The Cloning And Functional Characterizations Of GbEXPA2 And GbEXPATR During Cotton Fiber Elongation

Cotton fiber is the most important natural textile fiber. Cotton fiber, a highly elongated, thickened and unbranched trichome, differentiates from the single outer epidermal cells on the seed. Cotton fiber cell wall properties determine qualities, hence, it is important to study genes related to cell wall from Gb. Not only is the research area of cell wall enriched, but also the new breeding materials are provided. In previous studies, Gb EXPA2 and Gb EXPATR, two α-expansin genes, were identified from a normalized c DNA library of G. barbadense 3-79 fiber. Gb EXPA2 expression was detected in Gh and Gb varieties, however, unlike Gb EXPA2, Gb EXPATR expression was only detected in Gb varieties. Gb EXPA2 encoded a typical α-expansin protein including N-terminal signal peptide, domain 1 and domain 2, while Gb EXPATR encoded a shorter protein containing only signal peptide and domain 1. Base on the researches, we further analyzed the functions of these two genes.1. Gb EXPATR which is specifically expressed in G. barbadense fiber encoded by the AT genomeComparisons of distinctive SNP loci with the diploid and Gh genes showed that the SNP loci of Gb EXPA2 was closest to Gr-contig12 and Gh-contig29-2, which encoded by the DT genome of tetraploid Gb. The SNP loci of Gb EXPATR was closest to Ga-contig4 and Gh-contig29-1, which encoded by the AT genome. As the Gh, Ga and Gr homologs do not have the same deletion, this must have occurred at or soon after the polyploidization event that species formed Gb.2. pro Gb EXPA2 and pro Gb EXPATR are preferentially expressed in trichomes, and pro Gb EXPA2 was regulated by GA and ABAThe 839 bp and 1405 bp sequences upstream of the Gb EXPA2 and Gb EXPATR translation start codon “ATG” was isolated by a genome-walking strategy from G. barbadense 3-79, respectively. Sequence analysis revealed that the DNA fragment of pro Gb EXPA2 was similar to the sequence of pro Gb EXPATR. The pro Gb EXPA2::GUS and pro Gb EXPATR::GUS constructs were generated and transformed, respectively. The promoter pro Gb EXPA2 and pro Gb EXPATR were able to express GUS to high levels in elongating fibres and Arabidopsis rosette leaf trichomes, but not in the root, stem, or leaf. GUS activity of pro Gb EXPA2 always could be detected in the trichomes of very young cotton leaves. A deletion analysis of pro Gb EXPA2 revealed that the 461 bp fragment was sufficient to drive GUS expression in cotton fibres and Arabidopsis rosette leaf trichomes. It was indicated that the regions of the Gb EXPA2 promoter from-461 to-258 bp were crucial for trichome-preferential expression. To determine whether pro Gb EXPA2 is regulated by GA and ABA in vitro, the vegetative tissue of six-day-old seedlings of transgenic Arabidopsis lines with different promoter lengths(P-839, P-705, P-588 and P-461) were treated with GA3 and ABA. The GUS activity in Arabidopsis trichomes could be strongly up-regulated by GA3 and, in contrast, down-regulated by ABA.3. Down-regulation of EXPA causes shorter fiber lengths and thicker cell wall, however, up-regulation of Gb EXPATR results in longer fibers coupled with thinner cell wallTo characterize the functions of EXPA, five EXPA-RNAi transgenic lines were identified. The EXPA-RNAi lines had shorter fiber lengths, higher micronaire values, and lower strength compared with wild type. Further, RNAi lines caused a thicker cell wall and higher crystalline cellulose content. To study the functions of Gb EXPA2 and Gb EXPATR in cotton fiber, two over-expression vectors were constructed and transformed into G. hirsutum YZ1, and obtained three different transgenic lines, respectively. Gb EXPA2 over-expression lines had no effect on mature fiber length, but produced fibers with a slightly thicker wall with increased crystalline cellulose content. Gb EXPATR over-expression lines resulted in longer, finer and stronger fibers coupled with significantly thinner cell walls. Homozygous lines of the EXPA-3’ UTR RNAi plants were crossed with the highest expressing 35::Gb EXPATR homozygous line. The short fiber phenotype of EXPA-3’ UTR RNAi were completely restored in the F1 lines, indicating that Gb EXPATR can substitute for the full-length EXPA in enhancing fiber elongation.4. Through delaying the expression of secondary cell wall-related genes and then changing cell wall composition, Gb EXPATR could promote fiber elongationDigital gene expression profiling of 10 DPA fiber from EXPA-3’ UTR RNAi lines did not detect any meaningful gene expression differences between the RNAi and wild type lines at this developmental stage, other than the silenced α-expansins. The expression levels of CTLs, CESAs and TLP genes at 15 and 20 DPA were not consistently altered in RNAi lines compared with controls. In contrast, a significant reduction in the expression levels of all three types of secondary cell wall genes was often observed at 15 DPA in Gb EXPATR and Gb EXPA2 over-expression lines. At 20 DPA, the transcripts of these secondary cell wall genes also showed slightly decreased expression in the Gb EXPATR over-expression lines, but not in the Gb EXPA2 over-expression lines. To investigate whether the EXPA RNAi and over-expression lines contained altered wall polymer composition, monosaccharide linkage analysis was performed on 20 DPA fibers. Cell wall polysaccharide composition estimation revealed that at 20 DPA EXPA-RNAi and Gb EXPA2 over-expression lines increased cellulose levels; and decreased levels of other polysaccharides compared with wild type. In contrast, the amount of cellulose in Gb EXPATR over-expression lines was reduced in parallel with low cellulose crystallinity. These outcomes suggested that the two homoeologous Gb EXPA2 and Gb EXPATR genes can play similar in primary cell wall and distinct in secondary cell wall roles in fiber development to affect fiber quality.