Functional Analysis Of GhPHOT2-1 On The Regulation Of Chloroplast Movement In Plants

Cotton is an abundant source of fiber and oil, and it is one of the most important economic crops worldwide. However, despite its great economic value, it is still urgent to improve the architecture of cotton via molecular breeding. Gossypium hirsutum L.（AADD） is a tetraploid plantwidely cultivated in America and Asia, Gossypium arboretumL.（AA） and Gossypium raimondii（DD） are diploid and are grown in India or in South America. Whole genome sequencing of Gossypium arboreumL.and Gossypium raimondiiis completed earlier than Gossypium hirsutum L., so cotton gene research is mainly rely on gene data of G.raimondii and G.arboreum L. In recent years, genome sequencing of tetraploid Upland cotton was completed, and this enables the study of Gossypium hirsutum L. and molecular breeding of tetraploid Upland cotton possible.Light mediates various developmental aspects of plant. Phototrophins, phytochromes, cryptochromes and UV8 are respective photoreceptors for the blue, red, and purple light, and plants integrate the light signals via these receptors. In the model plant Arabidopsis, there are phototrophins 1（PHOT1） and phototrophins 2（PHOT2）, they mediate signaling of the bule light on the controling of movement toward light in plants. In Arabidopsis thaliana, At PHOT2 controls phototropism under high bule light, the chloroplast movement under high bule light, stomatal opening, and leave expansion. There are three domain in the amino acid sequence of PHOT2 s, namely LOV1（light oxygen voltage sensitive domain 1）, LOV2（light oxygen voltage sensitive domain 2）, and Ser/Thr protein kinase domain. LOV area induced by blue light, FMN and cystine 39 to form a covalent compound to change the conformational in the protein. Thereby activating the protein kinase domain receptors in plants caused autophosphorylation, causing a series of plant physiological and ecological responses. In this reseach, we focused on the chloroplast avoidance movement function of PHOT2.In this study, we performed a BLAST in Cotton Research Institute, Chinese Academy of Agricultural Sciences Upland data（http://cgp.genomics.org.cn/page/species/index.jsp）, using amino acid sequence of Arabidopsis AtPHOT2 as probe. We found that there are nine genes of the GhPHOT2 subfamily in cotton, namely CotAD₀9562（A genome of 9 chromosomes）, CotAD₁2100（D genome of 13 chromosome）, CotAD₁3007（A genome of 8 chromosome）, CotAD₂9566（D genome of 3 chromosome）, CotAD₃3323（location unknown）, CotAD₃3403（A genome of 13 chromosome）, CotAD₅8277（location unknown）, CotAD₆0197（A genome of 5 chromosome）, and CotAD₇0827（A genome of 8 chromosome）. After bioinformatics analysis, we selected the Upland CotAD₅8277 as the research object, this gene has two PAS domains and a protein kinase domain, and we named it GhPHOT2-1.We found that expression of the PHOT2 between Arabidopsis and Gossypium hirsutum L. under blue light intensity is different. Leaves aperture light transmissive experiment of cotton and Arabidopsis, and GhPHOT2-1 expression and protein transfered showed us that GhPHOT2-1 reduced tendency were the same, may be this is resulted from evolution between two species.We constructed related vectors and used Agrobacterium mediated and pollen tube injection and other experimental technology methods to obtain different categories of transgenic Arabidopsis and Upland cotton. In Arabidopsis recover-materials, we obtained recover-materials with Upland cotton gene GhPHOT2-1 into Arabidopsis mutants phot2 and phot1 phot2 double mutant, with these recover-materials, we confirmed that the cotton gene GhPHOT2-1 in Arabidopsisis substantially consistent with the function of Arabidopsis gene AtPHOT2. In Virus Induced Gene Silencing experiment, the control group and the empty vectors group show that chloroplast avoidance movement, and the gene silencing groupdoes not appear the same reaction that proved the chloroplast movement function of GhPHOT2-1 in cotton.