| Cotton(Gossypium spp.)is the most important textile and oil crop worldwide.About 30%of the seed coat epidermis cells will differentiate into fiber cells,whereas the embryo will synthesizes oils and proteins.With about 21%oil and 23%protein by weight in mature cotton seed,however,this makes cotton the fifth largest oil crop in the world and the second most important potential source of plant proteins.Meanwhile,as part of the cotton seed,the cotton fiber development is partially controlled by the cotton seed development.An in-depth understanding of metabolic events in cotton seeds is therefore of vital importance for improving the yield,quality and ultimately the value of seed products and opens the possibility for providing genetic and molecular basis for improving cotton fiber yield and quality.A putative full-length cDNA which encodes a protein comprised of 619 amino acids was cloned from a G.irstutum fiber cDNA library,and we named it as GhKOR1(GenBank accession no.AY 574906).This gene encodes a glycosyl hydrolase which contains a predicted 23 amino acids transmembrane domain.Protein alignment with homolog in other plants showed that the deduced GhKOR1 protein contained the conserved polarized signal peptide targeted to the plasma membrane,glycosylation sites,and residues essential for catalytic activity.The GhKORl gene shares a similar gene structure with AtKOR1,both of which contain the same number of exons and introns with similar nucleotide number in the exons.The above analyses strongly indicate that GhKOR1 encodes a KOR protein in cotton.The expression of GhKOR1 in various tissues was investigated by RT-PCR and qRT-PCR.The analyses revealed that GhKORl gene was expressed in root,stem,leaves,developmental fibers,ovules,seed coat and filial tissues.These results indicated that GhKOR1 gene is constitutively expressed and could exert housekeeping functions in cotton plant.The functions of GhKOR1 in cotton seed and fiber development were analyzed using the anti-genetic technology.The over-expression construct and RNA interference construct of GhKOR1 gene were developed using pCAMBIA-1301 and pCAMBIA-1302 vectors,respectively.Then,these two vectors were transformed into the upland cotton WO genome by the Agrobacterium-mediated transformation to do the functional analysis.All transgenic plants were detected by PCR analysis using hygromycin gene and promoter-gene specific primers.Primarily,8 RNAi transgenic lines and 3 over-expression lines were obtained.The expression level of GhKOR1 in developmental fibers of transgenic lines and WO was investigated by qRT-PCR.Six out of eight primary RNAi lines and one out of three primary over-expression lines showed down-regulated GhKOR1 gene expression.Three transgenic lines were selected for the further functional analysis and we renamed them as RNAi line1,RNAi line5 and Co-S linel.First,we investigated the functions of GhKOR1 gene in cotton seed development.RT-PCR and qRT-PCR analyses showed that the transcript level of GhKOR1 gene was significantly down-regulated in the seed coat and filial tissue of transgenic lines compared with that in wild type.In contrast,the mRNA level of GhKOR2,the only remaining gene encoding membrane-anchored endo-1,4-beta-glucanase besides GhKOR1 in cotton sequence database,was only marginally affected in transgenic tissues.The seed growth was inhibited in the GhKOR1 gene down-regulated transgenic lines.Both the embryo and endosperm development were delayed and disrupted in the transgenic lines.Cellulose content analysis showed that the cellulose in the 20DPA seed coat of the transgenic lines was significantly reduced.Further sectioning and staining results exhibited that the endosperm cellularization in 10DPA seed of transgenic lines was disrupted,the endosperm cell number was reduced and the cell wall of the endosperm cell was disorganized.In addition,the embryo development in 10DPA seed of transgenic lines was delayed and the embryo became smaller compared to that in wild type.Analysis on 15DPA seed coat sections revealed that the callose content in the transfer cells was significantly reduced,while the cellulose content did not exhibit changes.The expression level of GhKOR1 gene in 15DPA seed coat of transgenic lines was significantly down-regulated.Meanwhile,the transcript level of one endo-beta-1,3-glucanase gene was also down-regulated.Sequence alignment showed that GhGlu3 cDNA shared high homology with the GhKORl fragment used in the RNAi construct in some regions.Co-suppression between GhGlu3 and GhKOR1 fragment could occur and the expression level of GhGlu3 gene was down-regulated in the transgenic lines.Afterwards,the seed and seedling vigor was tested.It has been shown that the mature seed weight was significantly reduced,the germinating time of the transgenic seed was significantly prolonged and the seedling vigor of the transgenic plants was significantly reduced.The transcript level of GhKORl gene in root and above ground tissue of transgenic seedling was also down-regulated.These analyses indicated that the reduced seed and seedling vigor could be result from the compromised seed development in the transgenic lines.Second,we examined the possible roles of GhKOR1 gene in cotton fiber development.RT-PCR and qRT-PCR analyses revealed that the mRNA level of GhKOR1 was low in early stages but was dramatically increased at～10 DPA onwards and maintained at a high level up to at least 25 DPA.Further expression analysis showed that GhKORl gene was significantly down-regulated in transgenic 10DPA and 20DPA cotton fibers.The fiber length and cellulose content in the transgenic lines was reduced.In addition,the fiber weight per cotton boll and fiber weight per seed was also decreased.These analyses indicated that GhKOR1 gene played important roles for cotton fiber elongation and secondary cell wall cellulose biosynthesis.In summary,our research demonstrated GhKOR1 gene is required for cotton seed and cotton fiber development.This is the first time to provide evidence for the essential role of KOR in the development of reproductive tissues. |
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