| Two kinds of protein phosphatase (PP2C-type protein phosphatases, PP2C) are a kind of protein phosphatase with serine/threonine residues, its existence was monomer forms in cells, and enzymatic catalyses activity depended on Mg2+or Mn2+. PP2C played negative role in controlling signaling cascade system through protein kinase phosphorylation, involving in the cell cycle, stress signal transduction, gene transcription, protein translation and post-translation cellular activities process. PP2C activity can be adjusted by H2O2, unsaturated fatty acid, Ca2+/CaM, lipid signal molecule, et al. Being aimed at the function of HAI-1gene and AtCAL gene, the research results were obtained as follows:(1) The genome sequence was provided with TAIR (www.arabidopsis.org) website, and the bioinformatics analysis was performed. The cis-regulatory elements are the same DNA molecular role which have special functions of transcription factor, DNA binding sites and other control base sequence, such as CAAT-box, ABRE, CGTCA-motif, G-box, and TGACG-motif appeared higher frequency among them. Signal peptide prediction based on HAI-1protein sequence, we can clearly draw conclusions that it has no signal peptide, and it may not be secretary protein, but possibly is organelles protein classes (such as chloroplasts, mitochondria, etc).(2) The HAI-1gene sub-cellular localization were analyzed through constructing HAI-1::GFP fusion expression vector. The instantaneous expression analyses with its fusion expression vector bombarded epidermal cells in onions by gene gun; the results were found green fluorescent protein in the nucleus, which account for HAI-1protein belonging to that nuclear protein. The promoter::GW-GUS expression vector of HAI-1gene was constructed through Gateway method, which was transformed in Arabidopsis thaliana flower buds by electric conversion, harvested To generation seeds, and got positive plants by screening and identification of homozygous. By GUS histochemical staining, the experiment results showed that the cloned1681bp upstream and222bp downstream from the start codon of HAI-1gene fragments had the complete promoter activity. Its expression had specificity of time or space. At the same time to illustrate HAI-1gene on germination and growth process, its expression can be induced by ABA hormones and damage.(3) The results of HAI-1gene mutant seeds germination experiment were showed that the seed germination of wild-type and hai-1mutant was almost the same with1μM ABA, and cultivated under continuous white light for7days, but that of over-expression transgenic plants was obviously lower than that of wild-type and hai-1mutant seeds at4days. Low temperature treatment at7days, all of the green cotyledon percentage were100%in the absence of ABA hormone MS culture medium plate. With ABA concentration elevated, the green cotyledon percentage of hai-1mutant seeds was higher than that of the wild type and over-expression plants seeds. The green cotyledon percentage of all the mutant seeds was zero, when ABA reached to0.6μM.The NaCl stress experiments of seed germination and green cotyledon percentage were showed there was no obvious difference between hai-1mutant seeds and others. However, the mutant seeds were almost germinated100%, but hai-1mutant seeds were obviously higher than wild type and over-expression transgenic plants in green cotyledon percentage at4days using20mM NaCl stress.(4) For the results of the long-term dehydrate experiment, the degree of withering was almost between the mutant plants and wild-type plants, and showed more sensitive to drought. After dehydration for12d, there were no obvious withering symptoms, which explained to have certain patience to drought. From the results of short-term dehydrate experiment; its conclusion was approximately the same as the results of long-term dehydrate experiment. In dry air after four hours, the heaviest leaf fresh weight was hai-1mutant plants, and then was wild-type plants, the last was over-expression transgenic plants respectively.(5)8-d-old seedlings as materials, and extracted their RNA, reversed transcription into cDNA. Using Q-PCR method to study the ABA stress response genes in different organization parts (the whole plant, leaf and root) of wild-type plants and over-expression transgenic plants, the results were found that their expression was different, and had no obvious rule. The whole plant expressions of ABI1gene in over-expression transgenic plants were significantly higher than that of wild-type plants, no matter in the whole plant, leaf or roots. And the whole plant expression of ABI2gene in wild-type plants was higher than that of over-expression transgenic plants. But in roots and leaves, the ABI2gene expression in over-expression transgenic plants had significantly higher than that in wild-type plants. This showed the expression of ABI2gene in other tissues that the expression of the wild type plants than that of over-expression transgenic plants. Other ABA-related genes quantitative results are similar.(6) According to the stomata opening statistics of outer epidermis under leaves to wild type plants and over-expression transgenic plants, the results were found at different concentration of ABA stress that their stomatal openness was different. In no hormone ABA stress, the porosity degree of stomatal of over-expression transgenic plants was slightly higher than that of wild type plants. But as applied ABA, it brought about some changes. When the ABA concentration was1or10μM, wild type plants’outer epidermis of the porosity degree of stomatal was obviously higher than that of mutant plants. That showed mutant plants performance to the super sensitive, made its porosity degree of stomatal to the mutant plants less than those of the wild type plants.(7)About vegetative growth of mutant plants, Rat2/Ratl RNAi double mutants were apparent late-flowering phenotype. For germination experiment of each mutant seeds, as the NaCl concentration increased, the mutant material germination rate was also lower. The lowest germination rate among the mutants was Rat2/Ratl RNAi double mutants. Constructed the fusion plasmid of AtCALl::GFP and AtCAL2::GFP, and bombarded into onion epidermis. Fluorescence microscopy revealed that there is green fluorescent protein to stimulate in onion epidermal cell nuclei, which proved AtCAL1protein and AtCAL2protein were located in the nucleus. |
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