Molecular Characterization And Function Of The Cotton Annexin Genes

Cotton, the most important economic crops in the world, plays an important role in the national economy of China. Cotton fiber is the main products of the cotton production, its yield and quality have a direct impact on the output and benefit of cotton production. It has great theoretical and practical significance to isolate and study the functions of the genes related to the yield and quality of cotton fibers. In recent years, although a lot of cotton genes correlated with fiber development have been identified and studied in detail, little is known about the expression and roles of annexins in fiber development so far. The annexins are a multigene family of Ca2+-binding and phospholipid-binding proteins which spread widely in the tissues and cells of animals and plants. Plant annexins not only play important roles in plant development but also in the response to environmental stresses.To investigate the roles of annexins in fiber development, four cDNAs encoding novel annexin proteins were isolated and characterized from cotton (Gossypium hirsutum) fiber cDNA library. The expression patterns, promoter activities as well as the Ca2+-binding activities, subcellular localization and functions of these genes were further studied. The main results are as following:1. Isolation, characterization of AnnGh cDNAs and structure anlysis of their encoding proteinsBy screening cotton fiber cDNA library, four cDNAs encoding novel annexin proteins were isolated and designated as AnnGh3, AnnGh4, AnnGh5and AnnGh6. Comparing with other known cotton annexins, the four genes share high sequence homology with them not only at nucleotide acid level but also at amino acid level (54%-95%identity). Protein structures predicted showed that they all have a typical annexins domain which consists of four repeats (Ⅰ to Ⅳ). In the first and fourth repeat, there is a type-Ⅱ Ca2+-binding site (G-X-G-T-{38}-E) respectively. In addition, a potential phospholipid binding site and a potential heme-binding domain were also found in the first repeat. In other three repeats, there are several other conserved sites important for annexin function, such as an F-actin-binding site (IRI) in the third repeat and a GTP-binding site (DXXG) in the fourth repeat. These results suggested that the four new cDNAs encoded typical annexins, possessing the classical characteristics of the cotton annexin family proteins.2. AnnGh proteins are localized in cytoplasm of cotton cellsIn order to investigate the subcellular localizations of AnnGh proteins, AnnGh3:eGFP and AnnGh6:eGFP fusion expression vectors were constructed and introduced into cotton. The Stable transformed cotton callus cells were observed under the confocal laser scanning microscopy. And the results indicate that the two AnnGh proteins both localized in the cytoplasm of cotton cells. 3. Analysis on the Ca2+-binding activity of AnnGh proteinThe pMal-AnnGh3prokaryotic expression vectors were constructed and MBP-AnnGh3fusion proteins were obtained successfully through E.coli system. Fluorescence quenching experimental result shows that AnnGh3protein can combine with calcium ion in vitro indicating that it has Ca2+-binding activity.4. The expression patterns of AnnGhs in cotton fiberQuantitative real-time RT-PCR results show that the expressions of AnnGh genes in cotton are tissue specific/preferential. Among the4AnnGhs, three AnnGhs (AnnGh3, AnnGh4、AnnGh5) are preferentially expressed in cotton fiber while AnnGh6is predominantly expressed in roots. The expressions of AnnGh3/4/5are fiber-developmentally regulated. The transcripts of AnnGh3/4/5were mainly accumulated in3to12DPA fibers, while in fibers of15DPA to20DPA, their expressions were declined to very low levels quickly. Except fiber development, their expressions in fibers are also regulated by some phytohormones and the [Ca2+] in vitro.In fiber cells development early stage (0to2DPA), the results obtained by using in situ hybridization show that strong hybridization signals of AnnGh3/5transcripts were detected in the ovule epidermis and fiber cells. The data above indicate the two annexin genes may be involved in fiber initiation.5. Expression activity assays of AnnGh3promoterThe793bp promoter fragment of the AnnGh3gene was isolated by Genome walking PCR. The chimeric AnnGh3p::GUS vectors were constructed and transformed into Arabidopsis using the floral-dip method. The results of GUS staining analysis show that the promoter of AnnGh3has strong expression activity in the cotyledons, hypocotyls and roots of3to10day-old seedlings. With Arabidopsis plant growing, its activity decreased gradually. In mature Arabidopsis plants, the promoter has strong expression activity in the sepals, filaments, peduncle base and weak expression activity in anther. In addition, the activity of the promoter was significantly increased in transgenic seedlings after IAA, dark and H2O2treatment. However, the activity of this promoter in cotton tissues especially in cotton fibers still needs more studies in detail.6. Overexpression of AnnGh3in Arabidopsis promoted trichome’s differentiation and developmentTo evaluate the role of AnnGh3protein in fiber development, AnnGh3gene was overexpressed in Arabidopsis trichomes. A significant increase of leaf trichomes’ density and length was observed in transgenic plant. Comparing with wild type Arabidopsis, the density of leaf trichomes of transgenic plants increased8.3-32.8%and the trichomes’average length also increased18-32μm, rose 5.8-10.4%. These data indicated that AnnGh3may contribute a lot to the initiation and elongation of trichomes. Acorrding to the differentiation and developmet of trichomes and cotton fiber are regulated by similar molecular mechanisms, above data demonstrated that AnnGh3play important roles in fiber initiation and elongation indirectly.7. Modification of resin semithin section staining method in plant tissuesTo establish a more suitable staining method for the resin semi-thin section of plant tissues, young roots, young stems, leaves and ovules of cotton (Gossypium hirsutum) and stems of maize (Zea mays) were manufactured by the methods of resin semi-thin section. And the staining methods (including safranin-fast green staining, hematoxylin-eosin staining, FeCl3-haematoxylin staining) were improved and applied in the preparation of the plant semi-thin section. Our experimental results indicated that clear-staining and integral-structural semi-thin section of plant tissues were obtained by using the modified methods of resin semithin section. This new resin semi-thin section staining method provides another ideal microsection technology for related researches..