Analysis Of Temperature-related Gene Expression In Ammopiptanthus And Research On Cold Induced Amduf1517Gene’s Cloning And Function

Temperature is one of the important environmental factors affecting plant growth, development, andalso can affect crop yield. As an evergreen shrub in China’s temperate desert, Ammopiptanthusmongolicus can endure drought, cold, high temperature, and poor soil, and has developed a uniquetolerance mechanism in the long-term stress adaptation. So Ammopiptanthus is the ideal material tocarry out the study of plant’s tolerance to environmental stresses. In our study, we screenedtemperature-related TDFs based on the cDNA-AFLP results and carried out functional identification ofnew genes, aiming to help to provide new genetic resources for resistance breeding. Results are asbelow:1. Screening on Ammopiptanthus temperature-related TDFs. Bioinformatics analysis was carried outon Ammopiptanthus adversity transcriptional expression profiles.43TDFs upregulated by temperaturewere chosen for semi-quantitative PCR analysis, using β-actin as a control. Finally, we identified16positive TDFs, among which2are related to signal transduction,3related to cell metabolism,4relatedto photosynthesis and energy,2related to transcription,1related to cell rescue, and4function unknown.Rapid amplification of cDNA ends (RACR) was used to clone full-length of three cDNA sequences, andnamed them AmRubisco Activase, AmDUF1517and class II small AmHSP according to theirHomologous genes from NCBI. Cold related gene AmDUF1517was chosen for further study.2. Genetic characteristic of AmDUF1517. This gene has an open reading frame of906bp, encoding301amino acids, of which the C-terminal is a conserved region, being high homology with ArabidopsisDUF1517domain (DUF1517superfamily). This gene has an intron of969bp. Chromosome walkingwas used to get AmDUF1517’s promoter sequence of about1.0kp, which has the typical eukaryoticpromoter sequence characteristic at the site of-127bp to-78bp upstream, and a TATA box at about-50bp upstream, the result also conformed us that we had got the whole length of AmDUF1517. Southernblotting detected there is a single copy of AmDUF1517in the genome of Ammopiptanthus. Subcellularlocalization showed AmDUF1517protein locates on chloroplast. Real-time PCR showed lowtemperature of4℃can induce AmDUF1517’s expression, and two peaks appeared in the early phase oftreatment on the expression curve. Real-time PCR detected after treatments of ethylene, NaCl, ABA, Vc,GA, SA and MaJA, ethylene, GA and SA could induce the expression of AmDUF1517to2fold or moreof control, NaCl made to2.5fold.3. Experiments of over expression and mutant reply verified AmDUF1517gene function.AmDUF1517was linked to plant expression vector and transformed into Arabidopsis of wild-type andmutants. Transgenic lines of mutant351,353,357and361, and transgenic line of wild type were got.Electrolyte leakage rate, free proline content, soluble sugar content, and MDA content of eachArabidopsis line were measured before and4℃for2h, and besides351’s transgenic line, other mutantreplies showed a significant increase to cold. For the Wild-type, soluble sugar content and free proline content showed enhanced resistance in transgenic line.4. Comparison of Arabidopsis germination under stresses of salt and drought was carried out, andthere was no significant difference among wild type, overexpressed type, mutant and mutant reply underthe same stress. Recovery of1week after-20℃2h was carried out on Arabidopsis seedlings. For the3d old （when did the cold treatment）seedlings, root length of mutant was significantly shorter than thewild type, overexpressed type and mutant reply. For the10d old seedlings, there was no significantdifference in root length among the4lines, but the mutant had more fibrous than the other3lines, andsome leaves of the mutant turned yellow. These results indicated that mutant’s resistance to coldtolerance is lower than the other3lines. Experiment of-20℃2h on Arabidopsis leaf protoplasts alsoshowed that AmDUF1517could increase the stability of chloroplast membrane structure inoverexpressed type and mutant reply compared with wild type and mutant.