Molecular Cloning And Functional Characterization Of MdCIPK6 Reveals Its Involvement In Multiple Stresses Tolerance In Apple

The calcineurin B-like proteins (CBLs) are recently identified to be a kind of plant specific calcium sensors. CBL interacts with CIPK (CBL-interacting protein kinase). CBL and CIPK were proved to play important roles in responding to stresses and regulating certain developmental processes in plants. CIPKs are involved in the responsiveness of plants to abiotic stresses such as high salinity, drought and low temporature. However, although researches on CIPKs have made much progress in Arabidopsis, rice and other model plant species in the recent years, only few studies have been made and thus related information is limited in fruit tress. As less and less cultivated field, and the global climate change makes it worse for fruit trees to grow under deteriorating environment. Cloning CIPKs in apple not only shows theoretical meanings, but also benefits to practical application, providing potential candidate genes for improving stresses torlerance by genetic transformation in apple. Here, we isolated MdCIPK6, a CIPK gene from apple. Its structural features, expression profiles and fuctions have been analized. The main results were as follows.1. The full length of MdCIPK6 cDNA was obtained by using RACE method and it is 1565bp in length. Its ORF was 1299bp and encoded a polypeptide containing 433 amino acid residues. The predicted protein was 8.98 in PI and 48.50 kDa in molecular weight. It contained a serine/threonine kinase domain in N-terminal region and a NAF domain in C-terminal region.3. The MdCIPK6 constitutively expressed in all kinds of tissues tested. Among these tissues, root generated the least MdCIPK6 transcripts. However, MdCIPK6 transcripts were dramatically induced in roots by high salinity and ABA. In addition, the expression patterns of MdCIPK6 were also tested in shoots in response to abiotic stress,plant hormone and exogenous Ca²⁺. It suggests that MdCIPK6 may be involved in the response of apple to abiotic stresses.3. Yeast two-hybrid (YTH) assay showed that MdCIPK6 physically interacted with AtCBL4 (AtSOS3). In parallel, complementation test showed that both MdCIPK6 and MdCIPK6T175D partially complemented the function of arabidopsis mutant sos2 in salt tolerance. Ectopic expressions of either MdCIPK6 or MdCIPK6T175D enhanced the tolerance to abiotic stresses in Arabidopsis. It suggests that MdCIPK6 functions in stress tolerance partially via SOS pathway as AtSOS2 does in Arabidopsis.4. To identify the function of MdCIPK6 in stress tolerance in apple, MdCIPK6, MdCIPK6T175D and antisense MdCIPK6 were transformed into apple, respectively. Transgenic apple shoots overexpressing MdCIPK6 or MdCIPK6T175D were much tolerant to abiotic stresses than the WT control and antisense transgenic lines. Therefore, MdCIPK6 functions as a positively regulator to confer tolerance to abiotic stresses in apple.5. To further characterize its functions in crops, MdCIPK6 gene was transformed into tomato. Transgenic tomato lines showed higher tolerance to abiotic in different stages including seed germination, seedling and adult plants. Our findings indicate that the biological function of MdCIPK6 is conserved among different plant species.