Sieve and Molecular Identify of Arabidopsis Autophagy Family Eight Transgenic Plants
|School||Shandong Normal University|
|Keywords||ATG8 PCD autophagy homozygote transgenic|
In general, plant can not maintain the normal physiological function because of lacking nutrition in environmental stress.The nonbiologic stress such as low temperature, drought, ABA, high salinity is the disadvantageous environment which can widely affect crop production and plant growth.When the plants are under environmental stress, they can regulate the expression of their stress-related genes by theirselves and bring forth related-tolerance. At the present, a lot of stress-related genes had been cloned, and were used in anti-stress gene engineering.At the same time, we agained some transganic plants which can grow normally in some environmental stress.All exhibit extent prospects.Programed cell death (PCD) is a kind of death which is orderly and independent and is controled by gene.The aim is to keep the steady of the inside environment. It involves a series of effects which is the activation, expression and regulation of gene. Programed cell death plays an important role in plants development, immune regulation, and general cellular homeostasis of embryonic. So it is physiological and selective. In general, cell death has been divided into necrosis and programed cell death (PCD). The former is a passive death because of pathology. Chemic or physical injure factors and lack oxygen or undernourishmend all can cause this type cell death. And the later is a programmed and an active cell death process which is definited by depending on the caspase protein family. But a lot of researches indicated that there was a kind of cell death which was non-dependent caspase.We preliminary knew such way such as autophagy.Autophagy exists widely in eukaryotic cell and it is a degradable way which is depend on lysosome.It can cause long-live proteins of cells degrading,and it takes part in the normal growth,development and metabolism homeostasis in cells. Under some stress, such as damage, nutritive deficiency, hunger and so on, autophagy induces plants use cells component by cycling that can help cell live better and it even plays important roles in some cancer cells. Autophagy is a life phenomenon which is widely in eukaryotic cell, it runs through growth, development and Pathophysiology of normal cell.It can prevent decline and some illness (such as neurode generation, tumor, myocardosis and so on) and it also can prolong life. So the research of autophagy and autophagy-death is very useful to not only the theory significance but also application meaning. The find of autophagy gene helps us know the molecular level of autophagy, but the origin and signal transduction of autophagy and its affect to the cell survival have not been fully known. With the mechanism of autophagy in-depth study, we expect that we can control of cancer and the development of neurodegenerative disease, prolong life and improve the quality of life by regulating the level of autophagy in cells.In recent years,because of molecular biology,genomics and other experimental means improving, especially mutant strains of autophagy in Yeast,the research of autophagy molecular basis was made a great progress.ATG8 is the most important component of autophagic lysosomal degradation pathway. Inhibition of autophagy by chemical compounds or knocking down the expression of key autophagy which is called ATG8 can inhibit autophagy, and prevent ROS accumulation and cell death. These findings unveil a molecular mechanism for the role of autophagy in cell death.This experiment using RT-PCR method amplified ATG8e, ATG8f, ATG8h.Using of plant expression vector pCAMBIA3301 (which carried separatly promotors of CamV35S, rd29A, SEN1) and Agrobacterium tumefaciens GV3101 and introduced into Arabidopsis thaliana by in planta transformation method. Because transformants have the ability of resisting basta, we can acquire transformants by spraying basta even obtain their homozygotes.And then we validated their molecular identification and observed their phenotype when they were under environmental stress.The molecular identifications of these homozygotes are as below:1 PCR result showed that all homozygotes plants had strong positive signals, which is the gene of Bar and nearly 500bp specific bands, and no signal was shown in wild type plants.2 The RNA of homozygotes plants and wild type plants were extracted and reverse transcription, then the cDNA were made to RT-PCR. The results showed that comparing with the wild type plants the ATG RNA rised in homozygotes plants when they were dealed with stress condition.(e.g. NaCl,low temperature,ABA).This further explained ATG gene has been integrated in arabidopsis genome and transcriped normally.And this caused multiplication of ATG RNA.At the same time, the homozygotes plants and the wild type plants were all under environmental stress,such as:drought,low temperature,ABA and NaCl,and observed the phenotype of all plants when they were under different environmental stress.When homozygotes and the wild type plants growed under drought,low temperature condition,autophagy played the role of anti-cell death.So homozygotes growed better than the wild type plants.When they growed under the condition of hight salt, autophagy played the role of pro-cell death.So homozygotes growed worse than the wild type plants. All these illustrated that autophagy and programed cell death had close relationship.