| Nematode in the four pathogens which caused cucumber infectious diseases is gradually becoming one of the important diseases of the plant pathogen,which is the third after fungi, bacteria. Cucumber nematode disease has been prominent issues on cucumber production. Methods of control of nematode include the cultivation prevention,chemical prevention and anti-nematode species breeding. Anti-nematode species breeding has been a perfect way which the breeders search for.But it is difficult to get resistant nematode cucumber varieties only through current resources and conventional methods because of the lack of germplasm resources.Biotechnology-assisted breeding is an effective way to solve the lack of resistance,which has been applied in cucumber breeding. RNA interference (RNAi) is one of the hot spots in the molecular biology ,and it is a phenomenon of gene silencing which is triggered by a homologous sequence. because cucumber tissue culture is difficult,the regeneration rate is low, regeneration cycle is long, very few workers choose cucumber as a genetic transformation of the receptor. This test carried out the research on high effective regeneration system of cucumber ,the construction of RNAi and the research on agrobacterium-mediated transformation of technology and pollen tube pathway into cucumbers, the result are described below:(1)Screened optimized cucumber regeneration system.Cotyledons of two days seedlings are the most suitable age; nearly petiole parts of the terminal is the best explants; culture mediums with MS + 6-BA 1 mg L-1 + ABA 1 mg L-1 + Ades20 mg L-1 can obtain high regeneration frequency; during the early stage of culturing, the exiting of Ades and ABA can decrease the concentration of POD, but promote the regeneration of the adventitous shoots.(2) Binary vector is constructed. By RT-PCR the conserved nematode 16D10 gene was obtained fron the infected cucumber root and interted into T vector pMD-18, sequencing show that 16D10 gene is highly conserved during the four main nematode, namely Meloidogyne incognita, Meloidogyne javanica, Meloidogyne arenaria and Meloidogyne hapla.A double strand vector which is named pBLUE-22-21 is constructed, and the double-stranded gene is inserted into cloning vector pRTL2 with the 35S promoter ,named pRTL2-22-21, then the fragment with the promoter and terminater and double-stranded is inserted into the expression vector pBIN-19, expression vector pBIN-22-21 is received.(3)The impact of Agrobacterium-mediated genetic transformation factors is studied.Genotype affects greatly on anti-Kan shoots regeneration, variety Lu 8th is better for transformation; cotyledons of different stage has different effect to anti-Kan shoots regeneration ,explants are cutting when the two cotyledons leave from the seed coat and part of the cotyledons is green , more anti-Kan shoots can be formed ;cucumber is sensitive to Kan; the method of a delay in screening is used ;500mg/L Carb or 300mg/L Cef have no obvious effect to the coming out of cucumber adventitious shoots,both can be selected as an antibacterial agent.(4) Pollen tube pathway gene transforming is researched.The frequency of remaining growing cucumber was affected greatly by different method of injection. The best method is cutting off stigma after pollination 24hours then injecting foreign purpose of DNA . |
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