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Role Of Nutrition-related Genes(fdr3 & TaNRT1)

Posted on:2001-06-04Degree:MasterType:Thesis
Country:ChinaCandidate:Y X BaiFull Text:PDF
GTID:2120360002950423Subject:Botany
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Fdr3 is one of the differential screened genes from eDNA expression library of Fe-deficinet maize root. Innduced by IPTG, the expression product of fdr3 recombinent plasmid was isolated by SDS-PAGE, and then had western blotting with Fe-related antiserum. As a result, there was a hybrid zone. And one soluble protein around 4OkD from maize root could also cross-react with the same antiserum. In the analysis of DNA sequence, a 1 068bp ORF was found in the foreign fragment. Having homology searth in GenBank indicated that Fdr3 is an unknown gene. Kyte-Doolittle hydropathy analysis of its amino acid sequence showed there were several hydrophobic domains in it, which suggested that it was a protein of function. Using restriction digestion we got a 800bp fragment from the ORF. As a probe, the 800bp fragment was then had southern blotting with the genomic DNA of maize ,wheat, tobacco and an unicellular green alga, C.reinhardtii.The result indicated that the fragment was a component of maize ,wheat and C.reinhardtii genome DNA, and it was not a component of tobacco genome likely. AF(TaNRTI) is a cDNA fragment(about 657bp) of high-affinity nitrate transporter gene , which was obtained by RT-PCR from total RNA of wheat roots. It was first cloned into pBluescript-T vector.Restriction endonuclease analysis and sequencing confirmed that the foreign fragment was indeed the AF fragment. Then, by restriction digestion, we cleavaged the AF from the T-vector and cloned it into pGEX- 1 ,2T,3X. Restriction endonuclease analysis and sequencing(pGEX-2T-. TaNRT1 only) confirmed the corrected construction. Induced by IPTG, the molecular weight of the expressed product of pGEX-2T- TaNRT1 was smaller than the expected 52kD fusion protein. So we can only regularize the induce conditions again to get the aim fusion protein.
Keywords/Search Tags:nutrition-related
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