Development And Evaluation Of Transgenic Rapeseed With Broad-Spectrum Herbicide Resistant Gene

Rapeseed is one of the most important oil crops in China.Weed damage is one of the major biological stresses affecting the growth and yield of rapeseed.Due to the existence of both monocotyledonous and dicotyledonous weeds in rapeseed fields,and the amount types of herbicides must be carefully selected,and the application time is also strictly limited.However,use of selective herbicides are often difficult to achieve desired results.Therefore,rapeseed varieties with broad spectrum of non-selective herbicide-tolerance are highly required to improve the efficiency of rapeseed industry in China.At present,there is no broad spectrum non-selective herbicide resistant rapeseed variety with intellectual property?IP?in China.The aim of this study was to transfer the novel glyphosate-resistant genes I.variabilis-EPSPS^*and the glufosinate-resistance gene RePAT^*with own IP to Brassica napus J9707 by means of Agrobacterium-mediated genetic transformation to develop transgenic rapeseed glyphosate or glufosinate-tolerance.The main findings are as follows:?1?Using rapeseed transformation technology with hypocotyl as explants,130putative I.variabilis-EPSPS^*tolerance plants were obtained under the selective pressure of 30 mg/L glyphosate.PCR detection using the target gene sequences demonstrated that the positive rate of resistant plants was as high as 97%.?2?The results of the resistance treatment of transgenic I.variabilis-EPSPS^*rapeseed at T₀ generation showed that they could grow normally under the conditions of spraying with 100-fold diluted of 41%glyphosate isopropyl-amine.?3?Forty-three possible single-copy inserted transgenic materials were identified from 96 transgenic plants of I.variabilis-EPSPS^*by molecular hybridization?Southern blot?.?4?Tail-PCR and Inverse-PCR methods were used to analyze the flanking sequences of 43 single-copy I.variabilis-EPSPS^*genetic material.A total of 31flanking sequences of insertion sites were identified.There are 14 different types of insertion events in these 31 materials.?5?To evaluate the resistance of T₁ generation of I.variabilis-EPSPS^*-plants,seven lines with different copies were treated and they can grow normally under the spraying condition with 100-fold diluted glyphosate isopropyl-amine.The negative control also failed to survive at a dilution of 200-fold.The ratio of surviving strains to dead strains after treatment with a single copy transgenic line was consistent with a 3:1 single gene segregation ratio.?6?The gene expression and protein level of six T1 generation.I.variabilis-EPSPS^*transgenic lines were analyzed by RT-PCR and Western blot methods.The expression of the target gene I.variabilis-EPSPS^*in transgenic plants was expressed,but the expression levels varied among different materials.The expression of the protein was detected in the transgenic offspring materials,but there were significant differences in protein expression levels between different materials,and protein levels and gene expression levels were consistent.?7?Comparison of glyphosate resistance of transgenic I.variabilis-EPSPS^*materials and CP4-epsps genetic material from three different transformation events.The results show that two types of materials containing different resistance genes have similar tolerance levels of glyphosate.?8?Using rapeseed transformation technology with hypocotyls as explants,93putative RePAT*transgenic plants were obtained under 2.5 mg/L glufosinate selective pressure,but the positive rate was only 8.6%.The resistance treatment of transgenic plants of T₀ and T₁ generations of transgenic plants with RePAT*revealed that some plants could survive under 100-fold dilution of 18%glufosinate.