Establishment And Application Of Transient Expression System Of Cotyledons In Cotton

Cotton, a special seed fiber which belongs to Malvaceae Gossypium, is one of the most impotant crops and native to subtropical countries. In recent years, research genes’ function of cotton has developed to deeper and wider period with cotton genome sequencing been finished. In order to further study, genetic modified method usually is used to analyse gene function. But cultural transgenic cotton takes long time for transferring and subculture. Meanwhile it needs more labour and material with getting lower transfer efficiency. Transient transformation system of plant can supply a convinent and rapid way to research promoter activity, protein-protein interaction, subcellular localization, and so on.Cotton cotyledon was chosen as plant material to establish cotton cotyledon transient expression system by using GUS expression vector and Agrobacterium tumefacien mediated injection. We worked out orthogonal experimental design to optimize species of Agrobacterium tumefacien, bacteria density, incubation time, leaves growth age and volume of transformation liquid. Based on this system, we checked the activity of induecd promoter D-7and subcellular localization of two PPR proteins which were coloned from cotton. The main results are as follows:1. Cotton cotyledon was chosed as plant material. GUS expression levels were detected by injecting plant expression vector with gus gene into cotyledon via Agrobacteriu mediated transformation. Orthogonal experimental results show that6d cotton cotyledon is injected with50μl OD600Agrobacterium tumefacien transformation liquid, and co-incubating about2-3d, exogenous gene displays the highest expression efficiency.2. Cotton cotyledon is better than euphylla to establish transient expression system, and transformation mediated by Agrobacterium tumefacien LBA4404is prefer to GV3101. Using this method only needs8-12d to getting results from seedling germination to observation. Meanwhile the new cotton cotyledon transient expression is easy and convenient to manipulate, which provids great value to research genes function come from cotton.3. We used cotton cotyledon transient expression system to analyse the activity of promoter D-7. The results show that D-7promoter is a weak stress-inducible promoter. D7can drive egfp to express green fluorescent when the cotton seedling treated with drought, salt, ABA, low temperature and high temperature to6to9h. This result is similiar with that Arabidopsis steady transformed with D-7-eGFP and detected D-7promoter activity. All these results show that cotton cotyledon transient expression system can be used to analyse promoter activity. And stronger promoters are easily to observation and detection.4. Bioinformatics analyse shows Ghppr1may locate in nucleus. A1875bp sequence, named Ghppr1-p was amplificated from Ghppr1by PCR reaction. A plant expression vector, drived by a constitutive promoter CaMV35S, was constructed using pEASYTM-T5Zero、PZP21and Ghppr7spE as middle vector. The new expression vector is named Ghppr1-eGFP.5. We observed subcellular localization of Ghppr1and GhpprH2by cotton cotyledon transient expression system and scanned by Confocal laser scanning microscope. Experimental result indicated that chloroplast revealed obviously green fluorescent after3d co-incubation with cotyledon injected LBA4404carrying vector GhpprH2-eGFP. We could predict GhpprH2may locate in chloroplast. The cotton cotyledon transient expression system can be affirmed to study subcellular localization.