Molecular Cloning, Expressional And Functional Analysis Of A Prp19Ubiquitin Ligase Gene (GhMAC3) In Gossypium Hirsutum L.

The fuzzless-lintless cotton mutant of Xuzhou142(fl), a spontaneous mutant derived from a cotton cultivar of Xuzhou142(WT), produces few fibers on its ovules. Therefore, it is an ideal material for fiber initiation development research. In our previous proteomics study, a protein (best match to EST of gi|164307915) was expressed in-3and0days post anthesis (DPA) ovules of WT, but was not expressed in fl or Ligon lintless-1mutant ovules, thus, it is reasonable to believe that this protein is a significant protein for normal fiber development. However, this gene has not been molecularly cloned, and the function remains unknown.In this study, first, the full-length of the encoding sequence of this protein was cloned by using RACE-PCR method firstly, as the protein sequence shows83%similarity with that of Arabidopsis ubiquitin ligase gene of AtMAC3, so it was denominated as GhMAC3temporarily. Then, the expression pattern and subcellular localization of this gene were analyzed. The GhMAC3interacting proteins were further screened and identified by means of Yeast two hybridization, furthermore, the over-expressed GhMAC3transgenic Arabidopsis was obtained by using floral-dip Agrobacterium-mediated transformation, in combination with phenotype identification of the AtMAC3knock-out Arabidopsis lines, the function of GhMAC3was preliminarily analyzed. The main findings are as follows:1. According to the EST sequences (gi|164307915), we cloned the full length of the cDNA of the gene from cotton ovules by3’and5’RACE respectively. The length of the ORF is1575bp, which encodes524amino acids, containing a U-box, a Prp19, and a WD40function domain respectively.2. The expression levels of GhMAC3in different tissues were examined using real-time fluorescence quantitative PCR, the results inidicated that no significant difference was observed between different tissues of WT or between WT and fl, thus, the expression of this gene may be modified by post-transcriptionanl or post-translational mechanisms. 3. The transient expression analysis in onion epidermal cell by gene bambardment showed that GhMAC3protein was localized in the nucleus.4. A recombinant plasmid of pGBKT7-GhMAC3was constructed, and six proteins were screened and identified to be interacted with GhMAC3protein with Yeast two hybridization system, these proteins include ribosomal protein small subunit4e, exosome complex exonuclease RRP46homolog, cytochrome P450like TBP, auxin-responsive protein IAA35-like, transmembrane BAX inhibitor motif-containing protein1-like, and cytochrome b-c1complex subunit8.5. The GhMAC3gene was cloned into plant expression vector of pBI121, then transformed into Arabidopsis by flower-dip technique. By kanamycin resistance screening and PCR,27positive transgenic GhMAC3Arabidopsis plants were obtained. We found that the WT roots are more vigorous than those of the knock-out lines of1and2of Arabidopsis seedlings.