| In order to achieve timely, effective expression of the target gene in transgenic plants, it is necessary to design an effective "gene switch" system for the control of target gene expression.Based on the "gene switch", an artificial gene regulation system can be constructed, which will be valuble for gene expression and research in plant. The Cre/lox recombination system is composed of Cre recombinase of 38.5 kD and 34bp lox site.The Cre recombinase will catalyzes the excision of DNA segments when the loxP sites occur as direct repeats or the inversion of DNA fragments if the flanking loxP sites are placed in the opposite orientation.The recombination of Cre/lox system showed highly efficient and stable in bacteria, fungi, animals and plants, and was used widely in the field of plant transgenic research and gene function identification.In addition,the chemically induced gene expression system make it is possible for the artificial regulation of gene expression. Including tetracycline inducible expression system, steroids activate system, copper-induced system and ethanol-induced system have been applicated successfully in plant.The ethanol-inducible system was derived from Aspergillus nidulans filamentous fungus.Transcription factor AlcR in the system is active only in combination with ethanol molecules, the activated AlcR then binds to the promoter of the target gene such as alcA(alcohol dehydrogenase I), then the downstream target gene will express. The ethanol-induced system has almost no physiological and toxic effects on plant growth within inducing alcohol concentration range,and the inducing manner is flexible, convenient,cheap.Furthermore,the ethanol-induced system can be used in laboratory and field.Based on the characteristics of the ethanol-induced system and Cre/lox recombination system,a combined system of Cre/lox recombination system and ethanol-induced expression system was constructed in this paper.In the combined system,the expression of Cre recombinase gene is under the control of ethanol application, which give rise to a new regulation approach for gene expression in plant. Two plant expression vectors were generated and were transformed into the tobacco by agrobacterium-mediated co-transformation.One is Cre recombinase expression vector, transcription factor AlcR gene control by 35S promoter will constitutive express,and the expression of Cre recombinase gene was under the control of alcA:35S heterozygous promoter which derived from the fusion of alcA promoter and-46 to 5 region of 35S promoter. Another is GUS/Bar vector, which was used to track the recombination process induced by ethanol.In this vector, GUS gene flanked by two direct repeat lox sites was placed the downstream of 35S promoter,and a herbicide gene Bar without promoter was placed downstream of GUS gene.So, once the Cre gene is activated, the GUS gene will be excised,the Bar gene will access to the 35S promoter and the plants will obtain resistance to herbicide Basta. The main results are as follows:1、PCA13alcRCre and PCA23GusBar were transformed into tobacco by agrobacterium-mediated leaves disc co-transformation.The shoots generated on the medium supplement with kanamycin and hygromycin.2、Total 37 transgenic plants were tested the herbicide resistance on the medium with 10 mg/L ppt in it.There were 11 plants that the leave disc were no any differentiation, indicated them were herbicide-sensitive.And the rest plants were herbicide-resistant, these plants leave discs differentiated callus and shoots on the medium with 10 mg/L ppt. The further GUS staining indicated the GUS express well in those herbicide-sensitive plants,but all plants which were herbicide-resistant did not detect any GUS activity except for No.3,No.4 and No.23 plants. Subsequently PCR analysis showed the expected 1.8kb GUS gene fragment appeared in all herbicide-sensitive plants, but it absent from all the herbicide-resistant and GUS activity negative plants.3、The No.10,No.11, No.12 and No.32 plants were treated with ethanol.60 ml 4.0% concentration of alcohol was used to induce the exprssion of Cre gene by root irrigation.And the GUS activity was detected once again before the ethanol inducing,the results showed the GUS expressed well,after 48 hours treated with ethanol,the GUS activity could not be detected anymore,and the leave disc could differentiated on on the medium with 10 mg/L ppt.The results showed the GUS gene had been deleted by the ethanol induced Cre activation.4、RT-PCR analysis about the expression of AlcR gene and Cre gene in auto-inducible plants without any ethanol treatment was performed. We detected the expression of AlcR gene in these plants, at the same time, the evident level expression of Cre was also detected even there was no any ethanol inducing. The result indicated the background expression is existent in some transgenic plants of the ethanol-induced Cre/lox recombination system.5、The RT-PCR and Q-PCR analysis results indicated there were always higher level expression of Cre in auto-inducible plants than artificia inducible transgenic plants when 60ml 4% concentration of ethanol was used to root irrigation.The Cre expression of auto-inducible transgenic plants increased with the time after ethanol induced, and the expression level reached a peak after 24 hours. The Cre expression of artificial-inducible transgenic plants showed low level expression after ethanol induced, although we could found the expression level also rose with the time like the auto-inducible plants. The expression peak appeared after 48 hours of the inducing in artificial-inducible transgenic plants, and there was a lowest level expression after 24h hours of the inducing.6、The remain sequence after the excision mediated by the ethanol induced Cre activation was analyzed by PCR and sequencing. The results showed the recombination event was conservative, without loss or alteration of the lox sequence or its flanking DNA. |
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