Construction Of Forward Subtractive Library And Analysis Of Differentially Expressed Genes During Secondary Cell Wall Thickening Stage Of Upland Cotton(Gossypium Hirsutum) Fiber

Cotton is an important economic crop, and is also the major source of natural fiber for textile industry. In addition, cotton fiber cells are also an ideal model material for studing the mechanism of cell elongation, cellulose biosynthesis, and secondary cell wall development in plants. In recent years, with the use of various high-throughput research strategies, the genetics mechanisms of cotton fiber development has been investigated at a deeper lever than before, and a succession of significant research achievements have been obtained. Nevertheless, the most of the researches were focused on the periods of initiation and elongation of cotton fiber development, and the stage of secondary cell wall thickening has been seldom characterized. As a result, it has greatly hampered the steps of using the genetic engineering methods to improve the cotton fiber strength, which is an important character of cotton fiber. So, it is very necessary to carry out more thorough studies on the mechanisms of secondary cell wall thickening of cotton fiber so that it could provide more available gene resources for cotton molecular breeding.In the present study, one of excellent recombinant inbred line (69307) with high fiber strength was selected as material, which developed from an F6:9population of upland cotton (Gossypium Hirsutum L.) cross of sGK9708x0-153. sGk9708is the commercial transgenic cultivar resistant to budworm, and0-153with high fiber strength. A forward subtractive cDNA library for the stage of secondary cell wall thickening of upland cotton fiber had been constructed via Suppression Subtractive Hybridization (SSH) technology. Meanwhile, the analysis of differentially expressed genes had been conducted. The results are as follows:1. A forward subtractive cDNA library for the stage of secondary cell wall thickening of upland cotton fiber had been constructed, in which the mRNAs isolated from15Days Post Anthesis (DPA) fibers were used as driver, and the mRNAs from20Days Post Anthesis (DPA) fibers as tester. The subtracted library contained1152positive clones, and the recombination ratio was97.04%, titer was1.89×l04cfu/ml. 2. The1152positive clones had been screened by means of Reverse Northern Dot-Blot. Finally,340clones expressed differentially had been selected, and represented115non-redundant genes, which involved35contigs and80singlets. Bioinformatic analysis revealed that these differentially expressed genes were extensively involved in metabolism of carbohydrate, lipid, amino acid and other substances, also involved in biological process, such as cellulose biosynthesis, cell wall formation and modification, oxidation and reduction, signal transduction,etc.3.18representative genes were selected from the115differentially expressed genes for the expression pattern analysis via Real-time PCR. The results showed that the expression level of the18selected genes were higher in20DPA fibers than that in15DPA fibers, which indicated that the results of the subtracted library were real and reliable. Meanwhile, it suggests this genes may be related with the secondary cell wall thickening development of cotton fiber. The18genes included aquaporin protein, arabinogalactan protein4, receptor-like kinase, fasciclin-like arabinogalactan protein1, fasciclin-like arabinogalactan protein16, chitinase-like protein1, Fblate-2, cellulose synthase1, cellulose synthase2, pectin methylesterase5, cytochrome c oxidase, Cobra-like4protein, NADH dehydrogenase subunit, plant glycogenin-likestarch initiation protein3, secretory carrier membrane protein, function unknown1-A10, function unknown3-E3, function unknown10-F6.4. The Simple Sequence Repeat Identification Tool(SSRIT) was used to search for the simple sequence repeat(SSR) sites from the115ESTs. As a result,16sequences which contained simple sequence repeat(SSR) sites were obtained, and16primer pairs were designed.5. After the analysis of16EST-SSR molecular markers polymorphism, one EST-SSR molecular marker with co-dominance was detected. This marker was located on the16chromosome of upland cotton, and linked closely with CGR5621S marker, which the genetic distance was only0.6centimorgan.