Gene Cloning, Vector Consrtucting, Prokaryotic Expressing Of Two Unknown Genes Of Arabidopsis Thaliana And Arabidopsis Transgenic Research

Abscisic acid (ABA) plays a major role in regulating plant growth and development including seed maturation, germination and dormancy, as well as mediating adaptations to environmental tresses such as cold, drought and salinity. It has been proven that the ABI1 and ABI2 were key factors in ABA signal transduction. As ABA plays a major role in regulating physiological properties such as stoma closure and so on, it deserve researching ABA biosynthesis mechanism and new signal factors of ABA signal transduction which made the plants adapt to environment when it changed. The establishment of the yeast two-hybrid system provides efficient method to detect protein-protein interactions and screen interacting protein for known signal factor, consequently foreseeing unknown proteins' function in the signal transduction. In this research, 2 proteins which have interaction with ABI1 and ABI2 were identified from Arabidopsis cDNA libraries using the yeast two-hybrid system. Analysis of DNA sequences revealed that two genes (GenBank AB025622, AC023628) are unknown, thereby forecasting expressive product of two unknown genes are the modulatory factors of ABA signal pathways and named temporarily pcr7(AB025622 ) and pcr9(AC023628 ) according to discovered order.A key component of my study involved cloned these genes from yeast vector pGAD10 and blast with Arabidopsis thaliana cDNA libries, and then inserted the coding sequence in both sense and anti-sense ways, into the Agrobacterium-carrying plant expression vector pBI121. By using floral dip, a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, and driven by a high efficiency promoter, these transformed the target genes to create transgenic plants, so as to obtain two groups of Arabidopsis thaliana plants, either sensitive to Aabscisic acid or totally not.According to Arabidopsis thaliana cDNA libraries in GenBank, we blast the unknown genes, the results reveal that pcr7 is the full length coding sequence, while the pcr9 is a consistent sequence with cDNA libraries except the start codon. Using the Primer5.0 software to design primers of pcr7. Due to the requirement of later experiment, we design one start codon,one SmaI site and protecting base at 5' end of the primer of pcr9. By using pGAD10 which has two unknown genes as the template for PCR, the fragment was amplified with full length 600bp and then cloned into pMD18-T vector of Takara Co. in sence and anti-sence. The sequencing result indicated that the fragments maintained significant same to genes in GenBank in plant and contained no intron, which suggests the fragments can be expressed in E. coli by establishing prokaryotic expression vector.We inserted the fragment of pcr7 and pcr9 into pBI121 plasmid in both sense and antisense ways and obtained two kinds of pBI121 recombinants with pcr7 and pcr9 sequence in different directions, which can be used to transformed higher plants through Agrobacterium. Then we transferred above-mentioned recombinated pBI121 vectors into competent Agrobacterium EH105 and did transgenic research of Arabidopsis thaliana by floral dip method to further investigate the function of pcr7 and pcr9.After using SmaI digested pMD18-T-pcr7R and pMD18-T-pcr9R, two sequence were cloned into the prokaryotic expression pGEX-2T vector. Induced by IPTG, proteins were expressed under 28℃with a approximately relative molecular weight of 44.18kD(PCR7),46.9 kD(PCR9) and could be recognized by GST antibody in a Western-blotting analysis. We selected 0.1mM IPTG and induction at 28℃as the condition of the fusion protein purification. Generally, this research took the first step of more advanced research aimed at function and gene engineering of pcr7 and pcr9.