Cloning Of A Super-dehydrin-like Gene Family And Putative Function Analysis Of Abiotic Stress-related Genes In Ammopiptanthus Mongolicus

Ammopiptanthus mongolicus is the only evergreen broadleaf shrub endemic to the Alashan desert, northwest sand area of China, where the climate is arid, the site is desertification and salinization and the temperature is blow-30℃in winter, up to 40℃in summer. Situating long in adverse environments, A. mongolicus has gradually evolved typical superxeromorphic structures and molecular metabolic characteristics to adapt the adversity, such as drought, salinization, cold, heat and other stresses. Tolerance to various adversities makes A. mongolicus an excellent model of woody plants for the study of molecular mechanisms underlying abiotic stresses tolerance. However, few studies were focused on molecular biology of A. mongolicus. Hereby, we carried out a systematic study on the functions of some A. mongolicus abiotic stress-related genes to explore molecular mechanism of stress tolerance, and to provide data and technical support for stress tolerance and resistance breeding of crops. The main findings in this study are as follows:1. Identification of A. mongolicus a KS-type dehydrin-like protein AmCIP and a super KS-type dehydrin-like protein family.1.1 Identification of protection role of a KS-type dehydrin-like protein AmCIP from A. mongolicus under cold stress.Herein firstly, we report function analysis of a dehydrin-like protein AmCIP unified with a distinct K-segment of HKEGLVDKIKDKVHG similar to the 15-mer consensus sequence EEKKGIMDKIKELPG of typical dehydrin. Expression of AmCIP in transgenic tobacco leaded to enhanced tolerance to cold stress for both geminating seeds and seedlings. Over-expression of AmCIP in transgenic E. coli significantly improved freeze-thawing tolerance of host E. coli cells. Moreover, the recombinant AmCIP kept soluble pre-and post-10 min boiling treatment, and conferred stabilization of the LDH under freeze-thawing stress, consistent with the characteristic of dehydrins. YFP-AmCIP fusion protein was subcellular localized in the cytoplasm and nucleus in onion inner epidermal cells, suggesting that AmCIP might function in the cytoplasm and nucleus in cells. The combined results indicated that AmCIP with a distinct K-segment was a hydrophilic dehydrin-like protein and closely associated with resistance to cold stress. This research offered the valuable information for molecular mechanism of dehydrins and stress responses in A. mongolicus.1.2 Cloning, sequencing and functional analysis of a super KS-type dehydrin-like gene family in A. mongolicus.A super AmCIP homologous gene family was obtained through PCR by primers pairs according to DNA sequence of AmCIP. The total present members of this AmCIP homologous gene family account for 126 plus AmCIP. The coded proteins of these genes range from 106 to 290 amino acid (aa) residues, which share high degree of homology with AmClP and between each other, while share low degree of homology with other typical dehydrins. And this dehydrin family has not been reported before. These proteins are named through AmP+protein length+different English letters to present different members, such as AmP106A, AmP106B, AmP106C, et al. The calculated molecular mass of these proteins ranged from 11.661 KDa to 30.688 KDa. Glycine, glutamate, asparagines, glutamine and histidine in each of these protein accounted for more than 60% of the total number of amino acid residues, in which the content of glycine is the highest. As predicted, almost all the members of this A. mongolicus KS-type dehydrin-like protein family are acidic proteins, except for AmP200P which is a basic one.These proteins have a 33 aa conserved N-terminal region, and a 71 or 73 aa conserved C-terminal region. Between these two conserved regions, there are 0～8 conserved repeated amino acid fragments, the length of which is 22 or 24 or 26 aa. And these repeated amino acid fragments also have high degree of homology. Each of these proteins also has a distinct K-segment similar to the 15-mer consensus sequence EEKKGIMDKIKELPG of typical dehydrin, and an S-segment in the conserved C-terminal region, but has no Y-segment. So this group of proteins was called KS-type dehydrin-like protein family of A. mongolicus. These proteins are all hydrophilic and flexible proteins with 3-11 casein kinaseⅡphosphorylation sites,1-16 N-myristoylation sites, suggesting these proteins may be involved in signal transduction activities during abiotic stress. Some members of this family also have cell attachment sequence. Phylogenetic analysis indicated that the members of the KS-type dehydrin-like protein family of A. mongolicus are closely related to each other, and this group is most closely to AEJ20970.1 of Caragana jubata, AEV59620.1 and AEV59619.1 of Oxytropis campestris subsp Johannensis, AEV59613.1 of Oxytropis arctobia which are all cold responsive dehydrins.Expression of this A. mongolicus KS-type dehydrin-like protein was analysed through western blot by using antiserum agains AmCIP. Results showed that this A. mongolicus KS-type dehydrin-like protein family was constitutively expressed with low level, and accumulated at the late stage of cold, drought, salt and heat stresses.The combined results indicate that this A. mongolicus KS-type dehydrin-like protein family plays important roles in abiotic stress responses. Prior to this, no such big dehydrin family has been reported, and the discovery of them is valuable for future researched on evolution of dehydrin and anti-stress mechanism of A. mongolicus.2 Functional identification of A. mongolicus defensin gene AmDFAmDF was obtained through solid-phase subtractive hybridization from cold stressed A. mongolicus. Sequence analysis showed that the overall length of AmDF gene was 414 bp, and the cDNA of AmDF contained a 207 bp ORF encoding a polypeptide of 68 amino acids with a calculated molecular mass of 7.66 kDa and a theoretical pI of 8.92. AmDF contained eight conserved cysteine residues which could form four disulfide bonds, and a Knotl structure which is common in y-thionin and knottin with antimicrobial ability. Phylogenetic analysis indicated that AmDF was most closely related to Plantago major defensin CAH58740.1. Real-time quantitative PCR analysis showed that AmDF was constitutively expressed, up-regulated by cold, drought, salt and heat stress, and specifically accumulated at the late stage of cold treatment. YFP-AmDF fusion protein was subcellular localized in the cytoplasm and nucleus in onion inner epidermal cells, suggesting that AmDF might function in the cytoplasm and nucleus in cells. However, the cold, drought and salt tolerance of AmDF transgenic tobacco was not improved. The combined results indicate that AmDF protein plays some roles in anti-microbial activities under cold stress to elevate the survival rate of A. mongolicus under cold stress, which need to be confirmed by further study.3. Expression analysis of abiotic stress-related functional genes in,4. mongolicus3.1 Reference gene selection for A. mongolicus under cold, drought, salt and heat stressAbiotic stress tolerance of plant is not determined by single gene, but resulted from products of great many genes. The mechanism of stress tolerance could be hardly explained by analysis of one or a class of genes. However, a comprehensive research on expressed genes is advantageous in revealing the complex stress tolerance mechanism, which is more and more based on gene expression. qPCR is more widely used for its multiple advantages of accuracy, sensitivity, reproducibility, and high throughput. Nevertheless, the accuracy of qPCR was significantly influenced by the reference genes used. By now, no report has so far described the selection of reference genes to get stringent normalization for qPCR in A. mongolicus.We identified reliable reference genes for normalization of qPCR data in A. mongolicus under abiotic stresses from 14 reference gene candidates (UBQ, Tub1, Tub2, Abel, Ubc1, Ubc2, Ubc4, Ubc5, elF1, elF2, eIF3, eIF4, EF1, EF2). We set a series of 22 experimental samples covering the control and different time points under cold, dry, salt, and heat stresses. According to geNorm and NormFinder, the combination of elF1 and eIF3 was more sufficient to confer an accurate normalization across all the treatments, which was confirmed by normalizing qPCR data of AmHsp90. In contrast, Tub1, Abel, and EF1 were ranked poorly and should be excluded as reference genes.3.2 Expression analysis of abiotic stress-related functional genes in A mongolicus.55 anti-abiotic stress genes were chosen from an EST data of cold-and drought-stressed A. mongolicus, including AmSOD01～04, AmCAT01, AmAPX01, AmAPX02, AmGPX01, AmPrx01, AmPrx02, AmTrx01～06, AmFd01～04, AmFNR01, AmGrx01～05, AmHsp70-1～3, AmHsp90-1, AmDnaJ01, AmDnaJ02, AmLEA01, AmLEA14, AmLOX01, AmLOX02, Am1433-01～03, AmCAB01～07, AmFBP01～02, AmFBA01, AmPGK01～02, AmEno01, AmMT01～03, AmFerOl, which are known to be involved in anti-stress mechanism. The expression of these genes was analysed in the same above 22 samples by qPCR through normalizing against elF1 and elF3. Results showed that these genes are all constitutively expressed in A. mongolicus, suggesting that they had basic functions under normal growth conditions. The balance and coordination of them provides highly efficient machinery to maintain normal growth and metabolism. The 55 anti-stress genes exhibited diverse expression levels and differentially up-or down-regulated expression patterns under abiotic stresses, indicating different anti-stress protection roles of them under abiotic stresses. The anti-oxidative genes are up-regulated at various stages of cold, drought, salt and heat stresses, suggest a powerful control for generating oxidative damage under abiotic stress. Photosynthesis associated genes AmCAB01～07, and gluconeogenesis and glycolysis associated genes AmFBP01～02,AmFBA01,AmPGK01～02 and AmEno01 were down-regulated at the late stages of abiotic stresses, indicating that A. mongolicus may reduce its demand for energy and assimilates at the late stages of abiotic stresses to survive the long-term hard environment. The combined results suggested that these genes may play positive roles in mechanism of A. mongolicus abiotic stress tolerance. The diversity of expression patterns of these genes under abiotic stresses provides a preliminary impression of the complicated stress tolerance mechanisms in A. mongolicus. These results are valuable for future research on gene expression and abiotic stress tolerance in A. mongolicus.In conclusion, A. mongolicus has evolved a complete set of defense mechanism to survive in the extreme environment of desert. The findings in this study confer a better understanding of the special anti-stress system of A. mongolicus, and provide important guidance for future researches on plant anti-stress activities.