Construction And Validation Of A Lethal System For F₁ Generation Transgenic Pollen And Egg Cell

Transgenic technology has become an important approach of the modern plant breeding research by the attention of the horticultural and agronomic breeding researchers for its strong purpose in the modification of gene,genetic resources application from any species,modification and innovation in agronomy characters and accelerating in the process of breeding.There are several candidate strategies to protect the foreign genes in transgenic plants from drifting by pollen and seed such as male sterility and seed sterility.But male sterility and seed sterility could not be the good choices for the crops that produce seeds and fruits as their mail productions.Therefore,the technologies of transposon-mediated approaches,site-specific recombinase system and homologous recombination were utilized in the safety-control of transgenic plants to solve the contradiction between preventing transgene flowing via pollen or seed and seed obtaining.But the technologies of homologous recombination,transposon-mediated approaches have some disadvantages such as low efficiency that can not satisfy the need of safety control in the large-scale cultivation of transgenic crops.The technology of site-specific recombinase system is intensively studied in the research of the cancel of the foreign genes in transgenic plants.Cre/LoxP system is studied the most intensively and widely in the recombination systems for its advantages such as efficiency,fast and accurate.Presently,there have been large amount of Cre/Lox P systems controlled by tissue-specific promoters in transgeinc plants to create the pollen,seed and fruit without transgene.But those systems do not solve the problem of transgene-free pollen,fruit and seed in F₁ plants.Based on the above problems,in this research,the Cre/Lox P recombination system controlled by the AtPKL seed germination specific promoter or TAGiLAT stamen/pistil specific promoter via the medium of tetracycline/heat shock control gene expression system was integrated into the alien chromosome of the T0 parents by the type of split form.After the integration of co-transformation,the separated pollen/egg cell specific promoter and Barnase lethal gene were recombined for the components such as the nptII gene between them were removed during the process the T1 seed germination of the transgenic plants contain the AtPKL seed germination specific promoter or during the process of stamen/pistil development of the transgenic plants contain the TAGiLAT stamen/pistil specific promoter.And then,the pollen and egg cells contain transgenic foreign genes would be killed to get the marker free,non-transgenic,and partly fertile progeny plants.This method may deal with the transgene drift of the F₁ transgenic crops which provide seed and/or fruit as their main production after being further improved.The main results are as follows:1 Establishment and verification of the heat-shock-on/tetracycline-off control systemConstructed a binary expression vector pVCT2041 which contain the heat-shock-on/tetracycline-off control system.The vector p VCT2041 was introduce into tobacco.The result of GUS histochemical staining,quantitative enzyme activity analysis and qRT-PCR showed that under the condition of heat shock,the reporter gene expression under the control of this system reached the level similar to the gene under the control of the CaMV 35 S promoter.Meanwhile,the GUS gene expression could be effectively inhibited under the condition of heat shock when tetracycline was added.2 Cloning and verification of the arabidopsis germination promoter AtPKLArabidopsis germination specific promoter AtPKL was cloned according to the sequence published in the GenBank.The GFP gene controlled by the AtPKL promoter in the transgenic tobacco plants expressed 1 h after the seeds absorbed water,and and continued to express during the process of the development of the transgenic plants in every part of the plant.But the GFP gene did not express during the seed development or in the dry seeds.3 Establishment and verification of the lethal system of transgenic pollen and egg cell controlled by heat shockA pollen-egg cell specific promoter Pol/Egg was constructed by combine the tomato pollen specific promoter Le LAT52 with the arabidopsis egg cell specific promoter DD45 that published by GenBank.Set a GFP reporter gene under the control of Pol/Egg to construct the binary expression vector pVCT2331.The result of microscopic identification and RT-PCR showed that,The GFP gene controlled by the Pol/Egg promoter in the pVCT2331 transgenic tobacco plants expressed only in the pollen and the embryo sac cells,in the embryo sac,it expressed last from unfertilized period to the torpedo embryo stage.Construct the binary expression vector pVCT2324 to set the Cre/Lox P recombination system under the control of heat shock promoter HSP70 m.Under the condition of heat shock,Cre gene expression can lead the recombination of the Pol/Egg promoter and the LoxP-?-Barnase::T35s sequence to kill the pollen and egg cells that take the foreign gene.The pollen of the p VCT2324 transgenic plants collapsed after heat shock recombination,the average number of pollen in a single flower of PVCT2324 transgenic tobacco plant were 2.3×105±0.94×105,2.2×105±0.66×105,2.4×105±0.87×105 and 2.3×105±0.86×105 in the 4 transgenic lines before recombination and were 1.4×105±0.65×105,1.3×105±0.43×105,1.2×105±0.54×105 and 1.5×105±0.59×105 after recombination;the average number of seeds in a single fruit of PVCT2324 transgenic tobacco plant were 733.4±2.7,656.4±2.3,720.6±5.4 and 679.3±5.4 before recombination and were 323.2.±2.5,334.6±3.6,420.6±2.8 and 383.4±3.6 after recombination;the rate of the seed of pVCT2324 transgenic plants that taking foreign gene was 98.03%,96.07%,96.7% and 93.31% before heat shock recombination,it declined significantly to 18.73%,28.22%,19.52% and 23.18% after heat shock recombination.4 Establishment and verification of the separable transgenic pollen and egg cell lethal systemthe Cre/Lox P recombination system controlled by the AtPKL seed germination specific promoter or TAGiLAT stamen/pistil specific promoter via the medium of tetracycline/heat shock control gene expression system was integrated into the alien chromosome of the T0 parents by the type of split form.After the integration of co-transformation,the separated pollen/egg cell specific promoter and Barnase lethal gene were recombined for the components such as the nptII gene between them were removed during the process the T1 seed germination of the transgenic plants contain the AtPKL seed germination specific promoter or during the process of stamen/pistil development of the transgenic plants contain the TAGiLAT stamen/pistil specific promoter.And then,the pollen and egg cells contain transgenic foreign genes would be killed.Binary expression vector pVCT2419,pVCT2420 and pVCT2421 were constructed,pVCT2419/pVCT2420 and pVCT2419/pVCT2421 were introduced into tobacco separately.7 transgenic lines of tobacco plant introduced by pVCT2419,4 transgenic lines of tobacco plant introduced by pVCT2420,4 transgenic lines of tobacco plant introduced by p VCT2421,4 transgenic lines of tobacco plant introduced by pVCT2419/2420 and 3 transgenic lines of tobacco plant introduced by pVCT2419/2421 were obtained by PCR identification of Kan resistant tobacco.The result showed that the average numbers of pollen in single flower and seeds in single fruit of the pVCT2419/pVCT2420 and the pVCT2419/pVCT2421 co-transgenic plants were significantly lower than that of pVCT2419,pVCT2420 and pVCT2421 transgenic plants.The result of GUS histochemical staining also showed that,the rate of the progeny of the pVCT2419/pVCT2420 and the pVCT2419/pVCT2421 co-transgenic plants were lower than that of pVCT2419,pVCT2420 and pVCT2421 transgenic plants.