Cloning And Functional Verification Of Brassinosteroids Biosynthese Gene GaCPD From Gossypium Arboreum L.

Brassinosteroids (BR) as the sixth largest plant hormone, plays a key role in plant growthand development. BR effects plant type, flowering, seed size, fertility of plants,photomorphogenesis, stress response and so on. Constitutive photomorphogenesis and dwarf（CPD） is a key rate-limiting enzyme in the synthesis of brassinosteroids, as C3oxidasecatalyzes reaction.The current functions of the gene expression patterns in Arabidopsis, rice（Oryza sativa）, tomato（Lycopersicon esculentum Mill.） had been related researched, andno CPD-related research in cotton had been reported. This article was a study of geneexpression patterns in cotton CPD and verify their functionality, for the first time from theAsian cotton (Gossypium arboreum L.) were cloned into the two homologous ArabidopsisCPD cytochrome P450mono oxidase gene GaCPD1, GaCPD2（GenBank code：KJ183066,KJ183108）,construction of plant expression vector，by transgenic technology toimport these vectors and the corresponding wild-type Arabidopsis mutants obtainedtransgenic lines were analyzed and compared by morphological science, molecular resultswere as follows:1.Two homologous Arabidopsis CPD cytochrome P450mono oxidase genes(GaCPD1,GaCPD2,GenBank accession number: KJ183066,KJ183108) were cloned from the Cotton(Gossypium arboreum L.) by RT-PCR, the bioinformatics analysis showed that:1) The sequence length CDS of GaCPD1, GaCPD2were1419bp and1413bp, encoding471amino acids,470amino acids, the amino acid sequence identity between84%;2) GaCPD1and GaCPD2theory of relative molecular mass were117.88kD,53.98kD,theoretical isoelectric point were5.01,9.40, mainly composed of β-fold, α-helix, extendedchain and random coil.3) Signal peptide prediction analysis showed GaCPD1a signal peptide, GaCPD2two signalpeptides, both the cell membrane proteins.4) GaCPD1and GaCPD2encoded proteins and Arabidopsis CPD, respectively,79%,84%amino acid identity, they had common conserved domains. 2.The use of Real-Time qRT-PCR analysis expression levels of different tissues andorgans of GaCPD2and GaCPD1in Asian cotton, the results showed:1）GaCPD1, GaCPD2were expressed in root, stem and flower, the highest expression of thefiber of10days after flowering was about10times0DPA.2）Expression of GaCPD2in cotton was higher than GaCPD1’s.3. The vector of35S::GaCPD1,35S::GaCPD2were introduced into Arabidopsis cpd91,were obtained3(R-G1-L1/2/3),3(R-G2-L1/2/3) transgenic lines. On representativetransgenic lines of morphological, genetic studies showed:1）35S::GaCPD transgenic lines had completely restored or partially restored mutantphenotype. Fully restored strain on behalf of R-G1-L1, R-G2-L1, compared with Arabidopsismutants,they presented longer roots, rosette leaves fully expanded, lighter color, increasingindividual plants, flowering early, improved fertility, increased leaf length, petiole elongation,leaf width increased, the leaf area increased, the pods grow, increasing the number of pods;partially restored lines on behalf of R-G1-L2, it got oval rosette leaves, plant height, leaflength, leaf width, leaf area, root length, pod length were lower than the wild type, highercpd91mutant.2）With the same period, In the light conditions,35S:: GaCPD transgenic lines and cpd91mutant comparison, hypocotyl elongation, root elongation; under dark conditions,35S::GaCPD transgenic lines and cpd91mutant comparison,the cotyledons closed, the top hook.4. The vector of35S::GaCPD1,35S::GaCPD2and GaCPD1::GUS, GaCPD2::GUS wereintroduced into wild-type Arabidopsis, were obtained transgenic lines of35S::GaCPD1(6lines),35S::GaCPD2(7lines), GaCPD1::GUS (6lines), GaCPD2::GUS (8lines). Onrepresentative transgenic lines of morphological, genetic studies showed:1) OX-G2-L2compared with wild-type Arabidopsis, leaf width, leaf area, plant heightincreased; petiole length OX-G2-L1, the leaf length, leaf width and leaf area were associatedwith the wild-type Arabidopsis essentially flat; the observed indicators OX-G2-L1than thewild-type Arabidopsis, the presence of inhibition.2) GaCPD::GUS staining results showed high genetic roots and cotyledons at the seedlingstage in the expression of GaCPD.