| Watermelon (Citrullus lanatus) is one of the most popular fruits among people, and our country has the biggest growth area and yield in the world. The origination of watermelon is southern Africa. As we all know watermelon is very sensitive to cold stress. During the early spring facilities culitvation, watermelon usually suffers from cold stress and result in weak seedings which will seriously influence its quality and production. Studies on the functions of cold related genes among watermeoln will provide a theoretical basis for its molecular breeding. This research focus on the function of ClAOX and its polymorphic loci and CICASPL gene under cold stress.Only one ClAOX gene was found in watermlon genome through degenerate primers cloning and genomic database searching. Gene structure and phylogenetic analysis revealed that ClAOX has 4 extons and 3 introns, belongs to AOX2 subfamily. Subcellular localization, cold-inducible expression and tissue-specific expression reveal that ClAOX localized in the mitochondria, could be induced by cold stress, and have different expression in watermelon roots, stems, leaves, flowers and fruits. GUS staining with ClAOX-promoter-gus transgenic lines showed tissure and developmental specific expression of ClAOX. Those results confirm the conclusion of ClAOX has both AOX1 and AOX2 subfamily characters.Abundant polymorphism of ClAOX was found among different watermelon sub-species and species through gene cloning and alignment. There is only one single nucleotide Polymorphism (K96, N96) among C.lanatus Var. vulgaris and C.lanatus sp. lanatus. Further experiments with transgenic plants showed that ClAOXK has a longer primary roots and larger fresh weight compared to ClAOXN under 10? treatment. chlorophyll fluorescence data also revealed better cold resistance ClAOX* compared to ClAOXN. DAB, NBT staining of cold treated plants showed that ClAOXN accumulates more ROS ClAOXk. Pre-treatments with cold stress could improve the oxidative resistance of all plants. Plants growth under 10? showed the most reasistant under oxidative stress while plants growth under normal condition were the most sensitive. ClAOXK showed better oxidative resistance compared to ClAOXN under both 10? and 15? stress. Those results indicate that the difference of cold resistance among ClAOXK and ClAOXN have some closely relationship with ROS signaling pathway.CICASPL was obtained from a reward subtractive cDNA library which was constructed using SHAM-treated plants as driver and control plants as tester. Its orthologous gene AtCASPL4Cl in Arabidopsis thaliana belongs to CASP (UPF0497) gene family. Watermelon ClCASP gene family contains 30 members, and each of them have special express patterns among roots, stems, leaves, flowers and fruits. Subcellular localization analysis showed that CICASPL is localized in the plasma membrane. Expression analysis using a ?-glucuronidase (gus) reporter reveals that AtCASPL4C1 is widly expressed in a variety of organs and cold inducible. In AtCASPL4C1 konck-out or CICASPL over-expression plants the formation of Casparin strip staining did not show any significant difference. Both CICASPL and AtCASPL4C1 could be induced by cold stress. Phenotype of AtCASPL4C1 konck-out and CICASPL over-expression plants under normal and cold conditions indicated negatively alters growth and cold tolerance of those two genes. Studies use Arabidopsis mutants showed that the lost function of AtAOX2 could cause significantly suppressed expression of AtCASPL4C1. Those results provide us basic information for the watermelon cold breeding, the connection between alternative oxidise pathway and Casparin strip. |
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