Cloning And Functional Characterization Of RAV1Gene In Cotton (Gossypium Hirsutum)

Abiotic stresses such as high salt and drought is a major limiting factor affecting plant growth and agricultural development, Thus, by means of various biological mechanisms and regulation of plant growth under stress conditions, thus improving the efficiency of agriculture, it is particularly urgent and important. Cotton (Gossypium hirsutum) is an important crop in the world economy, which produces fiber and cotton seed oil and is closely related to people’s daily lives, but because of Chinese domestic soil structure and cotton less able to resistant stresses, so their growth and development and crop yields are often affected due to biotic stress. Currently, about cotton in the stress response and ABA signaling mechanisms reported even less.RAV-like protein family belongs to a small class of AP2-like proteins, which plays a key regulatory role in responses of stresses and plant hormones aspects. AP2protein family is divided into two subfamilies according to the number of domain, including AP2subfamily (containing two AP2domains), and EREBP subfamily (containing only one AP2domain). The RAV like proteins are a class of plant-specific subfamily proteins which contain an AP2domain and a B3domain, belonging the EREBP family.Our laboratory isolated a cDNA sequence from the cotton gene cDNA library, which encodes an RAV class protein, named GhRAV1. Preliminary studies indicate that, GhRAV1protein structure is same to the other RAV proteins, containing the conserved AP2and B3domains, possessing the nuclear localization feature. GhRAVl is higher expressed in nutrition tissues and fibers at the mRNA level, and also GhRAV1is induced by drought, high salt, and ABA. On the other hand, the expression level of the GhRAV1gene is also decreased by the BL, which may indicate that the function of GhRAVl is associated with the BR signal. This paper explains the function of GhRAVl from the following respect, including the gene expression patterns, protein subcellular localization, transcriptional activation feature, stress response and signal correlation with other aspects of BR. Achieved results are as follows:1. Isolation and phylogenetic analysis of GhRAVlA gene coding protein RAV, named GhRAVl, was isolated and identified from the cotton cDNA library. The cDNA open reading frame (ORF) of GhRAV1is1074bp, encoding a358amino acids protein, with a molecular weight of37.8kD. GhRAV1protein structure comprises an AP2domain and a B3domain belonging RAV subfamily members. Phylogenetic analysis showed that, GhRAV1protein is higher homo logy with tobacco protein (NtRAV), Arabidopsis protein (AtRAV2), Capsicum annuum protein (CaRAV1), which appears close genetic relationship. 2. Expression analysis of GhRAV1gene in cotton tissues under treatments of ABA, NaCl, PEG and BLThe analysis of GhRAV1expression level in different cotton tissues displays that, GhRAV1is expressed higher in cotton roots, cotyledons, leaf,9DPA fiber, the expression of GhRAV1in hypocotyl, anthers, petals, ovules, ODPA fiber,3DPA fiber,6DPA fiber,12DPA fiber,15DPA fiber,18DPA fiber lower.First, we respectively used ABA, NaCl and PEG to treat the wild-type cotton (Coker312), which grows5days in the MS liquid medium. Then, used real-time quantitative PCR (RT-PCR) to analysis the expression level of GhRAV1. The results showed that, the expression of GhRAV1was induced under the treatment of ABA, NaCl and PEG. Meanwhile, we used epiBL to treat the10DPA fiber of wild-type cotton (Coker312), the results of quantitative real-time PCR analysis indicated that the expression of GhRAV1was decreased under the treatment of epiBL.3. Self-transcription activity and subcellular localization analysis of GhRAV1proteinWe linked the cotton gene GhRAVl with the pGBKT7vector, and then transformed it into the yeast strain AH109and Y187to study whether GhRAV1protein has the transcribed activation activity. The results show that, GhRAV1proteins do not have self-activation of transcription activity.Using eGFP as a reporter gene, we built pBI121-GhRAVl:eGFP fusion expression vector, then injected it into the tobacco leaf epidermal cells, observed the green fluorescence under the confocal microscopy after the48hours. The results showed that GhRAV1protein was located in the nucleus.4. Stress resistance analysis of GhRAV1overexpressing ArabidopsisTo study the function of GhRAV1protein in plants, we built pMD-GhRAV1OE overexpression vector, and then transformed into Arabidopsis, analyze the resistance to stress of the positive plants. Plants start the Stress response pathway mainly dependent on the two ways, including ABA-dependent and non-dependent ABA two pathways. Therefore, during the seedling stage, we choose ABA, NaCl, PEG to simulated stress environment to analyze the GhRAV1gene resistance to stress, as well as the plants growing up stage.The results showed that, GhRAV1overexpressing plants increased sensitivity to salt and drought, which is consistent with the response sensitivity of ABA. This may indicate that GhRAV1function depend on the way of ABA-dependent response pathway.For now, many genes involved in stress response pathway have been reported in Arabidposis, including RAB18, RD29B, ABI1, ERD15, KIN1. We analyze these genes expression level by real-time PCR analysis in GhRAVl transgenic Arabidopsis. The results indicated that RAB18, RD29B, ABI1, ERD15, KIN1upregulated and Corl5a, RD29, RCI downregulated in transgenic Arabidopsis, which suggested GhRAVl may be involved in the stress response pathway.5. GhRAV1overexpression transgenic Arabidopsis responds to BR signalingTo study the function of GhRAV1protein in plants, we treated GhRAVl overexpressing Arabidopsis with exogenous BL. It was found that under BL processing conditions, GhRAV1overexpressing Arabidopsis root length elongation faster than the wild type, and the resin chips were carried out with an initial analysis of the root section. Speculately, GhRAV1may be involved in the BR signaling pathway.6. GhAP2RNAi transgenic cotton plantsPlant the GhAP2RNAi transgenic cotton plants subcultured by XiaHui senior sister apprentice, and then extract and identify the GDNA and RNA of the planted TO generation transgenic cotton. Further, we choose the1DPA ovules from the positive plants after flowering. The results showed that, the GhAP2RNAi transgenic cotton in vitro embryo influenced by ACC, but the effect of tissue culture progress for the TO generation plants may also be the reason. Therefore, further experiments and planting are continuing.Considering that the tissue culture progress may hard receive seeds, other GhAP2could be affected by ACC, guessing that the gene GhAP2may participate the ethylene response, and further affect the seeds mature. Therefore, a well-built fusion expression vector pMD-GhAP2RNAi, built by XiaHui senior sister apprentice, was used to transform cotton more than one time, which was mediated by Agrobacterium. Up to now, the just transformed plants have been transferred to CB4, has get136Lines of callus, and then is continuing subculture.