DREB 1A Multi-clonal Antibody Preparation And Northern Examination On Resistant Disease Mechanism Of Dry Mycelium Of Penicillium Chrysogenum

Plant transcription factor DREB (dehydration responsive element binding protein), which plays an important role in signal transduction of drought, high-salt and cold stress, specifically binds to DRE element and controls the expression of several target functional genes involved in plant tolerance to drought, high-salt and cold stress under drought, high-salt and cold stress. Thus, it may be an effective strategy to achieve ideal, comprehensive effect for improving plant stress-resistance by DREB gene transfer (LIU Qiang et. al. 2000). The DREB 1A gene used in the experiment, in fact, is cDNA through reverse transcription of DREB 1A mRNA from Arabidopsis.The gene encoding transcription factor DREB 1A was amplified through PCR as the template of plasmid pCAMBIA 1301/DREB 1A. The recombinant plasmid pGEX-4T-1/DREB 1A was constructed by ligation of the target fragment amplified and the expression plasmid pGEX-4T-1. Firstly, E.coli cloning strain DH 10 B was transformed with the recombinant plasmid constructed above, the positive clones picked out were verified through such techniques as colony PCR, agarose gel electrophoresis, restriction endonuclease digestion, and the target insert sequencing as well. Then E.coli expression strain BL 21 (DE3) was transformed with the recombinant plasmid sequenced above, the fusion protein was expressed on a small scale, SDS-PAGE analysis showed that the expression amount of the fusion protein, which existed as inclusion body in the bacterium cytoplasm, accounted for more than 50% of the total protein. The fusion protein was expressed on a large scale, the inclusion body was purified, the fusion protein was digested by a kind of protease named thrombin, target protein strip was recovered on SDS-PAGE gel, and the gel strip recovered above was used as an antigen to immune rabbit in order to preparing the multi-clonal antibody of the transcription factor DREB 1 A. The results of ELISA showed that the titre of the multi-clonal antibody prepared reached up to 25,600 dilution times, which can be used to perform Western blot of DREB 1A for the transgenic plant progeny containing the DREB 1A gene. Dry mycelium (DM) of PeniciIlium chrysogenum (PEN) is a waste of the pharmaceutical industry. When it is used in the agricultural production as an organic fertilizer, it has been found that DM of PEN is effective in controlling a number of soil born fungal diseases, e.g. soil drenched with both 5% and 7% extract of PEN induced significant protection in cotton seedlings against either Fov (Fusarium oxysporum f.sp. vasinfectum) and Vd (Verticillium dahliae Kleb), but the mechanism of its resistance disease is unclear(JJJ ). In the experiments, the seedlings of cotton and water melon with cotyledon just expanded were drenched with 5% (W/V) aqueous extract of DM of PEN, the treatment periods of were 4,6,8,10,12,24h respectively, with the controls drenched with distilled water. the total RNA of cotton and water melon cotyledons were extracted, with eight kinds of cDNA fragments encoding pathogenesis - related (PR) proteins PR-1a, PR-1b, PR-2, PR-3, PR-4, PR-5 ,PR-A and PR-B used as probes of Northern blot, the transcription levels of the transcripts encoding eight corresponding PR proteins mentioned above were examined through Northern blot and autoradiography, so that the mechanism of resistance diseases of aqueous extract of DM of PEN would be expounded from the transcription level. The experimental results indicated that aqueous extract of DM of PEN increase the transcription levels of the transcripts encoding the rest six PR forementioned to some degrees, except that there was no effect on transcription levels of the transcripts encoding PR-4, PR-5.