Transcriptional Characterization And Functional Identification Of A Gene Encoding DEAD-box Helicase (AvDH1) From Apocynum Venetum

Soil salinity, by inhibiting growth and crop yield, is a severe and increasing constraint on agricultural productivity. And the salt stress responses and adaptations are complex mechanisms and still not clear. Therefore, it is essential to analyse the expression pattern and the function of stress-induced genes not only to understand the mechanism of stress tolerance but also to improve the stress tolerance of crops by manipulating such genes. Our laboratory has isolated a salt-resposive gene encoding DEAD-box helicases, designated AvDH1 from the halophyte dogane (Apocynum venetum) by using suppression subtractive hybridization and RACE PCR, and then introduced this gene into cotton by pollen tube pathway. Based on these researches, in this paper, we did the transcriptional analysis and the functional determination of the gene AvDH1 .The main results are as follows:1. Tissue distribution and gene expression of AvDHl under abiotic stresses in dogbane. Northern blot analysis suggests that the transcription of AvDH1 is salt dependent and the AvDH1 mRNA increases with salinity. The expression of the AvDH1 mRNA is constitutive in the root, stem, and leaf. So the AvDH1 protein seems to play a role in the basic activity of cells under salinity. The AvDH1 expression is also strongly up-regulated by low temperature, but there is no change in response to water stress due to PEG, in aerial parts of seedlings, compared with non-stressed plants. The transcript level is hardly affected after treatment with ABA, suggesting that AvDH1 follows in ABA-independent salt stress signalling networks.2. AvDH1 has been integrated into cotton genome and is genetically stable. AvDH1 was transformed into cotton cultivar LU613 by using pollen-tube pathway method. To determine whether AvDH1-imparted salinity tolerance is genetically stable, the transgenic progeny were characterized. T2 and T3 seedlings were screened by kanamycin selection, demonstrating that they segregated in the 3:1 ratio of Kanr/Kans and mostly transgenic lines could inherit steadily.The presence AvDH1 was confirmed in the positive plants by PCR. Randomly selected positive plants were amplified the same size of the fragments as plasmid was done. The integration of the gene was further confirmed by PCR-Southern hybridization in T2, and the fragment of AvDH1 was observed, in contrast no band was detected in the WT plants (LU613). These results suggested that AvDH1 has been integrated into cotton genome.3. T2-transgenic plants tolerate excess salinity. To determine whether AvDH1-imparted salinity tolerance is functionally, T2-transgenic plants were screened by pollen grains screening and leaf disk assay. The pollen germination percentages of the T2 by sitting drop culture are significantly higher than the WT plants in salt stress. In leaf disk assay, salinity-induced loss of chlorophyll was lower in T2 compared with those from the WT plants (LU613). The damage caused by stress was reflected in the degree of bleaching observed in the leaf tissue after 72 h, and the damage of the WT plants were more severe than T2, suggesting that the leaf of T2-transgenic plants has higher salt tolerance compared with the WT plants (LU613).4. T3-transgenic seedlings were characterized. T3 and WT (LU613) seedlings were morphologically similar when grown without NaCl. In the presence of NaCl, the WT seedlings showed growth retardation, whereas transgenic plants did not develop any sign of stress; SOD activity and electronic conductivity of the leaf were determined as indicators of salinity stress, and the SOD activity values of T3 are higher and electronic conductivity values are lower compared with those from the WT plants grown in the same condition, indicating that salt tolerance of T3 transgenic plants was enhanced. Above all, it was evident that T3-transgenic plants have a better ability to tolerate salinity stress compared with WT plants.5. The reading frame in the AvDH1-cDNA encodes a 447 amino acid polypeptide having a predicted molecular mass of 50 478 Da and a pI of 7.61. Using the CLUSTAL W program, comparison of the AvDH1 amino acid sequence with known proteins demonstrates a homology with the DEAD-box family. Nine conserved amino acid motifs normally found in members of the DEAD-box protein family are all present in the middle region of AvDH1 (amino acids 33–365). In the SAT (Ser-Ala-Thr) motif, however, the first serine residue is changed for threonine in AvDH1. In the C-terminal region (amino acids 365–447) there are two RGG boxes, a motif involved in RNA binding, and a nuclear localization signal, KKSRKEKK.