Studies Of Using RNA Interference Technology Created Maize Dwarf Mosaic Disease Resistant Material

Maize dwarf mosaic disease(MDMV)is one of the most widespread viral diseases in the world,caused serious harm to the maize production.The current agricultural practices and chemical control can not effectively control MDMV,breeding resistant varieties is the most fundamental way.However,the rough dwarf disease resistant germplasm resources of current breeding material are limited,caused great difficulties to the breeding of resistant varieties.Transgenic technology has brought new ideas and methods for the cultivation of resistant breeding.In this research,according to the principle of RNA interference,construct a maize dwarf mosaic virus RNA interference gene expression vector,by Agrobacterinm-mediated transformation to its maize genome,identified by future generations,won the maize dwarf mosaic disease resistant,stable transgenic genetic material.The main contents are as follows:1.The Nib sequences of sugarcane mosaic virus(SCMV)was searched in the NCBI database.Continuous removal of base pairing to reach 19 or more sequences,Nib sequence-specific selection process can be avoided in the gene silencing RNA interference in SCMV.A size of 530 bp gene sequence of SCMV was selected and artificial synthesized in vitro.The fragment was digested by restriction enzyme then sub-cloned into the intermediate vector NPPLT to form the inverted-repeat constructs with intron sequence.The inverted-repeat constructs with intron sequence was link into expression vector PNCXB,which including Cre/loxP recombination system,by appropriate restricuion enzyme digestion and the new RNAi vector was named as PNCXB-846.2.The immature embryos and immature embryo callus of Hi II,Hi IIB,Hi IIA,H99 are infected by Agrobacterium,comparative study on the impact of genetic transformation of various factors,optimized and efficient maize genetic transformation system.The results showed that: immature embryo size is L2-1.8mm,bacterial concentration is 0.3-0.4,infection time 10 min for the young embryos are the optimal infection conditions;subculture three times the pre-cultured for 6 days,the bacterial concentration of 0.5-0.6,AS concentration of 150 mg/ L is optimum conditions of infection callus.Agrobacterium mediated young embryo resistant callus and regeneration plant more,and simple operation,high efficiency,but young embryo induction of callus in good condition for a specified period of growth,also suitable for agrobacteriuxn infection.Using the optimization of genetic transformation system,we obtain the four genotypes receptor of genetically modified maize dwarf Mosaic disease resistance of plants.3.Using appropriate concentration PPT phenotypic identification,marker gene PCR,target gene PCR,the purpose of genetic testing four methods for Hi II,Hi IIA,Hi IIB,H99 4 genotypes T1 T2,T3 transgenic lines carried purposes detection and identification of the gene,results show that the phenotypic identification of suitable concentration PPT processing can exclude some false-positive plants resistant plants were obtained for the purpose of further gene PCR and viral inoculation investigation,both to reduce the workload and improve the accuracy of identification;inoculation disease identification identification results show that transgenic plants and non-transgenic controls obtained using RNAi technology has been significantly improved compared to resistance,four kinds of genetically modified material in T2 generation has 18(60.00%)strains Department of disease resistance than the corresponding non-transgenic plants,T3 generation of 23(76.67%)strains of disease resistance than the corresponding non-transgenic controls,one(3.33%)strains of disease control imimrne resistance to achieve one of four level;Ti generation of disease was 9.78%~15.00%,T2 generation of disease was 22.32%-29.79%,T3 generation of disease was 46.34%-55.86%.The results show that with the increase of foreign genes generations gradually moving towards normal Mendelian inheritance;degree of resistance to the good performance of transgenic plants T1,T2 generation offspring,T3 lines fiirther on behalf of inoculation,still showed a high degree of resistance,thus performance speculated that anti-antidwarf mosaic disease transgenic plants exhibited genetic stability;Southern hybridization results further confirm the anti-maize dwarf mosaic virus RNA interference gene 846 has been integrated into the maize genome.