The Establishment And Analysis Of Transcriptional Profile Of Ammopiptanthus Mongolicus Under Abiotic Stress Conditions

Abiotic stresses, including extreme temperatures (extremes of cold and heat), drought, salinity, oxidative stress and chemical toxicity, are serious threats to the plant growth and productivity and result in a series of physiological, morphological, biochemical and molecular changes. In view of the serious influences of abiotic stress on plant, it is important to carry out the research in molecular biology of stress-resistant plant and to gain the gene resourses from the plant with resistance. However, traditional breeding methods usually have little effection on the resistance to abiotic stresses because of the lack of effective gene resources. During the evolutionary, crops have gained certain resistance which can only be stimulated when stress occurs. The activation of cluster resistance-related genes transcription can effectively enhance the resistance of crops. Ammopiptanthus mongolicus, mainly distributing in the desert regions of Northwest China, which is capable of surviving unfavorable conditions such as cold/heat weather or lack of moisture, should be paid due attention as a highly adversity-resistant plant in the research of plant stress.In order to identify stress-regulated genes, A. mongolicus was chosen as the study object to estamblish its transcriptional profile under abiotic stresses, and some transcription derived fragments (TDFs) from the plant were identified and analysed. Based on the optimization of the conditions of seed germination and seeding stresses, a cDNA-based Amplified Fragment Length Polymorphism (cDNA-AFLP) approach was applied in this study. After optimizations of cDNA-AFLP such as the choice of primers combinations, the reaction conditions of polymerase chain reaction (PCR), ways of polyacrylamide gel electrophoresis (PAGE),recovery of the transcript derived fragments (TDFs) and the method of re-amplifications, the transcriptional profile on A.mongolicus with cold, heat and drought treating was then analyzed. A total of 1487 TDFs expressed in various stresses were gained in the study, of which 536 TDFs were cloned and sequenced. Among the sequenced TDFs, 287 had an up-regulated expression while 249 were down-regulated, and 201 which had significant homeology with the sequences in data bank were chosen to establish the transcriptional profile of A. mongolicus under abiotic stresses.Among the 201 TDFs, 85 could be found in low temperature stress, 175 could be found in high temperature stree, and 93 could be found in drought stress. According the Blast results of the sequenced TDFs, 201 showed significant similarity to the reported genes in the NCBI databases and were functionally annotated to be used in gene difference expression. The TDFs in each stress could be classified as 8 kinds: cellular metabolism, signal transduction, transporter, transcription, cell resecue/ stress-related /defence, cell growth and structure, photosyntesis and energy, and unknown function. The largest category of identified genes was in cellular metabolism.A semi-quantitative RT-PCR strategy was then carried out to verify the expression of some TDFs under stresses, which have significant homeology with the encoding genes of HSPs, photosystem II CP43 chlorophyll apoprotein, CCR4-associated factor. The results of semi-quantitative RT-PCR showed that the expression patterns of chosen TDFs were same with previous cDNA-AFLP, representing simultaneous up-regulation or down-regulation.