| Rapeseed(Brassica napus)is one of the four major oil crops in the world.However,there are a large number of monocotyledonous and dicotyledonous weeds in the whole growth period of B.napus.The weeds will compete with rapeseed for light,temperature,water and fertilizer,seriously affecting the growth of rapeseed,and will lead to the decline of B.napus yield and quality.There are many kinds of weeds in rapeseed field,which greatly limits the selection and use of herbicides.The common selective herbicides can only target one kind or one kind of weeds,and there are strict requirements for the use time and dosage of herbicides,so the field weeding often fails to achieve the desired results.As broad-spectrum herbicides,glyphosate and ammonium glyphosate have the advantages of low toxicity and high efficiency.Therefore,glyphosate-resistant and ammonium glyphosate-resistant B.napus can greatly solve the problem of weeds in the field.On the other hand,the frequent occurrence of extreme weather also seriously affects the growth of B.napus,and whether the resistance of transgenic B.napus will be affected.The expression of target protein in transgenic B.napus with bar gene and transgenic B.napus with CP4-EPSPS gene under different temperature and light intensity,as well as the influence of environmental temperature and light intensity on the resistance of transgenic rape were evaluated.The results are as follows:1.Positive identification of transgenic B.napus with bar gene and transgenic B.napus with CP4-EPSPS gene showed that the target gene had been inserted into the B.napus.2.The bar gene and CP4-EPSPS gene were confirmed to be inserted into the B.napus genome in the form of single copy by Southern Blot detection.3.The T-DNA flanking sequences of transgenic rapeseed with bar gene and transgenic rapeseed with CP4-EPSPS gene were analyzed by Tail-PCR method.The bar gene was inserted into the A02 chromosome of B.napus genome,and the CP4-EPSPS gene was inserted into the A02 chromosome of B.napus genome;The specific detection of transformation in different generations showed that the target gene could be stably inherited in different generations.4.Western blot method was used to confirm that the target protein could be stably expressed in different tissues(root,stem,leaf and seed)of transgenic B.napus.The expression of foreign protein in different tissues(root,stem,leaf,flower and seed)was detected by ELISA.The expression of bar protein in leaves was the highest,about92.84～133.67μg/g,and with the growth of rapeseed,the expression of protein in leaves will decrease,the expression of protein in stems will first increase and then decrease,and the expression of protein in roots will increase;EPSPS protein also has the highest expression in leaves,Between 6.25 and 21.5 μg/g,the protein expression in leaves increased with the growth cycle moving backward,and the expression in stems and roots was relatively stable throughout the growth period,with no significant difference.It indicates that exogenous proteins can be stably expressed in different tissues of transgenic B.napus,and the expression level of exogenous proteins in the leaves of transgenic B.napus are relatively high throughout the entire growth period,indicating that transgenic B.napus are tolerant to herbicides throughout the entire growth period.5.Analyzed the expression of bar protein and EPSPS protein at different temperatures(10,20 and 30 ℃)and light intensity(10000,20000 and 30000 LX).At10 ℃ and 30 ℃,the expression of bar protein was higher than that at 20 ℃,while the expression of EPSPS protein decreased with the increase of temperature;Under different light intensities,the expression of bar protein and EPSPS protein will increase with the increase of light intensity.It indicates that in low temperature or high light intensity environments,the expression level of resistant proteins is higher,and transgenic herbicide resistant B.napus has relatively higher resistance.6.The effects of different temperatures(10,20,and 30℃)and light intensities(10000,20000,and 30000 LX)on resistance to glufosinate-ammonium and glyphosate in B.napus were studied.The results showed that at 10℃,20℃,and 30℃,the highest resistance of rapeseed to glyphosate reached 32 times the recommended concentration in the field,4 times the recommended concentration in the field,and 8 times the recommended concentration in the field;Correspondingly,the highest resistance of glyphosate resistant rapeseed can reach 16 times,8 times,and 8 times the recommended concentration in the field.Under the light intensity of 10000 LX,The resistance of transgenic B.napus to glyphosate can reach four times the recommended concentration in the field;Under 10000 LX light intensity,the resistance of glyphosate resistant rapeseed to glyphosate can reach twice the recommended concentration in the field,while under 20000 LX and 30000 LX light intensity,its resistance can reach 8 and 16 times the recommended concentration in the field,respectively.The results showed that in low temperature environments,the resistance of genetically modified rapeseed to herbicides would be improved,and the stronger the light intensity,the higher the resistance of genetically modified rapeseed to herbicides.7.Through bioinformatics analysis of bar protein and EPSPS protein,it was proved that they had no toxicity,sensitization and antinutrition effects,and the safety of bar protein and EPSPS protein was preliminarily proved. |
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