| At present,maize(Zea mays L.)is one of the main sources of wealth and income for farmers.However,maize is usually affected by a series of diseases and insect pests,so as one of the most important factors,maize is greatly damaged in the growth process.In addition,the single disease-resistant and insect-resistant varieties can no longer meet the demand,so the use of genetic engineering technology to construct the expression vector of insect-resistant and insect-resistant bivalent genes to transform maize can not only improve the double-resistance ability of transgenic maize,but also obtain new germplasm resources.This research through genetic engineering technology to build both NPR1 disease resistance genes and insect-resistant genes Cry1Ab13-1 gene and bar bivalent plant expression vector for selection markers,mediated by agrobacteriumtume faciens,pollen tube channel and ultrasonic assisted pollen the guide these three transformation methods for the construction of well plant bivalent expression vector in maize genetic transformation.And the T2 generation transgenic plants were detected by PCR,Southern blotting integration,real-time fluorescence quantitative PCR transcriptional level detection,indoor and outdoor insect resistance and indoor disease resistance.In order to obtain the excellent characteristics of both disease and insect resistance of the new lines and to create a new variety of corn to lay a foundation.The main research results of this experiment are as follow:(1)Because there were no promoters and terminators in the polyclonal sites,the NPR1 gene was inserted into the functional elements to obtain the target fragment35S+NPR1 gene +NOS(1275bp).After cloning,an intermediate vector PB121-35SNPR1-NOS was obtained to preserve the target fragment,and then the above target fragments were linked into the polyclone using the constructed expression vector p CAMBIA3301-cry1AB13-1 as the base vector Between the site xbaⅠand SacⅠ.The recombinant bivalent plant expression vector P Cambia3301-Npr1-cry1Ab13 was constructed using Bar as the screening marker gene and carrying both Npr1 and cry1Ab13-1 genes.(2)Agrobacterium-mediated genetic transformation of maize inbred line "H99" was carried out 5 transgenic plants of T0 generation,and the seeds obtained from these 5 plants were seeded in transgenic fields.After screening,a total of 2 positive plants of T1 generation were obtained,and 2 positive plants of T2 generation were obtained after screening T1 generation and sowing with Glufosinate ammonium.The influence of pollen tube channel method on maize inbred lines 90493.S3002.MJ-1,W107,W108,349,Zheng58.After transformation,the T0-generation grains were recovered and sown in the transgenic field and screened by Glufosinate ammonium,a total of 5 plants of T1 generation and 6 plants of T2 generation were obtained.The maize inbred lines MJ-1,W108,349,Zheng 58,Bao D and Bao E were transformed by ultrasound-assisted pollen mass guide method.Finally,54 transgenic seeds of the TO generation were harvested.After transgenic field seeding,a total of 4 plants of the T1 generation and 6 plants of the T2 generation were obtained.(3)Southern Blotting results indicateed that gene NPR1 and Cry1AB13-1 were a single copy,and consolidate into the maize by single method.Real-time quantitative PCR consequence displayed that gene Npr1 and Cry1Ab13-1 were transmitted in different tissues,and the relative expression levels were higher than those of negative control.(4)Insect-resistant experiments in and out of the laboratory showed that the resistance level of the positive plants was increased from the susceptible level of the receptor to the medium resistant level.The laboratory test showed that the positive plants were highly resistant,while the acceptor plants were medium resistant;... |
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