| The fiber of natural colored cotton contains natural pigments, however what kind of pigments, genes involved, and the formation mechanism of pigment are still unknown. In this study, different cotton varieties with different color shades of fiber were used to assess the gene differential expression related with pigment synthese by cDNA microarray, real-time fluorescence quantitative PCR and other techniques, and we also have deeply researched on CHI gene characters. This provides a theoretical basis for the research of colored cotton pigment type, and critical period and molecular mechanism of pigment synthesis and accumulation, and fully elucidating pigment-related genes.It has greater significance for breeding new colored cotton variety which has fiber color stability, good quality and high yield by using of genetic engineering. The main results are as follows:1) Brown fiber cotton line Z128 and two white fiber cotton varieties Kuche T94-4 and Liao 96-23-30 were used as materials to screen pigment biosynthesis related genes by using the cDNA microarray technology. The result indicated that 821 genes differentially expressed were screen between white and brown fiber cotton varieties, these genes were divided into a total of 64 biological pathways in KEGG (Kyoto Encyclopedia of Genes and Genomes) biological pathways analysis,7 genes matched in flavonoid biosynthesis pathway (F3H, LDOX, TT7, ATCHS/CHS/TT4, AT5G66220, BAN, A11/CFI/TT5) which was the same gene number as that in ribosome pathway, followed by starch and sucrose metabolism and phenylpropanoid biosynthesis with the 5 matching genes. In addition, the differential expression genes related with brown fiber also included the biological pathways of the photosynthesis-antenna proteins, Glycolysis/Gluconeogenesis and Carbon fixation, etc. It showed that a number of genes belonged to different pathways in cotton fiber presented differential expression status. GO (Gene Ontology) analysis result showed that a gene participate in not only a single biological process, but also involved in some others, which reflected the versatility of a gene and the interaction regulation and influence among the biological processes in the plant organism.2) Six genes (CHS, CHI, F3H, F3'H, LDOX, ANR) which belong to flavonoid biosynthesis pathway were selected to study the gene differential expression in 6 different color fiber cotton by real-time quantitative PCR technology. For completely studying the flavonoid biosynthesis pathway, the other 3 genes (F3'5'H, DFR, LAR) which missed in microarrays result were also selected for Q-PCR analysis. The results showed that the related genes with the brown fiber might be closely related with the flavonoid synthesis pathway, and finally the condensed tannins formed in the cotton boll, thus to further form oxidized tannins in the open boll and then performance brown fiber. Charaters. However, the pigment synthesis displayed poor correlation with biosynthesis of proanthocyanidins in green fiber cotton. Compared with white fiber, the genes related with pigment synthesis in brown fiber began to express at+5 DPA, and reached to peak at+10 DPA. Some of genes also possessed higher expression level at+15 DPA.3) Gene fragment PCR and Genome Walker technology were used to amplify the complete CHI DNA sequence,4781bp length seuqence was obtained including 1442bp length 5'flanking promoter region. By GENSCAN software analysis,684bp length ORF (Open Reading Frame) was obtained which encoded 227 amino acids.4) The amino acid sequence analyzed by DNAstar software demonstrated that the sequence of CHI could form several secondary structures with a-helix, P-fold, curl the corner etc. and the cluster analysis for protein sequences of the genes in other species was done by using Mega 4.1 software. The results showed that the CHI gene had a far relationship with the legume, but clustered together with that of dicotyledonous plants for the major categories.5) The predictation and analysis of the regulatory elements of CHI gene promoter was carried out by using PLANTCARE software. The results indicated that MYSI and MRE regulatory elements were found in CHI gene promoter, and they are the MYB binding site and light response MYB binding site. The MYB is a regulating gene to encode transcription factor. This shows that CHI gene may be regulated by MYB gene, thereby affecting the synthesis of flavonoids and other substances. |
