Functional Analysis GHABF2and Cloning Of GhSRK2D In Cotton

Drought and salinity are the most frequently and adversely abiotic stresses affecting agricultureworldwide, and the phytohormone abscisic acid (ABA) is a key endogenous messenger for plantresistance to stresses and understanding ABA signaling. Tremendous efforts have been devoted toidentifying key regulators such as ABA-signaling-related genes likely act as candidate genes for theirapplication in the improvement of drought tolerance in crops through transgenic approaches, which willhelp developing agricultural production in arid regions. In this study, SnRK2（GhSRK2D）and ABF2（GhABF2）, the key regulators in cotton’s’ ABA signaling, were analyzed with the status of Currentresearch on plant stress. The following main results are obtained1. In this study, SnRK2homologous, GhSRK2D was isolated from the Y18upland cotton viaRACE-PCR. The full length transcript (3796bp) of GhSRK2D, containing1781bp cDNA, putativelyencodes a protein with361amino acids. Bioinformatics analysis results suggested that GhSRK2Dbelongs to SnRK2s III subfamily, containing the N-myristoylation sites and trans-membrane domains,and five core serine-residues sites. Additionally, Osmotic Box, SnRK2Box and ABA Box were alsofound the end of C-terminal conserved domain of protein GhSRK2D.2. Southern blot results showed that GhSRK2D gene at least had two copies in cotton Y18ecotypegenome. Real-Time PCR showed that GhSRK2D and GhABF2were expressed in various tissues andorgans of cotton, the highest-level expression of GhSRK2D gene was identified in leaf organs and thatof GhABF2gene in stems, leaves and floral organs. GhSRK2D and GhABF2also were induced by ABA,high salt, hypertonic (mannitol) and low temperature treatments.3. GhABF2had transcriptional activation, while GhSRK2D showed no self-activation in yeast.GhABF2also physically interacts with kinase GhSRK2D in yeast. The LacZ-activity investigation wasconducted into the fusion strains of GhSRK2D target protein and variousdefective-GhABF2-recombinant proteins, the results proved to be consistent with trends of theself-activation analysis4. The encoding sequence of GhABF2and GhSRK2D were isolated from cotton and the followingplant expression constructs: P35S:GhABF2, P35S:GhSRK2D and P35S:GhABF2-GhABF2wasdeveloped, respectively. The T3generation transgenic cotton plants with P35S:GhABF2and T0generation transgenic cotton plants containing P35S:GhSRK2D and P35S:GhABF2-GhABF2wereobtained through cotton Agrobacteria mediated floral dipping transformation method. The details offunctional analysis of P35S:GhABF2transgenic cotton plants are as follows.(1)Under normal conditions, overexpressing GhABF2transgenic cotton plants have not anydetectable phenotypes observed in contrast to the wild-type cotton. However, under salt stress,overexpressing-GhABF2transgenic plants showed a significantly-higher-tolerance to salt comparedwith wild-type control plants, indicating that GhABF2plays a key role in cotton in response to saltstress.(2)Under Exogenous ABA treatment, overexpressing-GhABF2transgenic plants were more sensitive to the ABA than that of wild-type.(3) In growth medium with high sucrose/low glucose,overexpressing-GhABF2plants exhibitive a significant-improvement in the absorptive capacity of lowglucose when compared to wild-type whose growth was suppressed. This implied that GhABF2genemay be associated with glucose metabolism.(4) Under mannitol-simulated drought stress,overexpressing-GhABF2transgenic plants had a remarkably-enhanced tolerance to osmotic stress whencompared to wild-type whose growth and development is severely inhibited.In this study, we isolated the GhABF2and GhSRK2D protein kinase gene from cotton anddetected the expression pattern of GhSRK2D and GhABF2in various tissues and various stresstreatments including ABA, high salt, hypertonic (mannitol) and low temperature treatments. Asindicated in GhABF2-transcriptional-activation analysis, not only does the transcriptional activity ofGhABF2depended on its N-terminal67amino acid peptide, but also GhABF2physically interact withkinase GhSRK2D in yeast living cells. Furthermore, over-expressing GhABF2in cotton can contributeto a remarkably-improved-tolerance to drought. Additionally, GhABF2may be related to glucosemetabolism. These results suggested that GhSRK2D and GhABF2likely involve in abiotic stressphysiological processes in cotton.