Expression Profiles And Functional Genes Exploitation During Fiber Development Of Sea-Island Cotton

Sea-island cotton (Gossypium barbadense L.) is one of the most valuable cotton species due to its silkiness, luster, long staples, and high strength, transferring the excellent fiber traits from G. barbadense as the secondary gene pool to the vastly cultivated G. hirsutum via traditional and molecular-aided selection is an attractive aim of breeders. The developmental mechanism of G. barbadense fiber versus the one of G. hirsutum has not been surveyed, only few ESTs involved in the G. barbadense fiber development are in GenBank. In this study we constructed a normalized fiber cDNA library (from -2 to 25 DPA) of G. barbadense cv. Pima 3-79 (the genetic standard line) by saturation hybridization with genomic DNA and analyzed the genes specifically expressed in fiber of G barbadense. The main results are as follows.1. We constructed four individual libraries of Pima 3-79 fiber at the developmental stages of-2 to 4 DPA, 5 to 10 DPA, 11 to 16 DPA, and 17 to 25 DPA. The fiber cDNA library (from -2 to 25 DPA) was normalized by saturation hybridization with genomic DNA. Most inserts of the normalized library were 0.8 to 1.5 kb, and the average length was 1.14 kb.2. We screened Pima 3-79 fiber RNA from five developmental stages (0 DPA, 5 DPA, 10 DPA, 15 DPA and 20 DPA) using a cDNA array including 9126 plasmids randomly selected from the library, and sequenced 929 clones that showed different signal intensities between any two stages. After sequenceing, 887 high-quality ESTs were obtained.3. The 887 ESTs were assembled into 645 consensus sequences (582 singletons and 63 contigs), of which 455 were assigned to functional categories using gene ontology. Almost 50% of binned genes belonged to metabolism functional categories.4. Based on subarray analysis of the 887 high-quality expressed sequence tags with 0-, 5-, 10-, 15-, and 20- DPA RNA of Pima 3-79 fibers and a mixture of RNA of nonfiber tissues, only 118 of 781 ESTs were found to be expressed constitutively.We analyzed the expression profiles of the 663 genes specifically expressed in fiber. Totally 596 cDNAs (67 ESTs were unclassified) were found to show seven expression types.5. Our results showed that many genes may play an important role during fiber development, such as GbF3H, GbPDF, GbGLP, GbBHLH, GbarCaM, GbERS, GbCTLs and GbTLPs.6. We also compared the different development stages' expression profiles between G. barbadense and G. hirsutum L. At 10 DPA, 76 clones had higher expression levels in G. barbadense and only 19 had higher expression levels in G. hirsutum. At 20 DPA, 134 clones had higher expression levels in G. hirsutum and only 19 had higher expression level in G. barbadense. The expression profiles of these cultivated tetraploid species may help us to decipher the differences in quality and yield between the two.7. The alignments of the protein sequences and nucleotide acid sequences of the genes in two species, sea-island cotton and upland cotton showed obviously different. Except several nucleotide acids different there were deletions and inserts of DNA sequences, especially the 3'-UTR, such as GbPDF and GhPDF1; expansins; GbGLP and GhGLP.8. We modified the vector pCAMBIA2301 by PCR and deleted Ncoâ… and Bglâ…¡sites upstreaming the NPTâ…¡, which was named 'pCAMBIA2301m' and then was used for over expression.9. We analyzed the expression of GbEX2 and GbEX3, and found the GbEX2 only expressed in the fiber of the sea-island cotton but GbEX3 expressed in the fiber of both island and upland cotton. We have got the regenerated plantlets transformed by RNAi and over-expression vectors of the two genes.10. We also have cloned the promoters of GbEX2 and GbEX3, and analyzed the core promoters and cis-acting regulatory DNA elements. By alignments of the nucleotide acid seguences of the two promoters we foud that the similarity was high but there were some nucleotide acids differences and deletions (inserts). We also had inserted the promoters in the vector, pBI121 and pBINm-gfp5-ER, and transformed them into YZ1.11. We assessed the gene expression of 7 frequently used housekeeping genes, including 18S rRNA, Histone3, UBQ7, Actin, Cyclophilin, Gbpolyubiquitin-1and GbpoIyubiquitin-2, in a diverse set of 22 cotton samples. For fiber developmental series the expression of all housekeeping genes had the same down tendency after 17 DPA. But the expression of the AGP gene (arabinogalactan protein) that has the high expression level at the later fiber development stage regulated up from 15 DPA to 27 DPA. 12. For the nonfiber tissues series, three best control genes Histone3, UBQ7 and GbpoIyubiquitin-1 were needed to use together for better normalization. And the relative absolute quantification should be an efficient and convenient method for the fiber developmental series.