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| Cloning, Expression Analysis and Functional Verification of Brassinosteroids Biosynthese Gene (GaCPD2) in Cotton (Gossypium arboreum L.) |
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Abstract Constitutive photomorphogenesis and dwarf gene (CPD) is a key rate-limiting enzyme in brassinosteroids(BRs) synthesis, gene expression patterns and functions of which in Arabidopsis, rice (Oryza sativa) and tomato (Lycopersicon esculentum) have been researched, and not in cotton(Gossypium arboreum L.). To clone and verify CPD gene function in cotton, a cytochrome P450 monooxygenase gene, GaCPD2 (GenBank accession: KJ183066) was cloned from cotton by RT-PCR. Expression pattern and functional bioinformatics analysis showed that GaCPD2 was 1 413 bp, which encoded a deduced protein including 470 amino acid residues with theoretical relative molecular weight of 53.98 kD and pI 9.40, mainly composed of alpha helix, beta turn, extended strand and randon coil. Multiple sequence alignment revealed that the most similarity was 84.0% between the amino acid sequence of GaCPD2 and that of other reported species, which had conserved domain of cytochrome P450. Real-time PCR revealed that GaCPD2 expressed in root, stem, leaf, flower and fiber, and had the highest expression level in 10 days post-anthesis (DPA)fiber, which was about 10 times that of 0DPA. Cloned promoter sequence of GaCPD2 contained many cis-elements related to light response by bioinformatic analysis, which implied that GaCPD2 was light regulated. Overexpression vector 35S :: GaCPD2 for GaCPD2 transformation restored the growth of cpd91, which is a brassinosteroids deficient dwarf mutant of Arabidopsis (Columbia background). The transgenic lines showed flattened leaves and restored fertility. These results revealed that GaCPD2 played an important role in brassinosteroids biosynthese. These results lay the base for the further reseach of BR regulation in cotton development, and will help to reveal the BR regulation of plant molecular mechanism of plant type and yield traits.
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Received: 10 December 2013
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