| Transcription factors combined with cis-element of downstream in the promotor to regulated gene expression which Influence the biological phenotypes. Transcription factors includes several types:bZIP, zinc finger protein, B-ribbon and helix-turn-helix. The zinc finger protein is one of transcription factor which has an important role of regulatory effect in eukaryotic cells. The plant zinc finger protein has an important role of growth and plants tolerance to adversity stress. According to the well-known reported, most plant C2H2-type zinc finger protein related with adversity stress, and it will offer a favorable condition to enhanced the plants tolerance to adversity stress by the bio-engineering. Soybean (Glycine max [L.] Merrill) is the important food and oil crops of China which is the important raw materials of medicine,food and industriy. Low temperature, drought and high salt are three major abiotic stress factors which seriously affect the plant’s growth and output. The transgeinic arabidopsis of SCOF-1improve the plant’s tolerance to the low temperature. This paper, three C2H2-type zinc finger protein gene (SCTF-1, STF-2and STF-3)were cloned with bioinformatics by our lab,and respectively construct the plant expression vectors. The zinc finger protein gene transgenosis tobacco by the agrobacterium-mediated Genetic Transformation, and transgenosis into soybean by pollen tube pathway transformation. And the function identification of zinc finger protein gene by abiotic stress. The results revealed:(1) SCOF-1was used as a information probe to make homology screen by tblastn, and we selecte3novel homologous gene which encoding C2H2zinc finger protein structure. We isolated it from soybean "jinongl8", and named as SCTF-1, STF-2and STF-3. Total RNA was extracted from4-leaves stage soyebean, reverse transcribed to cDNA, cloned these gene by RT-PCR. Then these gene were inserted into pMD18-T vector and gene sequencing,and respectively construct the plant expression vectors pBI121-SCTF-1, PBI121-5TF-2and pBI121-5TF-3, The plant expression vectors transgenosis tobacco (Nicotiana tabacum L.) by the agrobacterium-mediated Genetic Transformation. The RT-PCR and Southern blot results showed:transgenic plants have been successfully obtained, and the target genes integrated into chromosome of tobacco.(2) Analysis on the chromosome location of three zinc finger protein gene,we found SCTF-1located on the short arm of chromosome4(C1) and6(C2), with genetic distances of 23.8cM on chromosome4(C1),25.5cM on chromosome6(C2). STF-2located on the long arm of chromosome10(O), with genetic distances of117.2cM on chromosome10(0). STF-3located on the short arm of chromosome14(B2), with genetic distances of43.2cM on chromosome14(B2).(3) RNA of roots,stems,leaves and flowers were extracted from flowering stage soyebean "jinong18". The expression pattern of zinc finger protein gene was studied various soybean tissues by RT-PCR, and the results showed that all of them were expressed in roots,stems,leaves and flowers at adult stage. SCTF-1was expressed with high levels in flowers and leaves,but lower in roots and stems. STF-2was expressed with high levels in leaves and flowers, secondly in stems, and the lowest in roots. STF-3was expressed with high levles in flowers and leaves,secondly in roots,the lowest in stems.(4) Transient expression of the pBI121-GFP-SCTF-1, pBI121-GFP-STF-2and pBI121-GFP-STF-3in onion epidermal cell showed that SCTF-1, STF-2and STF-3protein was localized in cell nuclei with inverted fluorescence microscope.(5) The plant expressed vector pBI121-GFP-SCTF-1, pBI121-GFP-STF-2and pBI121-GFP-STF-3were transformed into soybean "jinong28","jinongl8" and "jinong27" by pollen tube pathway, and a total of820seeds were obtained. These PCR analysis results of Ti transgenic plant:transformation efficiency of the transgenic plants with pBI121-GFP-SCTF-1is1.99%; The transformation efficiency of the transgenic plants with pBI121-GFP-STF-2is3.93%; The transformation efficiency of the transgenic plants with pBI121-GFP-STF-3is5.86%.(6) The function identification of zinc finger protein gene by abiotic stress with low temperature, drought and high salt in the lab. RT-PCR and real-time PCR showed that overexpression of SCTF-1enhanced cold tolerance of transgenic tobacco than the control. SCTF-1expression increased with time in4℃low temperature, after36h the expression was decreased. The resistance identification of transgenic tobacco by different degree abiotic stress. The rusults showed:overexpression of SCTF-1enhanced cold tolerance of transgenic tobacco than the control at0℃and4℃, enhanced cold tolerance of transgenic tobacco in short time at-10℃.STF-2enhanced drought tolerance of transgenic tobacco than the control with drought stress for10d. STF-2expression increased with time in20%PEG6000, and high level expression during the12-36h. The resistance identification of transgenic tobacco by different degree abiotic stress. The rusults showed:Overexpression of STF-2enhanced drought tolerance of transgenic tobacco than the control under15%PEG6000. STF-3enhanced cold, drought and high salt tolerance of transgenic tobacco than the control. STF-3expression increased with time in4℃low temperature, after36h the expression was decreased; STF-3expression increased with time in200mmol/L NaCL, and high level expression during the12-36h; STF-3expression increased with time in20%PEG6000, and after36h the expression was decreased. The resistance identification of transgenic tobacco by different degree abiotic stress. The rusults showed:Overexpression of STF-3enhanced cold tolerance of transgenic tobacco than the control at0℃and4℃, enhanced salt tolerance of transgenic tobacco under200mmol/LNaCL, enhanced drought tolerance of transgenic tobacco than the control under15%PEG6000.(7) Growth characteristic analyse of transgenic soybean, we found that the transgenic soybean become dwarfing, and the fresh weight decreased in4-leaves stage. |
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