Functional Divergence Of The AmCIP Gene Family And Functional Characterization Of AmLEA-5from Ammopiptanthus Mongolicus

Ammopiptanthus mongolicus, the only evergreen broadleaf shrub endemic to the northwest desert of China, is a valuable species for plant abiotic stress research. Based on the former research results,we mainly focused on the following two parts of research in this thesis.In the first experiment section, the Ammopiptanthus mongolicus AmCIP gene family members and homologous gene were selected as the target genes. Among the AmCIP gene members, AmP110C, AmP132E, AmP180D and AmP200O had a conserved N-terminal region and C-terminal region. Between the two conserved regions, there were0,1,3and4conserved repeated amino acid fragments, their K-segment belong to FI type. AmP154E and AmP200Q have2and4conserved repeated amino acid fragments, their K-segment belong to LV type. AmP95_are not assigned to the gene family.The experiment of E.coli treated by NaCl demonstrated that7homologous genes of AmCIP declined the salt resistance of strains and there were non-regularity among these genes. The freezing protection to E.coli and LDH proved that7homologous AmCIP could enhance their freezing tolerance. The regularity was that the freezing tolerance of low tandem duplication protein was increased more than high tandem repeats. Besides, there was a little difference between FI and LV type AmCIP in Antifreeze effect. These findings indicate that tandem duplications contributed significantly to the expansion of genes in Plant, which play an important role in the acclimation.The second experiment was about AmLEA-5gene. Molecular characterization and abiotic-stress regulated expression patterns of AmLEA-5(AAW31666.1) which was cloned from cold-and drought-stressed Ammopiptanthus mongolicus by a modified solid subtractive hybridization method werecarried out. Bioinformatic analysis showed that the gene encoded a group5late embryogenesis abundant protein (LEA).The overall length of AmLEA-5was693bp, and the cDNA contained a297bp ORF encoding a polypeptide of98amino acids with a calculated molecular mass of10.6kDa. AmLEA-5, with more phosphorylation sites, was a kind of hydrophilic protein. The analysis of codon bias showed that AmLEA-5gene was bias toward the synonymous codons with A and T at the third codon position. Phylogenetic analysis indicated that AmLEA-5protein was most closed to Medicago tnncatula LEA (ACJ84182.1) in genetic relationship. qRT-PCR analysis demonstrated that AmLEA-5was up-regulated by cold, drought, salt and hot stresses. Specifically, transcription of AmLEA-5accumulated more at the late stage of cold treatment. Intracellular localization analysis revealed that AmLEA-5protein tagged with yellow fluorescent proteins (YFP) was more likely localized in the cytoplasm and nucleus. Besides, the transgenic tobacco T1of AmLEA-5gene proved that this gene can significantly improve the stress resistance. The combined results suggested that AmLEA-5may play important roles in abiotic stresses responses, especially under cold stress condition in A. Mongolicus.