Isolation And Characterization Of WRKY Genes In Cotton (Gossypium Hirsutum)

Cotton (Gossypium hirsutum), a worldwide important economic crop, occupies an important place in the national economy in our country. As important natural fiber, cotton fiber is the main raw material for textile industry, widely used in textile, paper, biofuel and chemical industry, the value of cotton fiber quality determines it as a commodity. However, Cotton are frequently affected by biotic stress, such as pest species, and abiotic stress stresses, high salt, low temperature, lack of nutrition drought, leading to a sharp drop in cotton production. So sending the functional genes to plant genome by means of genetic engineering will expand the wide adaptability of plant and reduce the adverse effect on crops.WRKY (pronounced’worky’) transcription factors are a large family of regulatory proteins in plants. WRKY transcription factor owes its name to the most prominent feature of these proteins, the WRKY domain, a60amino acid region that contain a conserved amino acid sequence motif WRKYGQK at N-termini and a novel zinc-finger-like motif at C-termini. It is reported that WRKY transcription factors play a very important role in many physiological processes, including the growth and development, plant senescence, biotic and abiotic stress (high temperature and high salt, drought, etc.). In addition, it also regulated the growth and development of plant organ and metabolism, such as sugar. But there was no report that whether the WRKY involved in the regulation of cotton fiber. Therefore, in the future it is an important research direction to clarify the regulation and mechanism of WRKY transcription factors in cotton defense network and whether regulate the development of cotton fiber. At the same time, to clarify the mechanism of WRKY, it has important theoretical and practical significance for cultivating adversity resistant crop.To isolate cotton WRKY genes, we randomly sequenced over4000cDNA clones from cotton cDNA showed that the four WRKY genes (GhWRKY31-34) have an advantage in cotton root and leaf. To obtain more GhWRKYs, a javascript was developed to interrogate the cotton EST database in GenBank. Finally22GhWRKYs genes were got. The expression profiles of the22GhWRKY genes in cotton tissues are shown in cotton tissues, GhWRKY12/16/21transcripts were accumulated at relatively high levels in fibers. In this thesis, our studies mainly focused on the structure feature and classification of genes, qRT-PCR, the transcriptional activity, subcellular localization of proteins and so on to analyze the probable function simply. In addition, we also studied the response to abiotic stress. The results are as follows: 1. The isolation and identification of26GhWRKYs and the structural feature of their encoding proteins.We randomly sequenced over4000cDNA clones from cotton cDNA and obtained four full-length GhWRKYs, named as GhWRKY31, GhWRKY32, GhWRKY33, GhWRKY34. By means of electric clone we obtained other22GhWRKYs. The length of the open reading frames of GhWRKY genes vary from495bp to1302bp, encoding WRKY proteins of165to434amino acids, respectively. Their encoding amino acids contained WRKYGQK sequences and had a typical zinc-finger structure C2H2or C2HC at the C-termini.According to the number of WRKY domain and the feature of zinc-finger, the GhWRKY proteins can be assigned to one of the three previously defined groups (I-III), respectively. Five belong to the group I, five belong to the group III, others are classified into group II. Because of the differences of amino acids, the group II are further classified into five subgroups:Ⅱa, Ⅱb，Ⅱc, Ⅱd and He.2. The expression pattern of GhWRKYs in cotton.To investigate the expression patterns of these WRKY genes, firstly we extracted the total RNA from different cotton tissues, and then reversed them to cDNA as the template for qRT-PCR. The results showed that the most of genes expressed in vegetable tissues preferentially, especially in roots, leaves, cotyledons and hypocotyls. Three genes, GhWRKY12/16/21were accumulated in stage of3to10DPA fiber, which indicate that they may regulate the elongate stage of cotton fiber.It is reported that WRKY play a vital role in abiotic stresses. To examine whether these26GhWRKYs involved in abiotic stresses or not. We transferred7-old young cotton seedlings to1/2liquild MS containing150mM NaCl,250mM Man or100um ABA for5h, then we extracted their total RNA and reverse them to cDNA as the template for qRT-PCR. The results showed that in cotton roots expressions of GhWRKY14/20/24/33/34were up-regulated in roots treated with NaCl, ABA and mannitol, and GhWRKY9/10were induced in roots under ABA and mannitol treatments, while transcripts of GhWRKY11/13were greatly enhanced in roots by NaCl and mannitol. Additionally, expressions of six genes (GhWRKYl2/15/16/21/30/32) were induced in roots only by NaCl, and two genes(GhWRKY17/29) only by mannitol, while two genes (GhWRKY19/23) only by ABA. In cotton cotyledons about58%of GhWRKYs were up-regulated in cotyledons treated with all three stresses. In cotton hypocotyls only GhWRKY11was up-regulated under three treatments simultaneously. Transcripts of GhWRKY11/13/16/20/24/26/29/30/33/34have a greater accumulation in hypocotyls under NaCl and ABA treatments. GhWRKY12/31/32was only expressed highly in hypocotyls under NaCl treatment. These results indicated that GhWRKY genes may play important roles in regulating plant response to abiotic stresses during cotton development.3. The localization of GhWRKY31and GhWRKY34We construct fusion expression vector containing GhWRKYs and report gene of green fluorescent protein, which driven by CaMV35S strong promoter. By means of transferring DNA by agrobacterium and dipping into Arabidopsis thaliana flower, we obtained transgenic seedlings of GhWRKY31-GFP and GhWRKY34-GFP. The T2generation transgenic seedlings were grown up for5days, and then observe them on the confocal fluorescence microscope, we found the green proteins at the tip of root, which showed GhWRKY31and GhWRKY34all localized in nuclear.4. The self-trans-activation activity of GhWRKYsTo examine the trans-activation activity of GhWRKYs, The pGBKT7-GhWRKY12/16/31/32/33/34construct was transformed into the yeast strain AH109and Y187. The AH109positive transformants were cultured on SD/-Trp/-Ade and SD/-Trp/-His medium, and the Y187positive transformants detected by X-gal colorimetric assay on filter paper. The results suggested that GhWRKY31/33protein has the self-trans-activation activity, but GhWRKY12/16/32/34hasn’t.5. The salnity tolerance analysis of overexpression GhWRKYs transgenic Arabidopsis thalianaConstrccting overexpression GhWRKY12/31/34vector and dipping into flowers, we finally got three GhWRKYs transgenic Arabidopsis thaliana. Through analysis of several generations (T1-T3), the overexpression plants are almost the same as WT under normal circumstances. qRT-PCR showed GhWRKY12/31/34are induced by salt, therefore we inferred that they may involved in the response to salt. Under treatment of0,100,150and200mM NaCl, we counted and measured the germination rate, the rate of green leaves, root length, the content of chlorophyll and the expression of NaCl-related Marker genes of the transgenic plant and WT. From above results, it was suggested that GhWRKY12/34transgenic Arabidopsis thaliana enhanced the resistance to salt comparing with wild type. GhWRKY31transgenic seedlings enhanced salt tolerance in the stage of seed germination but sensitive to salt in the period of seedling growth.6. GhWRKYs transgenic cottonConstructing GhWRKY12/16OE and RNAi vector and transferring by agrobacterium LBA4404to cotton hypocotyls, we finally obtained transgenic cotton embryoid and then transgenic seedlings for7to8months. To date lots of embryogenic callus appeared and some have differentiated into transgenic cottons more than thirty. We have transferred and cultured them in soil, futher experiments are on the way.