The Preliminarily Functional Studies On AIR12in Chorispora Bungeana

Chorispora bungeana is a typical alpine periglacial plants collected from mountains with high altitude under low temperatures, strong ultraviolet radiation and thin air. It can grow, flower and reproduce offspring in the harsh environment, especially at low temperatures, suggesting that it has good antifreeze mechanism. It belongs to the cruciferous plants which also include the model plant Arabidopsis thaliana for transgenic research and a number of economically important crops (such as rape, etc.), therefore, Chorispora bungeana is an ideal material for antifreeze gene cloning and research on special antifreeze mechanism of alpine periglacial plants. CbAIR12, an cold-induced gene, is screened from suppression subtractive hybridization library of Chorispora bungeana, it belongs to Aux/IAAs class of auxin response genes, Arabidopsis AIR12was first expressed in the course of auxin-induced lateral root formation, there is not too much research on AIR12currently. The project studied the function of CbAIR12by using the methods such as TAIL-PCR, Real-time PCR, analysis of transformed tobacco leaves and floral dip method. We got the following results:1. The sequence of CbAIR12gene was cloned and its sequence features were analyzed. The full-length sequence of CbAIR12was cloned for the first time, the length of CDS sequence is744bp, encoding247amino acids. It was found that the homology of AIR12in Chorispora bungeana and Arabidopsis was as high as72%, according to software prediction, CbAIR12protein contains a trans-membrane domain;2. Expression pattern of CbAIR12gene was revealed.-4℃and4℃low-temperature stress treatments showed that the gene responded to low temperatures. AIR12gene can also be up-regulated by IAA and ABA, indicating it may have some roles in these pathways.3. The tissue distribution and subcellular localization of CbAIR12were determined. Real-time PCR results showed CbAIR12gene distributed among leaf, petiole and root, particularly the petiole with the highest level. Over-expression fusion vector containing AIR12and GFP was transformed into tobacco leaves, and by observing confocal microscope, we confirmed that the fusion protein was localized on the cell membrane and nuclear membrane; the same result also observed in stably transgenic Arabidopsis.These indicated that the protein plays a role in membrane signal transduction pathways.4. Transgenic CbAIR12over-expression Arabidopsis was established. On the one hand, homozygous transgenic Arabidopsis lines were obtained by Agrobacterium-mediated floral dip method; on the other hand, transgenic tobacco callus was successfully induced, it remains to be differentiated into seedling for further studies.So based on the above studies, the character of CbAIR12gene sequence, expression patterns and regulatory mechanism were revealed preliminarily, it established the theoretical basis for further research on gene function. The stress-induced gene pool in Chorispora bungeana was enriched, and it contributes to the further study of plant stress resistance mechanisms, in the meantime, it offers great resources for new anti-stress plants by genetic engineering method.