Gene Expression Profile Analyses Of Upland Cotton (Gossypuim Hirsutum) Fiber During Secondary Cell Wall Deposition

Cotton fiber is the major natural textile fiber and also an excellent single cell model system for the study of plant cell elongation and the mechanism of cellulose biosynthesis during secondary cell wall modification. With the development of modern molecular biology technology, such as gene chip and high-throughput sequencing, the molecular mechanism of the cotton fiber development has been investigated and a series of important progress have been obtained. However, these studies have highlighted the stage-specific transcription of genes involved in fiber cell initiation and elongation. The switch from PCW to SCW and SCW deposition stage within the fiber has been less well characterized. Secondary cell wall synthesis (approximately from 15 to 40 DPA) is the key period of fiber strength formation. To study the molecular mechanism of secondary wall thickening will pave the way for cloning and identifying the genes related to the secondary cell wall thickening of cotton fiber, and also establish the foundation for mining important genes of cotton fiber quality, and modifying cotton fiber quality with these important genes.In this study, two recombinant inbred lines (RILs) that differ in fiber strength,69307 (excellent) and 69362(poor), were developed from an F2 population of Upland cotton (Gossypium Hirsutum L.) cross of 0-153 X sGK9708. sGk9708 is the commercial transgenic variety with Bt+CpTI genes resistant to budworm, and 0-153 with high fiber quality. Gene expression profiles of developing cotton fibers from two recombinant inbred lines (RILs) were compared by cotton cDNA microarray which constructed by Peking University and CapitalBio Corp and contained over 29,185 EST probers derived from cotton fiber developmental diverse time-points. Fiber gene expression was compared at four time points of spanning elongation (10 DPA), fiber transition (15 DPA) and secondary cell wall (20 and 23 DPA) biosynthesis. In order to explore the candidate genes associated with secondary cell wall thickening, we complied differentially expressed genes for various comparisons of the array data into GO categories and used Fisher s exact test for enrichment of GO terms. The results could be made as:1, Within each line, a large number of genes were found to be differentially expressed during fiber development. For high fiber strength line of 69307,3802 genes (13.3% of total genes on array) were differentially expressed (Ratio> 2 or Ratio< 0.5), and for the lower fiber strength 69362,7146 genes (24.49% of total genes on array) were differentially expressed. When we compared two lines at each time point, a total of 5228 genes were differentially expressed, occupied 17.91% of total genes on array.2, For independent corroboration of microarray results, nine genes were selected for Real-time PCR analysis of fiber gene expression. This included fiber elongation stage-specific gene a-expansin, and secondary cell wall stage-specific genes, cellulose synthase,1,3-B-glucanase and sucrose synthase. And the other significant differentially expression genes were validated by qPCR. These gene contained Racl3 GTPas. RNA-dependent RNA polymerase (SDE1) and aquaporin protein. This indicated that the comparative microarray results were reliable.3, The different expression patterns of elongation (GhExp) and secondary cell wall synthsis{GhCesA, beta-1,3-glucanase) stage-specific development marker gene, suggest that the development processes for the two lines maybe not synchronous, high fiber strength line of 69307 might have longer elongation stage and delay into the secondary cell wall synthesis, the contrary result for 69362.4, Functional classification and enrichment analysis of differentially expressed genes between the two lines revealed that the genes associated with small GTPase protein and RNA-directed RNA polymerase activity have significant enrichment and temporal differences in gene expression patterns. This showed that the gene of different expression patterns may play an important role in the fiber development. During the elongation, the genes associated with auxin responsive, lipid metabolism and cell wall loosening expressed at high level. Following the start of secondary cell wall synthesis, these genes showed reduced expression levels in fiber cells, and the genes related to carbon cycle, such as cellulose synthesis, showed an increased level of expression in this stage. These results suggest a specialization process of cotton fiber development toward cellulose synthesis after entering secondary cell wall synthesis. It is believed the key regulation gene which control carbon flow, may play an important role in the development transition from cotton fiber elongation into secondary cell wall biosynthesis. In addition to this, the genes associated with phenylpropanoid and flavonoid biosynthetic process have significant enrichment in 69362 at 15 DPA. These genes and phenylpropanoid compounds whether would affect the fiber quality and the relationship between secondary metabolism and cotton fiber development could be a target for further research.