| Cotton is the world's leading fiber crop and is a mainstay of economy in China, which is the largest cotton producer and consumer around the world. Fiber is the major product of cotton production and the main raw material in textile industry. Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44), one of the key enzymes in the first step of the phenylpropanoid pathway, catalyzes the NADPH-dependent reduction of cinnamoyl-CoA esters to their corresponding cinnamaldehydes. A gene encoding Gossypium hirsutum Cinnamoyl-CoA reductase 4 (GhCCR4) was isolated from cotton fiber. Here we examine the role of GhCCR4 in cotton development.1) We choose transient expression assay system for test of GhCCR4 function in cotton fibers by using cotton ovule culture and biolistic transformation techniques. The transient expression vector pGUS-CCR4 is driven by CaMV35S promoter with GUS reporter gene and GhCCR4 gene. The conditions for bombardment were optimized : the highest transformation efficiency of GUS was obtained when 0 day of anthesis (0DPA) ovules were applied, bombardment include helium pressures of 1350 psi, distance of 9 cm and bomb times of twice. Histochemical staining showed that high levels of GhCCR4 gene expression was detected during the fiber rapid elongation stage and the secondary wall thickening stage in cultured ovules. Measurement of fiber length in different developmentally stage showed that the fiber length of the transgenic plants at the stage (8 DPA) was not different as compared to that of the wild-type plants . The fiber length in the transgenic plants reduced 19% compared with wild-type plants at the stage of 27 DPA. The cell wall of the transgenic fibre at the stage of 27 DPA was thicked as compared to that of the wild-type fibre. Transmission electron microscopy demonstrated that the wall thickness of transgenic fibre was increased to 17% of that of the wild type. These findings suggest that GhCCR4 could play a critical role in the processes of elongation and secondary cell wall formation during fibre development.2) An improved transformation system of meristem via Agrobacterium-mediated transformation was established after studies on many influential factors. The transformation rate of anti-antibiotic reached 35% and the transformation cycle decreased to 2-3 months. Incorporated anti-antibiotic and molecular detection, Selective Strategy was upgraded after improving selection of transformation plants. Transgenic plants were obtained and some of them had been carried out their target-gene measurements in leaves. All the results showed that the state of the tip conditions and the Selective Strategy acted vital roles in transformation rate and cycle.3) GhCCR4 were mediated into the cotton genome in identical direction via Agrobacterium-mediated transformation. After pre-culture,co-culture,selective culture,sub-culture and root induction culture, neogenesis young shoots with kanamycin resistance were obtained finally. The PCR primers were designed to depend on the gene order of CaMV35S and NPTâ…¡. Results of PCR indicated that the target gene had integrated into the genome successfully. The detection of transcription level indicated that they have visible different between transgenetic plant and control. The efficiency of sense enhancement and RNA interference is high in them, and exogenous gene restrain the expression of CCR.4) The hot borate method for efficient isolation of cotton total RNA were improved in different cotton fibers. Cotton total RNA was isolated from different tissues of immature plants and expression of these genes was analyzed by RT-PCR. The expression of GhCCR4 gene was detected of every tissue in this article. GhCCR4 gene is expressed high in root and stipe. This experiments showed a differential expression pattern of mRNA levels in different cotton fiber. Expression of gene in other tissues and each tissue from different growth period will be further studied.5) Transgenetic palnts of both sense enhancement and RNA interference were compared and analyzed through appearance viewing, lignin and cellulose content mensuration. The result show that it have some difference between them in morphous. The content of lignin cut down. The possible reason is the exogenous gene repressed the CCR expression.Based on this data, we suggest that GhCCR4 may play an important role in the morphogenesis and secondary wall thickening of cotton by positively/negatively regulating the structure of cell wall in growing course. Our study presents important experimental evidence for the function of lignin and provides gene candidates for genetic improvement of cotton fiber quality.
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