Studies On Construction Of Antisense AhFAD2 Expression Vector And Efficient Factors Of Gene Transformation With Agrobacterium In Peanut (Arachis Hypogaea L.)

1. Cloning of a sequence encoding ahFAD2B from peanut and constructing of antisense ahFAD2B expression vectorThe peanut fatty acid consist mainly of oleic and linoleic acid. The rate of O/L is the main guide line of storage time.△12FAD(fatty acid desaturate ) is one of the most important enzymes for production of poly-unsaturated fatty acid (PUFA) in plants, which can catalyze oleic acid to linoleic acid.The primers were designed according to the published sequence of fatty acid desaturase gene on Genbank. Extracting genomic DNA from peanut 2005D029( A cultivar of introducing Chinese scholar-tree DNA into groundnut) infancy leaves. The fragment of ahFAD2B gene was obtained by PCR. The ahFAD2B gene was cloned into pGM-Teasy vector and then sequenced. Sequence analysis indicated that the cloned fragment was showing 97% homology to the sequence ahFAD2B reported in Genbank(AF248739). The recombinant plasmid pFAD2B and pBI121 were digested by BamHⅠand SacⅠ, recover the aimed 1200bp fragment and the longer of pBI121 resected GUS gene. The result proved that the ahFAD2B gene was reversely inserted into the normal vector of pBI121. And the antisense expression vector of ahFAD2B gene was constructed.2. Study on the factors influencing gene transformation with Agrobacterium in peanut (Arachis hypogaea L.)To conduct gene transgenic research on peanut using vector harboring γ-tmt and bar genes, and leaflet of fenghua2 as explants. Study the effect of different concentration tobacco extract liquid and different sonication treatment on explants when infecting, and the effect of acetosyringone (AS) when activating agrobacterium and cultivated with it. The results are as follows:(1) Sonication treatment on explants when infected can availability improve the rate of transformation in peanut.With different length time of 0,2,4,6,8,10,15min sonication, the results showed that suitable time of sonication (6min) can accelerated genetic transformation efficiency of peanut. The frequency of transformation was up to 1.30% distinctly to the CK.(2) Adding moderate tobacco extract liquid can availability improve the genetic transformation rate of peanut.Add tobacco extract liquid when infect the explants. The concentration were 0,37.5,75,112.5,150mg/ml. The highest frequency of transformation is 1.58% when adding 112.5 mg/ml tobacco extract liquid, which is 1.25 times to the CK. And the tobacco extract liquid can promote the differentiation of shoots.(3) AS can't improve the genetic transformation rate of peanut.Add AS when activating agrobacterium and cultivating explants with agrobacterium. The concentration of AS were: 0,50,100,150,200μmol/L .The results showed that adding 50 or 100μmol/L AS have no significant effect on enhance transformation efficiency. The rate of transformation descend obviously when the concentration of AS are 150 and 200μmol/L.