Cloning And Functional Characterization Of GhPHOT1 And GhPHOT2 Genes

Strong light and high temperature is suitable for cotton growth, and the cotton distribute widely all over the world. As an important factor for cotton normal growth, light has great influence on cotton yield and quality. Current research about the effect of light on cotton growth mainly focus on time, light intensity, and different light quality such as red, blue affect the cotton production, however, the research of cotton light receptors has not been reported. So far, existing studies have shown that the phototropism receptor mainly include phototropin, cryptochrome and phytochrome. As common light receptors, phototropin not only mediate phototropism, stomatal opening, chloroplast movements, but also participate in many other reactions induced by blue-light in higher plants. There are two kinds of blue light receptors in moss, Arabidopsis and maize PHOT1 and PHOT2. The C-terminal of the PHOT1 and PHOT2 contain Ser/Thr protein kinase domain, and the N-terminal contains two LOV domain which can combine with flavin mononucleotide. Under the weak light, PHOT1 protein can adjust chloroplast concentrated movement and improve photosynthetic efficiency, PHOT2 can regulate the chloroplast avoidance movement and leaf flatting movement under high blue light to reduce the harm to the plant. Recent research have shown that the PHOT2 gene involves in plant drought resistance, and it can enhance the drought resistance of the plant by regulating the stomatal opening and a series of other reactions.We compared the sequence of AtPHOT1 and AtPHOT2 proteins with protein database of Gossypium raimondii in Phytozome website, and we found two genes Gorai.003 g139300 and Gorai.008 g012600 with homologous of 73.9%, 72.4%. Sequences alignment analysis showed that these two genes are homologous genes of PHOT1 and PHOT2 in Therobroma cacao. Protein structure analysis indicated that these two genes not only have Ser/Thr protein kinase domain in the C-terminal, but also contained two PAS domains in the N-terminal. These are typical structure of phototripin. The result of The Kyoto Encyclopedia of Genes And Genomes database showed that the two genes was PHOT1 and PHOT2 of cotton, and named them GhPHOT1 and GhPHOT2.We verified the function of GhPHOT1 and GhPHOT2 genes not only in Arabidopsis, but also in cotton. Firstly, we constructed the 35S::GhPHOT2 recombinant vectors, transform them into phot1phot2 and phot2 mutant respectively by Agrobacterium-mediated transformation, then screen and identify the positive transgenic plant. Experiment with GhPHOT2 positive transgenic plant showed that the phenotype of the double mutant plant was covered, and GhPHOT2 regulated chloroplastic avoidance movement and stomatal aperture through the experiment of small hole transmission, chloroplast movement and stomatal aperture. The subcellular localization analysis indicated that GhPHOT2 was located in the cell membrane or cell wall which is as same as the location of AtPHOT2. Secondly, we silenced the endogenous gene of phototropin by VIGS technology, and identified the silent plant by RT-PCR. Compared with the control plants, we found that the stomatal aperture of silent plants that silencing the expression of GhPHOT1 or Gh PHOT2 was smaller under 100μmol.m-2.s-1 blue light. These results were the same as in Arabidopsis thaliana.