Cloning And Functional Characterization Of A Novel Stress-responsive Gene, AmHP40from Ammopiptanthus Mongolicus

Abiotic stresses are one of the severest environmental pressures for plants, commonly occurring under natural growing condition due to drought, salinity, cold and heat. Plants sensitively respond to these stresses by activating a set of genes, which encode proteins necessary to overcome the crisis. To carry out abiotic stress tolerance mechanisms, Ammopiptanthus mongolicus provides strong resistance of growing under extremely drought, high temperature and poor soil quality in the desert area of northwest China. In our study, we cloned and identified genetic function of new genes obtained from A. mongolicus, aiming to offer new genetic resources for breeding program of abiotic stress tolerance. The results are as follows:1. Genetic characteristics of AmHP40. AmHP40, a stress-responsive gene was isolated from A. mongolicus by using cDNA-AFLP and RACE-PCR methods. Its elongated fragment was321bp open reading frame (ORF) with a start codon ATG and a stop codon TAG. The BLAST from NCBI analysis revealed that this ORF encodes for the AmHP40protein of106amino acids residues. Sequence homology revealed the highest similarity to predicted protein which unknown function, whose proteins have not been characterized. AmHP40protein contained a typical domain of unknown function (MWHLIAAVRR) from position Metl to Arg10and no transmembrane domain. In addition, AmHP40protein appeared to be soluble protein. Its secondary structure includes helix, coil, and beta strand which is low in helix and rich in coil. We further showed that GFP-tagged AmHP40was preferentially distributed to the cytosol in Arabidopsis protoplast. In addition, expression of AmHP40was strongly induced by salt, freezing, heat, drought and gibberellic acid.2. Overexpression and physiological characteristics of transgenic plants. AmHP40was ligated into pBI121expression vector and transformed to Arabidopsis thaliana. Western blot analysis and GUS activity assays confirmed the integration into A. thaliana genome of AmHP40gene. GUS signal showed expression of AmHP40gene was strongly expressed in leaf, stem, silique, and root tissues of transgenic Arabidopsis plants. Transgenic Arabidopsis exhibited normal growth. No dwarfed phenotypes were observed. Interestingly, transgenic plants flowered earlier than WT after grown in soil about4weeks.3. Abiotic stress tolerance analysis. The tolerance ability was tested in three developmental stages; seed germination, seedlings and whole-plants of transgenic and WT. Transgenic Arabidopsis exhibited enhanced tolerance to abiotic stresses. Germination of transgenic seeds on medium containing100mM salt and300mM mannitol were higher than WT. Root length and dry weight of transgenic seedlings treated with100mM NaCl (salt),300mM mannitol (drought),-20℃for1h (freezing) or45℃for1h (heat) was less affected by treatments. An obvious difference in shorter root length of transgenic seedlings was observed under normal conditions. In addition, chlorophyll a, b and total chlorophyll of transgenic plants irrigated with100mM NaCl were higher than WT as well as the number of new leaves of transgenic plants treated with45℃for4h (heat) or-20℃for4h then4℃for2h (freezing) or no irrigation for10days. Furthermore, we found that accumulation of total soluble protein in transgenic seedlings was significantly increased under salt, freezing and heat treatment. 4. Expression of marker genes in abiotic stress response pathway. The transcript levels were changed between transgenic and WT plants after salt, cold and heat treatments, indicating that AmHP40mainly plays a role in enhancing salt, cold and heat tolerance pathway. Our data suggest that AmHP40improved salt, cold and heat tolerance by contributing accumulation of AmHP40proteins that may function as osmoprotectant. AmHP40probably use as a target gene for enhancing abiotic stress tolerance in important crops.5. Deletion analysis of AmDUF1517upstream region. The1,026bp of promoter sequence of AmDUF1517contains cis-acting elements responsive to abiotic stresses and plant hormones. To identify the functional region, serial deletions of the promoter and caMV35S were fused into pGL4expression vector and seven constructs were obtained.