Identification Of Plant Autophagy Protein ATG8e In Plant Aging Induced By Carbon Starvation

Under the conditions of biological stress and abiotic stress,Plants will aging quickly.The most obvious phenomenon of plant senescence is that the leaves are yellowing and the substances in the senescent leaves are degraded into small molecules for the recycling of new organs by autophagic pathways.Abiotic stress,such as carbon starvation can cause Arabidopsis plants premature luteinization in a short time and induce plants to produce ATG8e-PE to participate in autophagy.In this study,polyclonal antibody was prepared and the urea gel system was constructed according to the theoretical basis of autophagy study.And using the laboratory-built carbon starvation system to identify carbon starvation induced plant autophagy generated ATG8 e and ATG8e-PE.The main findings are as follows:1 ? Construct pGEX-4T-1-ATG8 e and pET28a-PBA1 prokaryotic expression vector.Expression of proteins by expression system of Escherichia coli.Rabbit was immunized after isolation and purification of the target protein.The purified rabbit antiserum was successfully prepared and the polyclonal antibody of ATG8 e and PBA1 was successfully prepared.2 ? On the basis of traditional urea glue,adds 6 M urea as a denaturant and the buffer system changes into Tris-Tricine.The new urea glue can distinguish small molecule membrane proteins ATG8 e and ATG8e-PE significantly.3 ? The accumulation of ATG8 e and ATG8e-PE in the short time was increased by the treatment of carbon-starved wild-type Arabidopsis thaliana plant with conanamycin A(final concentration of 1 ?M);Protease activity inhibitor E-64 d Concentration of 20 ?M)treatment is the same to concanamycin A;Arabidopsis thaliana plant growing 7 days using autophagic pathway-enhanced substance BTH(final concentration of 100 ? M),the amount of ATG8 e and ATG8e-PE increased in a short time;The plant protein of ATG8 e overexpressed and wild-type Arabidopsis thaliana was extracted.ATG8 e was increased in ATG8 e overexpressed plants.Phospholipase was used to treat carbon starved The wild type Arabidopsis thaliana and ATG8e-PE were reduced in the wild type Arabidopsis thaliana;Wild-type Arabidopsis thaliana and autophagic defeat mutant plant proteins were extracted,and no accumulation of ATG8e-PE was found in the mutant.These experiments demonstrate that the dark carbon starvation system induces plant senescence,and the autophagic processes may lead to ATG8 e and ATG8e-PE.