Cloning And Functional Analysis Of Three SAUR Genes In Cotton (Gossypium Hirsutum L.)

Cotton fibers are single-celled seed trichomes originating from the epidermis of the outmost integument of ovule. Cotton fiber development is a complex biological process regulated by different factors at different levels including gene transcription, translation, metabolism and plant hormones pathways. Phyhormones have been proved to play an important role in fiber elongation, for example, IAA can modulate fiber cell elongation by regulating expression of the auxin-responsive genes. Fiber elongation is one of the key periods determining fiber yield and quality. It is of theoretical and applied importance to identify and utilize the key genes involved in fiber elongation for elucidating the molecular mechanisms of fiber development and improving cotton fiber yield and quality.Ligon lintless 1 (Li-1) is a monogenic dominant mutant characterized by abnormal lint fiber development, bears very short lint fibers indistinguishable from the fuzz fibers. Li-1 fibers initiate and elongate in a similar manner to its isogenic wild-type line (TM-1), from 0 to 2 DPA until 3 DPA when Li-1 fibers appear to be slightly malformed, the fiber secondary wall deposition is extensively thickened as well, therefore, Li-1 was employed as a model to study fiber elongation and secondary cell wall synthesis. In a previous study in our lab, the differentially expressed genes in the 0 DPA ovules and 3 DPA,6 DPA fibers between Li-1 and TM-1 were detected by using microarray analysis, with those in TM-1, Auxin was found to be crucial for fiber elongation, among the differentially expressed genes, three SAUR (Small Auxin Up-regulated RNAs) genes showed to be down-regulated in Li-1 were identified. These SAUR genes in cotton were temporarily denominated as GhSAUR68, GhSAUR-likel and GhSAUR-like2 respectively, as their sequences are highly homologous to AtSAUR68 and two SAUR-like proteins (AT1G75590 and AT2G46690) in Arabidopsis. In this study, the three genes were molecularly cloned, and the expression pattern and subcellular localization of each gene was analyzed. In order to investigate the function of these three SAUR genes, transgenic overexpression in Arabidopsis and cotton were conducted by using Agrobacterium inoculation. The major results are as follows:1. According to the Unigene sequences of the three genes, we cloned the full-length of cDNA. The full length of GhSAUR68 cDNA is 553bp, the open reading frame (ORF) of GhSAUR68 is 444bp in length, and predicted to encode a protein of 148 amino acid residues with calculated molecular mass of 36.42 kDa. The full length of GhSAUR-likel cDNA is 638 bp, the ORF is 474bp in length, and predicted to encode a protein containing 158 amino acid residues with calculated molecular mass of 39.34 kDa. The full length of GhSAUR-like2 cDNA is 747 bp, the ORF is 369 bp in length, and predicted to encode a protein containing 123 amino acid residues with calculated molecular mass of 29.95 kDa.2. The spatio-temporal expression patterns of GhSAUR68, GhSAUR-like1 and GhSAUR-like2 in TM-1 and Li-1 were analyzed by qRT-PCR, and the results showed that the three genes were preferentially expressed in ovules and fibers compared to roots, stems and leaves, furthermore, the expression levels of them in 6 DPA,10 DPA and 20 DPA fibers are significantly decreased in Li-1 compared to those in TM-1, suggesting that the three GhSAURs may be involved in cotton fiber elongation and secondary wall thickening development.3. The subcellular localization of these three GhSAURs were detected by using onion epidermal cell bombardment transgenic transient expression experiment system, the results indicate that both GhSAUR68 and GhSAUR-like1 protein were located in the cell membrane and nucleus, GhSAUR-like2 protein was located in the nucleus. Suggesting they all may function at the downstream of auxin signaling pathway, GhSAUR68 and GhSAUR-like1 located in cell membrane may be involved in upstream auxin signaling pathway.4. The three GhSAURs were inserted into the expression vector pBI121 driven by the cauliflower mosaic virus (CaMV)-35S promoter, and subsequently transformed into Arabidopsis using floral-dip method.62.5% of the kanamycin resistant transgenic GhSAUR68 Arabidopsis plants have no or etiolated heart leaves.18 positive transgenic GhSAUR68 Arabidopsis plants showing normal plant phenotypes were obtained by kanamycin resistance screening and PCR validation, the result of GUS staining suggested that GhSAUR68 was specifically expressed in root and old leaves. The GhSAUR68 overexpressed plants displayed slow growth, delayed development, leaf presenility and more trichomes on leaf surface in comparison with the wild type.5 positive transgenic GhSAUR-like2 Arabidopsis plants were obtained by kanamycin resistance screening and PCR amplification. GhSAUR-like2 overexpressing Arabidopsis plants exhibited shortened taproots and increased lateral roots, and decreased number of leaf trichomes. These results indicated that GhSAUR68 influenced normal plant growth and development of Arabidopsis, but it was probably able to promote leaf trichome development; GhSAUR-like2 may have little effects on plant growth, however might negatively regulate trichome development in Arabidopsis.5. The plant phenotypes and responses to gibberellin (GA3) and auxin (IAA) of AtSAUR68 and AT2G46690 knocked-out Arabidopsis mutants were inspected respectively. Both mutants showed shortened roots compared to the wild type in normal hormone-free culture medium.5 μM IAA retarded root and hypocotyl development in both the wild type and the mutants, while 0.5 μM GA3 have little effects on root and hypocotyl development in both the wild type and the mutants, IAA and GA3 have no synergistic effects.6. SAUR-like2 driven by 35S, FBP7, a seed epidermis specific promoter, SCFP, a fiber specific promoter were transformed into cotton by tissue culture and Agrobacterium inoculation, the growth and development of callus at different stages were documented. The transgenic regeneration plant were obtained, which pave the way for further studies on the molecular mechanisms of cotton ovule and fiber development.