Analyses Of Expression And Function Of GhGalT1Gene In Cotton(Gossypium Hirsutum)

Cotton is one of the most important economic crops in the world, and cotton fiber is the main raw material for the textile industry. It represents an ideal experiment model for studying the mechanism of plant cell elongation and cell wall biogenesis. It will be of cardinal importance to isolate and analyze the key genes involved in cotton fiber development for improving cotton fiber yields and quality.The hydroxyproline-rich glycoproteins (HRGPs), as a superfamily of plant cell wall proteins, can be divided into three subclasses, namely highly glycosylated arabinogalactan-proteins (AGPs), moderately glycosylated extensins (EXTs), and lightly glycosylated proline-rich proteins (PRPs). They all have their Pro residues hydroxylated to Hyp by prolyl4-hydroxylases (P4Hs) and then are extensively O-glycosylated by glycosyltransferases in the Golgi or endoplasmic reticulum (ER).AGPs are the most highly glycosylated proteins in nature. AGPs consist of approximately90%carbohydrate with just10%protein in weight, which means glycans play an important role in the interaction of AGP and other proteins and defined the functions of AGP. However, precise roles of AGP glycosylation remain elusive and little is known about specific glycosyltransferases. Consequently, it is important to isolate and characterize some key glycosyltransferases genes involved in AGP biosynthesis.In this study, a GhGalTl gene encoding a putative β-1,3-galactosyltransferase (GalT), which mignt involve in the glycosylation of AGP protein backbone, was identified from cotton EST libraries and the full-length cDNA was obtained successfully. Gene expression pattern, subcellular localization and functions of GhGalTl were studied. The main results are as follows:1. A gene encoding a putative β-1,3-galactosyltransferase (GalT) was isolated from the cotton cDNA library, and designated as GhGalTl. This cDNA fragment included a1053base pairs (bp) open reading frame (ORF), a short5'-untranslated region (UTR) and a48bp3'-UTR. It encodes a protein of350amino acids, which belongs to GT31family. GhGalT1protein contained a putative N-terminal transmembrane domain, a galactosyltransferase (GalT) domain. Sequence comparison and phylogenetic analysis revealed that GhGalTl, PtGalT1, ATGalT1and ATGalT2are closely related, and these proteins share a72%to86%identity.2. Semi-quantitative RT-PCR experimental results showed GhGalTl expressed strongly in hypocotyls, less strongly in root, its mRNA can be detected in ovule and fiber. The results also showed that GhGalTl gene was highly expressed in2DPA (days post anthesis) fiber and15DPA fiber, which means that GhGalTl expression was fiber developmental stage regulated.3. To further investigate the tissue-preferential and developmental regulated expression of GhGalT1, a528bp fragment of GhGalT15'-flanking sequence was isolated by genome walking. Analysis of the isolated promoter sequence by PlantCARE program revealed it not only contained putative CAAT box, TATA box sequence, but also contained MYB, HSE and TC-rich repeats cis-acting element which involved in drought, defense and stress responsiveness. A GhGalT1promoter::GUS fusion expression vector was constructed and was transformed into Arabidopsis. Histochemical assay of transgenic Arabidopsis revealed that GUS activities were mainly accumulated in root tips of primary and lateral roots from5-to15-day-old seedlings, and less strongly in cotyledons and rosette leaves. The results of stress and hormone treatments revealed that the GhGalTl promoter is salt-/osmotic-/6-BA-/MeJA-/BL-inducible.4. Bioinformatics analysis predicted that GhGalTl protein belongs to type II membrane protein and it is localized in the Golgi. To study its actual subcellular localization, green fluorescent protein (GFP)-tagged GhGalTl protein was coexpressed with yellow fluorescent protein (YFP)-tagged GONSTI (Golgi marker) in leaf epidermal cells of tobacco(Nicotiana tabacum). Examination of the fluorescent signals revealed that GhGalT1exhibited a punctate distribution, a pattern matched with GONSTI, which is known to be localized in the Golgi and revealed that GhGalT1was localized in the Golgi.5. To evaluate the function of GhGalTl, we expressed GhGalTl in Arabidopsis, the GhGalT1ORF was inserted into the expression vector (pBI121) under the control of CaMV35S promoter and introduced into Arabidopsis. RT-PCR analysis showed that GhGalTl transcripts were highly abundant in the four homozygous transgenic lines, while undetectable in wild type. Line1(L1) and Line5(L5) have the highest expression of GhGalTl, so we choose L1and L5for further phenotypic analyses. We also purchased two T-DNA mutants of two Arabidopsis genes which share high sequence similarity with GhGalT1, Genotyping analysis showed that the two mutants both have two opposite T-DNA insertions and RT-PCR results revealed that the transcript level of respective gene was decreased in the mutant. During seed germination stage, the wild type and the transgenic lines overexpressing GhGalT1showd no obvious changes, but during seedlings growth stage, under normal growth conditions (1/2MS with10%sucrose) the transgenic lines grow better than the wild type. Especially on the medium without sucrose, the transgenic seedlings have much longer primary roots, higher chlorophyll content and higher photosynthetic activity. Under dark conditions, the transgenic seedlings have much longer hypocotyls. From the above results, it can be postulated that GhGalTl may be involved in carbohydrate metabolism.6. Overexpression of GhGalTl in Arabidopsis leads to increased tolerance to a certain concentration of D-galactose and D-arabinose. The growth of wild tye Arabidopsis seedlings was suppressed while the transgenic seedlings could be insensitive to exogenous D-galactose and D-arabinose.7. In order to investigate if the cell wall was altered in the transgenic lines, Over20genes involved in cell wall biosynthesis were selected. RT-PCR results showed that expression of three genes involved in pectin biosynthesis (GAUT8,GAUT9and xgdl) were up regulated dramatically in the transgenic lines relative to the wild type. GC analysis of monosaccharide composition of cell wall fractions from transgenic lines and the wild type also showed that the pectin was changed.8. GhGalTl overexpression and RNAi vectors were constructed and transfered into cotton by Agrobacterium-mediated method. Up to date,15lines of transgenic cotton plants overexpressing GhGalTl were obtained (add up to100) and8lines of GhGalT1-RNAi silenced transgenic plants were gained (amount to50). Preliminary PCR results showed most of transgenic plants were positive, further phenotypic analyses are undergoing.9. GhGalT1protein was expressed in Pichia pastoris, but SDS-PAGE results showed that the expressed protein has higher molecular weight which implied that GhGalT1may form a complex. Then we used yeast two-hybrid system to identify the putative proteins that can interact with GhGalT1, but no proteins were screened from the yeast two-hybrid system.