| ABI5 subfamily of transcription factors has been spontaneous for more than 20 years.As a leucine zip-type(b ZIP)transcription factor,nine highly homologous transcription factors were identified in the Arabidopsis genome,namely ABF1,ABF2,ABF3 and ABF4,At DPBF1,At DPBF2,At DPBF3 and At DPBF4,and At5G42910.Over the years,it has been found that Arabidopsis ABI5 subfamily transcription factors are involved in regulating multiple stages of plant growth and development,and play an important role in plants under abiotic stress.Due to functional redundancy and transcriptional regulatory regions,the division of labor and precise roles of ABI5 subfamily transcription factors are difficult to determine.Based on previous research in our laboratory,we recombined the conserved region II of At DPBF4,the most active transcriptional activation region,with the b ZIP region of ABF2,ABF4 and At5G42910,respectively,to construct overexpressed recombinant transgenic plants,hoping that in the absence of transcriptional activation inhibition region,To explore the specific functions of the above three transcription factors.In addition,we constructed the recombinant gene of Streptavidin-Horseradish Peroxidase(HRP)in Arabidopsis thaliana,and obtained stable genetic recombinant gene through genetic transformation.The objective of this study is to prepare a recombinant secondary antibody system for biological and medical detection by avidin binding biotinylation target and horseradish peroxidase catalyzed enhanced chemiluminescence.The main research results of this paper are:1.Using the hygromycin resistance and molecular identification methods(DNA identification and RNA identification)of transgenic plants,through the breeding and screening of TI generation transgenic plants,T3 generation homozygous superexpression recombinant transgenic plants were obtained,and the correct T3 generation super-expressed recombinant transgenic plants were identified by molecules Named A.thaliana A2,A.thaliana A4,A.thaliana 5G42910.2.Through the statistics of germination rate of transgenic plants and wild-type plants,it was found that all three transgenic plants had early germination phenotypes:statistics were counted in point species 35,36.5,38,39.5 h.The germination rates of were 46%,57%,64% and 75% for wild-type plants.Genetically modified plants A.thaliana A2 were 79%,85%,88% and 90%,genetically modified plant A.thaliana A4 is 82%,87%,90% and 94%,transgenic plant A.thaliana 5G42910 is 69%,77%,79% and 84% 。3.Three transgenic plants and wild-type plants were planted in 1/2 MS plates and containing 50 m M,100 m M and 150 m M Na Cl,respectively growing on 1/2 MS plates with concentration for 10 d,statistical root length changes showed that all three transgenic plants had different degrees of Na Cl Resistance.The wild-type plants grew on plates with different concentrations of Na Cl for 10 days,and their root length was inhibited by 31.3%,86.3% and 95%.A.thaliana A2 was 39.7%,65% and 85%.The transgenic plants A.thaliana A4 were 32.3%,72.7% and 93%.The transgenic plants A.thaliana 5G42910 were 24%,69% and 92%,respectively.4.Three transgenic plants and wild-type plants were planted on 1/2 MS plates containing mannitol concentrations of 50 m M,100 m M and 200 m M respectively for10 days.The statistical changes of root length showed that the three transgenic plants had different degrees of sensitivity to osmotic stress.The root length of wild-type plants was decreased by 19.7%,36.7% and 70.1% when growing on plates with different mannitol concentrations for 10 days.A.thaliana A2 was 21.9%,32.2% and 64.3%.The transgenic plants A.thaliana A4 were 38.6%,42.4% and 63.3%.The transgenic plants A.thaliana 5G42910 were 19.3%,34% and 63.3% respectively.5.Three transgenic plants and wild-type plants were planted on 1/2 MS plates containing 1%,3% and 5% sucrose,respectively,and grew for 10 days.The statistical changes of root length showed that the three transgenic plants had different degrees of sensitivity to sucrose.The root length of wild type plants was inhibited by 19%,21.7%and 21.1% when they were grown on different sucrose concentrations for 10 days.A.thaliana A2 was 21.2%,34.9% and 52.1%.The transgenic plants A.thaliana A4 were15.8%,26.6% and 36.7%.For A.thaliana 5G42910,19.3%,24% and 21.3% were found.6.Three transgenic plants and wild-type plants were planted on 1/2 MS plates containing 0.25 μM,0.5 μM,1 μM,and 2 μM ABA for 10 days,respectively.Statistical analysis of root length showed that all three transgenic plants were insensitive to high ABA concentrations.The root length of wild type plants was decreased by 0.6%,79.6%,93.8% and 97.3% after 10 days of growth on a plate with low to high ABA concentration.The transgenic plants A.thaliana A2 were 47.3%,67.8%,91.1% and95.2%.The transgenic plants A.thaliana A4 were 18.4%,77.8%,87.3% and 94.3%.The transgenic plants A.thaliana 5G42910 were 16%,73.3%,86.7% and 94.7%.7.Through the analysis of transcriptome data of A.thaliana A1 super-expressed recombinant transgenic plants,it was found that the differential genes of transgenic plants were mainly enriched in the two pathways of plant hormone signal transduction and phenylpropane biosynthesis,and by regulating the above two metabolic pathways,the transgenic plants showed multiple phenotypic and stress resistance changes.8.Plant expression vector p CAMBIA 1300-c SA-HRP with 6×His tag was constructed,and the T0 transgenic plant was obtained by Agrobacterium infection,and the correct transgenic plant was named A.thaliana 35S-SH after hygromycin screening and molecular identification.9.The total plant protein of A.thaliana 35S-SH was extracted by denatured protein extract and non-denatured protein extract,respectively,and detected by gel electrophoresis and Western Blot(WB).SDS-PAGE results of total plant protein extracted from denatured protein extract showed that there was a non-specific band near the theoretical molecular weight of the target protein(48 KDa)compared with the total protein of wild-type plants.Further WB test was conducted to confirm the WB results,and it was found that there was no imprint.The results of protein electrophoresis of total plant proteins extracted from non-denatured protein extract showed no specific bands compared with the wild-type plants,but only a fuzzy non-specific band was revealed after exposure to nitrocellulose membrane.The detection of c SA protein at the N terminal of fusion protein by dot hybridization with different methods was also not detected. |
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