resistant crops. |
