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1. 研究目的与意义、国内外研究现状（文献综述）
课题的意义、国内外研究进展、应用前景等（列出主要参考文献）Cytosolic glyceraldehyde-3-phosphate dehydrogenases in plants (GAPC2) is a key enzyme in glycolysis that catalyzes the first step of the pathway by converting D-glyceraldehyde 3-phosphate (G3P) into 3-phospho-D-glyceroylphosphate, essential for the maintenance of cellular ATP levels and carbohydrate metabolism by similarly binds DNA in vitro. In plants, various studies have demonstrate that cytosolic GAPDHs, referred to as GAPCs, meaning GAPC1 and GAPC2 may be localized in both the cytosol and the nucleus (Anderson et al. 2004, Holtgrefe et al.2008, Wawer et al. 2010, Henry et al. 2015, Peralta et al. 2016). An accumulation of AtGAPC1 in the nucleus has been observed in response to a peptide derived from bacterial flagellin, suggesting that the nuclear accumulation of GAPC is involved in pathogen-associated molecular pattern that triggered immunity (Henry et al. 2015). Similarly, AtGAPC1 accumulates in the nucleus in cadmium-stressed Arabidopsis roots (Vescoviet al. 2013). However, although this study suggests a role for plant GAPCs in oxidative stress signaling, it appears that the S-nitrosylation of GAPCs is not necessary for their translocation into the nucleus (Vescovi et al. 2013). ROS are generated as signals that alter various cellular and physiological processes in plant growth and development (Desikan et al., 2001; Apel and Hirt, 2004; Gechevet al., 2006; Shao et al., 2008). Hydrogen peroxide (H2O2) is the major and most stable species of ROS and plays a signaling role in plant response to stresses, such as mediating abscisic acid (ABA)–regulated stomatal closure (Peiet al., 2000; Zhang et al., 2001). H2O2 is thought to affect target protein activities through modification of thiol groups of Cys residues (Hancock et al., 2005). However, it is unclear how such oxidative modification affects a signaling cascade that leads to alteration of cellular function and plant stress responses. ROS affects the interaction between GAPCs and ATG3, indicating that GAPCs could influence ROS-mediated autophagy. Autophagy plays an essential role in plant immunity (Liu et al.,2005). This led us to hypothesize that GAPCs may also affect innate immunity. We therefore challenged the GAPC-silenced N gene-containing N. benthamiana (designated NN) plants with GFP-tagged Tobacco mosaic virus (TMV-GFP). At 3 d post infection (dpi), the number of TMV-GFP local infection loci showing GFP fluorescence, visualized under long-wavelength UV light in the GAPC-silenced NN, was reduced compared with that in TRV control plants.This results reveal that VIGS of GAPCs reduced TMV viral RNA accumulation and NN susceptibility to TMV infection. Furthermore, we analyzedthe effect of silencing of GAPCs on N-mediated hypersensitive response (HR) induced by N gene elicitor protein, TMV-P50 was analyzed. HR cell death in GAPC-silenced NN plants was enhanced compared with control NN plants indicated by trypan blue staining. These results suggested that GAPCs negatively regulate N gene-mediated resistance against TMV. Furthermore, an investigated was conducted whether GAPCs would have a general role in plant immunity against bacterial pathogens. In this regard, It was tested whether GAPCs silencing has an effect on a compatible pathogen, Pseudomonas syringae pv tabaci, as well as an incompatible pathogen, P. syringae pv tomato (Pst) strain DC3000, in N. benthamiana plants. We found that the growth of P. syringae pv tabaci was reduced ;14-fold at 2 dpi in GAPC-silenced plants compared with that in control plants. Studies also shows that the growth of Pst DC3000 was reduced ;54-fold at 3 dpi in GAPCsilenced plants compared with that in control plants. However, we did not find an obvious effect of GAPCs silencing on HR cell death induced by Pst DC3000. Taken together, these results suggest that GAPCs function in both R gene-mediated defense and basal defense. GAPC2 is reported to bind to PA and promotes proteolytic cleavage in Arabidopsis seedling. The GAPC2-PA may indeed provide a significant signaling in plants. As we know plants are mainly multicellular predominant photosynthetic eukaryotes of the kingdom Plantae, under stress, they are usually known to be quantification of environmental effects on plants health. In Arabidopsis GAPC2 is thoughts to play a role in oxidation signaling and cadmium treatment that cause the inactivation of GAPC enzyme activity and relocation of GAPC from cytoplasm to nucleus.The tomato originated in the Andean region of South America, was grown by Aztecs in Mesoamerica, and spread to Europe by early Spanish explorers. Today, hundreds of varieties are grown throughout the world, with the largest producers being China and the United States. Although the origin of the tomato is somewhat clouded, there is no doubt that the cultigen of today has had a long journey. Many wild relatives of the tomato such as Lycopersicon esculentum var. cerasiforme, L. chilense, L. peruvianum, L. hirsutum, and L. pimpinellifolium are among the richest genetic pools available for cross breeding. Almost all of the effective resistances to virulent tomato diseases have been found from wild species of Lycopersicon and Solanum. Few other crops are blessed with such extensive collections of wild forms and their derivatives." Not only are these wild relatives valuable sources of genetic material for disease control and prevention, but also for arthropod resistance, improving fruit quality, abiotic stress tolerance, and drought/cold resistance among many others.References1. Holtgrefe, S., Gohlke, J., Starmann, J., Druce, S., Klocke, S.,Altmann, B., Wojtera, J., Lindermayr, C., and Scheibe, R. (2008).Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications. Physiol. Plant. 133: 211–228.2.Katagiri, T., Takahashi, S., and Shinozaki, K. (2001). Involvement ofa novel Arabidopsis phospholipase D, AtPLDd, in dehydration-inducible accumulation of phosphatidic acid in stress signalling. Plant J. 26: 595–605.3.Guo, L., Devaiah, S.P., Narasimhan, R., Pan, X., Zhang, Y., Zhang, W., and Wang, X. (2012). Cytosolic glyceraldehyde-3-phosphate dehydrogenases interact with phospholipase Dd to transduce hydrogen peroxide signals in the Arabidopsis response to stress.Plant Cell 24: 2200–2212.4. Guo, L., Devaiah, S.P., Narasimhan, R., Pan, X., Zhang, Y., Zhang,W., and Wang, X. (2012). Cytosolic glyceraldehyde-3-phosphate dehydrogenases interact with phospholipase Dd to transduce hydrogen peroxide signals in the Arabidopsis response to stress.Plant Cell 24: 2200–2212.5. Hayward, A.P., and Dinesh-Kumar, S.P. (2011). What can plantautophagy do for an innate immune response? Annu. Rev. Phytopathol. 49: 557–5766. Muoz-Bertomeu, J., Bermúdez, M.A., Segura, J., and Ros, R. (2011). Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate dehydrogenase show alterations in abscisic acid (ABA) signal transduction: interaction between ABA and primary metabolism. J. Exp. Bot. 62: 1229–1239
2. 研究的基本内容和问题
.research objectivesTo understand the process and techniques used to clone tomato using gapc2 geneTo learn more about the dangers that may occur during cloningTo be aware of improvements and changes that may develop in the environment.ContentSolanum lycopersicum (tomato) is an important crop part that is a member of the nightshade family, Solanaceae, which includes a variety of agricultural crop plants (e.g. potato, pepper, eggplant, and tobacco). In plants, various studies have demonstrated that cytosolic GAPDHs , referred to as GAPCs, meaning GAPC1 and GAPC2 may be localized in both the cytosol and nucleus (Anderson et al. 2004, Holtgrefe et al.2008, Wawer et al.2010, Henry et al.2015, Peralta et al.2008). GAPCs have been regarded as two phosphorylating cytosolic forms of GAPDH, but our results show that GAPC2 and its cleavage product are present both in the cytosol and the nucleus. This raises questions on the function of the nuclear GAPCs. In mammalian cells, GAPDH, in addition to the role as a glycolytic enzyme, has multiple functions, such as the regulation of gene expression, cell signaling, and membrane trafficking GAPDH functions in both transcriptional and post transcriptional regulations of gene expression through diverse molecular mechanisms. GAPDH is able to regulate gene expression by maintaining DNA integrity in the nucleus and modulating mRNA stability in the cytosol, via protein-nucleic acid interaction, as well as by associating with transcription factors. Glycolytic activity is not required for many of these novel functions of GAPDH.Key issues to be solvedFirst the process Is too long and there is no guarantee that it may work.The sequences we get may be wrong.
3. 研究的方法与方案
Several experiment methods and techniques were applied. Firstly the process of extracting plant tissue RNA . In this process all the necessary components and solutions are used and to get a consistent mixtures . This process is one of the best techniques because It helps with isolation and purification of a product, It prevents ribonuclease activity ,Rapid denaturation of nucleases and stabilization of RNA, Scalable format, Organic extraction is the gold standard, and protocols are well-established and routinely used, making the procedure straightforward for novice researchers. Also percussions were taken into consideration like when adding solutions, add the right amount of grams needed and avoid sneezing or Coughing close to your sample because it may result into contamination.Secondly polymerase chain reaction were some of the samples are prepared before hand and added into a centrifuge tube. PCR is generally more sensitive than culture for detecting organisms of interest, it also detects non-viable organisms and many other things. When using this techniques we have to know the conditions needed, like the length of the target DNA sequences and it’s factor in the level of specificity. In some cases the target sequence may be too long , this will give difficulties to ensure that the target sequence will be copied in its entirety and accurately. The gel electrophoresis analysis , this means we insert our sample into the gel and wait about 20 minutes to get the results. After we do PCR purification, then linkage of the target gene to the vector, then finally transformation.
4. 研究创新点
特色或创新之处GAPC2 gene In tomato (Solanum lycopersicum) plays a very significant part as it is able to produce a clone of it. An accumulation of AtGAPC1 in the nucleus has been observed in response to a peptide derived from bacterial flagellin, suggesting that the nuclear accumulation of GAPC is involved in pathogen-associated molecular pattern that triggered immunity (Henry et al. 2015). Similarly, AtGAPC1 accumulates in the nucleus in cadmium-stressed Arabidopsis roots (Vescoviet al. 2013). However, although this study suggests a role for plant GAPCs in oxidative stress signaling, it appears that the S-nitrosylation of GAPCs is not necessary for their translocation into the nucleus (Vescovi et al. 2013). ROS are generated as signals that alter various cellular and physiological processes in plant growth and development (Desikan et al., 2001; Apel and Hirt, 2004; Gechevet al., 2006; Shao et al., 2008). As we have mentioned Hydrogen peroxide (H2O2) is the major and most stable species of ROS and plays a signaling role in plant response to stresses, such as mediating abscisic acid (ABA)–regulated stomatal closure (Peiet al., 2000; Zhang et al., 2001). H2O2 is thought to affect target protein activities through modification of thiol groups of Cys residues (Hancock et al., 2005). However, it is unclear how such oxidative modification affects a signaling cascade that leads to alteration of cellular function and plant stress responses. Recent studies indicate that phospholipase D (PLD) and its product phosphatidic acid (PA) play a role in ROS-mediated signaling (Sang et al., 2001; Yamaguchi et al., 2004; Zhang et al., 2009; Lanteri et al., 2011)There are many benefits of cloning tomato that makes this system of propagation preferable to seeding. There is no guarantee that a plant will look the way the seed-mother looks. Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in the glycolytic pathway. It catalyzes the major step of the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate,linking the energy-consuming steps of the pathway with the energy-producing steps and providing intermediates for cellular Metabolism. In addition, “moonlighting” functions ofGAPDHs have been increasingly discovered outside of glycolysis. In animal cells, Glyceraldehyde-3-phosphate dehydrogenase participates in multiple nonmetabolic processes, including apoptosis activation, S phase-dependent histone H2B transcription, DNA replication, DNA repair, and immune response to various diseases .In plants, Glyceraldehyde-3-phosphate dehydrogenase have been implicated in embryo development, pollen development, root growth, lipid metabolism, seed oil accumulation, and abscisic acid signal transduction.
5. 研究计划与进展
研究计划及预期进展IntroductionCytosolic glyceraldehyde-3-phosphate dehydrogenases in plants (GAPC2) is a key enzyme in glycolysis that catalyzes the first step of the pathway by converting D-glyceraldehyde 3-phosphate (G3P) into 3-phospho-D-glyceroylphosphate, essential for the maintenance of cellular ATP levels and carbohydrate metabolism by similarly binds DNA in vitro.Tomato plants are vines, initially decumbent, typically growing 180 cm (6 ft) or more above the ground if supported, although erect bush varieties have been bred, generally 100 cm (3 ft) tall or shorter. Indeterminate types are "tender" perennials, dying annually in temperate climates , although they can live up to three years in a greenhouse in some cases. Determinate types are annual in all climates. They are plants that are dicots, and grow as a series of branching stems, with a terminal bud at the tip that does the actual growing. When that tip eventually stops growing, whether because of pruning or flowering, lateral buds take over and grow into other, fully functional, vines.Problem statementStudies have shown many roles of GAPC2 in plants, but not only in plants, also in mammalians, tobacco and Escherichia coli. There is enough prove that suggest that GAPC2 is indeed have an impact in the seedlings growth of a plant however there are some issues that are needed to be paid more attention to, such as why in some cases only GAPC1 is able to impact the seedling growth but GAPC2 is unable to. Studies have shown that PA and GAPC binding was abolished by the cation Zn2 , whereas oxidation of GAPCs promotes the PA binding.Expected progress On the 25 February 2019 we extract plant tissue RNA, this process takes a day to complete On the 26 February 2019 we use the process of segregating of total RNA and we analyzed the purity of total RNA using ultraviolet spectroscopy , then gel electrophoresis lastly picturing. On the 27 February 2019 I’ll be focusing on reverse transcription. On the 02 March 2019 I do PCR and PCR purification On the 04 March 2019 I do transformation process.This in the coming future will be more effective and many companies and laboratory will have access to this kind of research.Doing these kinds of experiments is expensive, and getting results that are correct may takea while but in the near future technology will have change and new machines will be designed.
