Cloning And Function Analysis Of Cold-responsive Gene (COR15) And The Promoters From Capsella Bursa-pastoris

Temperature is among the most significant factors that restrict the distribution of plants, the growth and development and the quantity and quality of crop products. With the development of transgenic technology, a large amount of cold-regulated genes have been used in plant breeding for greater cold tolerance. But a putative phenomenon of growth retardation in transgenic plants due to the use of35S promoter was observed, which was unfavorable in obtaining cold tolerant plants with good quality. In order to avoid this adverse impact of35S promoter, cold induced promoters are more favorable. But there is dearth of research on this topic by now. This research focused on several aspects of the cold-regulated gene CbCOR15b, including cloning the promoter, analyzing the cold regulating characteristics of the promoter, analyzing the subcellular localization and cryprotective functions of the protein in cells, and the main results are as follows:1. Function analysis of CbCOR15b from Capsella bursa-pastorisThe expression pattern analysis displayed that CbCOR15b not only expressed in the leaf, but also in the root of Capsella bursa-pastioris. A fusion vector overexpressing CbCOR15b-GFP was constructed and transferred into tobacco and onion epidermal cells. By monitoring the GFP fluorescence under a Laser Confocal Microscope, CbCOR15b was observed to exist in the chloroplasts of tobacco mesophyll cells and in the cytoplasm of onion epidermal cells. The vector35S::CbCOR15b was constructed and transferred into tobacco plants. The three physiological indexes, including the electrolyte leakage, the relative water content and the glucose content were measured to detect the cold tolerance of the CbCOR15b tobacco transformants in cold assay. The results indicated greater cold tolerance in transformants than that in control plants, which indicated the cryoprotective function of CbCOR15b in transgenic plants. Moreover, the phenomenon of normal growth and development in transgenic tobacco plants was favorable in cold engineering breeding.2. Cloning and activity analysis of CbCOR15promotersWith the genome-walking method, we have cloned the promoters of CbCOR15a and CbCOR15b from C. bursa-pastoris. Two CRT/DRE and One CRT/DRE have been identified in the sequence of CbCOR15aP and CbCOR15bP respectively by exploring in the PLANTCARE webpage. Expression vectors CbCOR15bP::GUS and CbCOR15aP::GUS were constructed and transferred into tobacco plants. The promoter activity was studied by RT-PCR and GUS staining assay under cold treatment. The results showed that only CbCOR15aP was active both in the leaf and the root in normal temperature. The results also showed that activity of both promoters increased greatly after cold treatment. In conclusion, CbCORl5bP was more suitable for cold engineering breeding since it was totally cold induced with no activity in normal temperature.3. Sequence deletion analysis of CbCOR15promotersPromoter fragments containing different amount of CRT/DRE c/s-element have been obtained and constructed into pCAMBIA1301to drive the expression of GUS in Arabidopsis thaliana. The GUS staining assay indicated that the core cold responsive cis-element is CRT/DRE, which was highly conserved and contains the sequence of CCGAC, and the number of the CRT/DRE elements affected the promoter activity in response to low temperature.4. Comparing analysis of COR15promoters from A. thaliana and C. bursa-pastorisFour expression vectors, including AtCOR15aP::GUS, AtCOR15bP::GUS, CbCOR15aP::GUS and CbCOR15bP::GUS were constructed and transferred into A. thaliana in order to analyze the promoter activity under cold treatment. The results showed that CbCOR15aP displayed a slight activity in normal temperature in seedlings, mature rosette leaves, stem leaves, flowers and mature siliques, and after cold treatment, the promoter activity increased greatly in all tissues. AtCOR15aP was detected active only in anthers in room temperature. But after cold treatment, the promoter activity was greatly induced in leaves, flowers and siliques, but not roots. RT-PCR analysis revealed that CbCOR15aP displayed highest activity in leaves after cold treatment, CbCOR15bP displayed less activity and COR15promoters from A. thaliana displayed least activity.This study was focused on CbCORl5b, including its promoter cloning, promoter activity, subcellular localization and protein function, results of which demonstrated that CbCOR15b was a cold-induce protein which localized in the chloroplasts and the cytoplasm and carried cryoprotective function there. This study not only helps elucidate the cold responsive mechanism in C. bursa-pastoris, but also provides a potential gene and its promoter for the cold engineering in crop breeding.