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Expression and Functional Analysis of the Proteins of Ferredoxin/Thioredoxin System from Maize

Author: FengAiHua
Tutor: FanJun
School: Anhui Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Iron thioredoxin system Corn Recombinant expression Function
CLC: S513
Type: Master's thesis
Year: 2010
Downloads: 98
Quote: 0
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Abstract


Chloroplast photosynthesis redox signaling system is iron thioredoxin system, including thioredoxin (thioredoxin, Trx) and its reductase : Iron thioredoxin reductase ( ferredoxin / thioredoxin reductase , FTR ) and the ferredoxin ( ferredoxin , FD ) , after both catalytic Trx remain restore the state . This paper studies the nature of the restructuring corn TRX , FTR and Fd . Obtain the following results : 1. Purified recombinant maize m and f Trx isoelectric point of 4.6 and 5.9 . m-type Trx f Trx have a stronger ability to restore insulin disulfide mutant protein is almost no reduction ability . Cys residues specific labeled compounds AMS mark TRX , wild-type Trx redox state , and the mutant protein is only reduced state . SDS-PAGE electrophoresis display in Maize Leaves target protein the Trx mutant f immobilized than the m -type Trx mutant capture more diversity . Extraction in Maize Leaves Total RNA as template , FTR variable subunit and the catalytic subunit of RT - PCR clone encoding genes inserted into two E. coli expression vector , expression of the protein containing the His-tag , and HisSUMO - tag, wherein the the variable subunit removing the N -terminal 20 amino acid residues , two protein subunits are expressed in E. coli and purified by SDS - PAGE displayed to the purified protein as a band the HisSUMO - tag was removed , indicating that removal of the N -side the partial amino acid residues , variable subunit FTRA stability increases. 3.RT - PCR clone Fd1 coding genes were inserted into the pQE80 and p28SUMO carrier . The ferredoxin purified SDS-PAGE purified protein as a main band , protein reddish brown , UV - Visible spectroscopy , obvious absorption peak in the 315 nm , 415 nm and 459 nm . The electron paramagnetic resonance ( EPR ) analysis showed that the protein contains a [ 2Fe - 2S ] . A target protein immobilized Fd from the variety of their role in Maize Leaves target protein adsorption . In summary, the E. coli expression system , the recombinant maize iron thioredoxin system proteins and mutants , and analysis functions , purified protein was used to study the maize and other cereal crops in iron the thioredoxin system activity regulation of the target protein containing histidine-tagged protein and mutant Filter from corn leaves in the target protein and the role of iron thioredoxin system .

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CLC: > Agricultural Sciences > Crop > Cereal crops > Corn ( maize )
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