The Cultivation Of Transformated NR Antisense Gene Tetratetraploid Antirrhinum Majus

Exerting the physiological role of the ethylene in Plants is through the biosynthetic pathway of ethylene and ethylene signal transduction pathway.Just as the ETR1 gene,NR gene exists as an ethylene receptor,which's reception of the ethylene signal transduction and directly impact on plant physiology,growth and the resistance of plant.NR gene not only controls the mature of fruit,but also is closely related to other physiological and plant metabolism,growth and development. Through the NR antisense transgenic tetraploid gene acquisition,it can study the relationship between the NR gene and the physiological metabolism of plants,plant growth,development and fruit ripening,and identify the function of NR gene,it also can acquire new varieties or breeding materials which are able to bear or endure resistant storage,and strong resistance.Antirrhinum majus are scrophulariaceae perennial flowers.They has many varieties,the color are rich and beautiful and the flowering period is long.They are generally used to disposal parterres and cut flowers.In recent years,Antirrhinum majus has become the important classic materials of genetics and molecular biology,especially the transposon,flower pigment development and the study of flower type development.The gene conversion of Antirrhinum majus was reported smaller.Due to the non-bacterium Antirrhimun majus is very weak growth,in addition the effect of Agrobacterium infection,when the hypocotyl explants are for Agrobacterium Ti plasmid-mediated genetic transformation method, explants were cultured in the death of almost all stages.So that the regeneration rate and conversion rate are descended enormously,which increases the difficulty of genetic transformation of Antirrhinum majus.Therefor,we combinate two kinds of technique,the polyploid inducement and genetic engineering.Before the genetic transformation,induce polyploid Antirrhinum majus with the method of adding colchicine into culture medium,acquire polyploid regeneration seedling which diameter is severalfold coarser than the hypocotyl.The transformation of foreign genes adopt Agrobacterium tumefaciens carrying NR antisense genes cultivate together with the stems of polyploid Antirrhinum majus regenerated plants,transfer the NR antisense exogenous gene into Antirrhinum majus's genome,and through adventitious bud's resistance markers selection,detect some genetical Tetratetraploid plants with PCR,Southern and Northern detection,anti-kanamycin polyploidy Antirrhinum majus are acquired successfully.The results are as follows:With the diploid varieties Antirrhinum majus of materials,the increasing thicken medium is 1/2MS+BA1 mg/L+IAA0.2 mg/L,tender stems'ifferentiation medium is MS+BA2.0 mg/L+NAA0.1 mg/L,rooting medium is I/2MS+IBA0.5mg/L.Induce polyploid Antirrhinum majus with the method of adding colchicine into culture medium, induced concentration and time are 0.1%,4 d.Detect parts of mutants with PCR detection and identification of chromosome ploidy,the result is that the tetraploid Antirrhinum majus is induced successfully.Subculture after repeated selection,Antirrhinum majus Tetratetraploid homogeneous are isolated.Firstly transformed the NR gene into Antirrhinum majus and parts of transgenic mutants were got.The high quality system was that:taking the tender stem of Tetratetraploid Antirrhinum majus as explants aRer 21 d dark cultivation in the regenerated media that infected by Agrobacterium with OD550 of 0.4,co-cultivated for 2 d in the regenerated media without Kanamycin pressure,and then turned into the selected media(MS+BA2.0 mg/L+NAA0.1 mg/L+Kanamycin50 mg/L +Carbenicillin500 mg/L).Resistant buds were transgenic mutants trader the pressure selection.Detected transgenic plants with PCR,we found one different strip in the electrophoresis.The result showed that exogenous gene was already integrated into the genome of Antirrhinum majus.Examined the mutants with Southern,both 35S promotor DNA and transforming plants DNA had cross-breeding signal,but untransforming ones had no signal.The results showed that NR antisense gene was correctly plugged into the Antirrhinum majus nucleus genome.Checked out the mutants with Northern,the mRNA expression quantity of the transformed was more feebler than untransforming plants.The results showed that NR antisense gene was validly expressed in the transgenic plants.The florescence of transgenic plants is 25-30 days,and the cut flowers fresh period is 10-12 days,while the florescence of non-transgenic plants is 15-20days,cut flowers fresh period is 5-6 days.The results indicate that NR antisense gene transfered into Antirrhinum majus can greatly extend the florescence and cut flowers fresh period.