Screening And Expression Analysis Of The Temperature Responsing Protein Which Can Remove Low Temperature Stagnancy Of Germination In Lepidium Apetalum Willd.seeds

Lepidium apetalum Willd. is a widespread plant, belonging to the family of Brassicaceae. Interestingly, the Lepidium living in northern Xinjiang province, which can withstand the cold air in early spring useing the snowmelt to germinate and grow,is a typical early sping ephemeral plant. Previous studies found that the seeds of Lepidium living in northern Xinjiang germinate under low temperature to a certain stage, there will be germination stagnation and may relieve stagnation after treatment for a short time by high temperature, then the seeds could continue to grow under low temperature.Therefore, the proteomic technology was applied to screen related proteins from releasing Lepidium seeds, the peptide sequences of mass spectrometry identification was retrieved the transcriptome database to obtain partial nucleic acid sequences; the expression of these genes corresponding with each protein at different stages of Lepidium seed germination under low temperature was analyzed and key gene related to germination stagnation relieve was cloned. All the above provide a basis proof for clarifying the mechanism that Lepidium living in northern Xinjiang can tolerate the cold air to germinate during early spring, meanwhile, it also provides a theoretical basis to reveal the widely distributed feature of Lepidium.1. Establishment of Two-Dimensional Electrophoresis System of Proteins in Lepidium SeedsThe effect to Lepidium seeds protein of three kinds of commonly used plant protein extraction method were compared, TCA/acetone precipitation method was finally uesed to extract Lepidium seeds protein. Optimization of the way of sample loading, focusing procedure, concentration of separation gel and other protein separation conditions were combined to establish a two- dimensional electrophoresis system, which is suitable to Lepidium seeds protein and could obtain 2-DE maps which are repeatable and has high resolution.2. 2-DE Maps Analysis of Differentially Expressed Protein in Lepidium Seeds Removed Germination Stagnation and Stayed Germination Stagnation Under Low TemperatureThe 2-DE maps of protein in Lepidium seeds releasing low temperature germination stagnation or not are compared, 37 distinct proteins were screened, 6up-regulated protein of them is dealt with LC-MS/MS mass spectrometry. According to the function of six protein, it is divided into three categories. The first category isthe molecular chaperones, including Cpn60, Hsp17.6I, Hsp70; second category is peroxidase, including Per12 and Per28, third category is the cell division control protein that is Cdc48 E.3. The Corresponding Gene Expression Analysis of Key ProteinThe nucleic acid sequence information of corresponding gene of Hsp17.6I, Per12 and Cdc48E were searched from Lepidium seeds transcriptom database. The expression on transcription level of the three protein is analyzed. The expression of CDC48 E, HSP17.6I and PER12 is significantly increased when Lepidium seeds at the period of germination stagnation are treated by 25℃ for 30 min, and the 25 ℃treatment within 45-55 min, the expression are increased over time. After treatment for60 min by 25℃, the expression are becoming stable and never significant grow.4. Cloning and Sequence analysis of CDC48 E GeneThe conserved cDNA of CDC48 E gene in seeds of Lepidium apetalum Willd.removed germinaion stagnation under low temperature was cloned by homologous cloning technology. The size of conserved cDNA sequence was 1123 bp.Bioinformatics software was applied to predict and analyze the nucleic acid sequence of conserved CDC48 E gene in seeds of Lepidium apetalum Willd. removed germinaion stagnation under low temperature. The results showed that: there was three conserved domians in the sequence, two AAA conserved domain, the first one is between 163 bp and 567 bp, the next one is between 166 bp and 576 bp, and one CDC48 conserved domain is between 753 bp and 1007 bp.This study started from the level of translation, two-dimensional electrophoresis,mass spectrometry and bioinformatics were combined to screen the response protein related to remove germination stagnation in Lepidium apetalum Willd. under low germination, and the screened protein expression patterns were analyzed by real-time quantitative RT- PCR technology(the result obtained was consistent with it in the transcriptome), and one of the key gene was cloned and analyzed to predict the structure and function of its. All the above had provided some reference to understand the the role of some related proteins on plant germination under low temperature and the signal mechanisms, and made a foundation for further study of plant adaptation to the environment, as well as the molecular mechanisms to resist low temperature to germinate.