| The karst ecosystem in southwest China's karst areas is a fragile ecosystem because of double-layer hydrogeologic structures. Thereby, it is very difficult to recover the vegetation and restore damaged ecosystems. Many reaseachers have testified that the plants in southwest China's karst areas have a common characteristic of high CA activity. The research on economic crops with high CA activity is significant for restoring damaged ecosystem and improving economic actuality in karst areas.Carbonic anhydrase (CA; EC 4.2.1.1) is a zinc metalloenzyme that catalyzes the interconversion of CO2 and HCO3-. CA plays an important role in photosynthesis and some researchers found that CA can also respond to environmental stresses. The screening of oilrape with high CA activity and gene cloning of CA from oilrape were important for increasing the yield and stress resistance of oilrape in non-karst areas, and could also lay a basis for solving the problem of drought resistance and yield of oilrape and other economic crops in karst areas. Therefore, the following aspects were studied: Firstly, the CA activities of different oilrapes, different parts of the same oilrapes at different growth stages were inspected. We placed emphasis on the correlation between CA activity and zinc content, as well as, CA activity and photosynthetic rate. Secondly, the CA activities in oilrape at the bolting stage were increased by spraying exogenous zinc ion on the surface of leaves, which led us to find out if increasement of CA activity helped to enhance drought resistance and yield of oilrape. Lastly, the CA gene from oilrape with high CA activity was cloned, analyzed, and heterologously expressed. The main results were as follows:1. Considering different kinds of oilrapes, CA activities in leaves were descending with the range as B. napus, B. campestris, B. rapa, and B. carinata. CAs were distributed extensively in root, stem, and leaf of oilrape. CA activity was much higher in leaves than that in roots or stems, while CA activity in roots was the lowest. During the whole growth period, CA activity increased at the bolting stage, and then decreased later. CA activity reached its maximum at the bolting stage. Correlation between CA activity and zinc content is significant at the 0.05 level, and its correlation coefficient was 0.95. Correlation between CA activity and net photosynthetic rate is significant at the 0.01 level, and its correlation coefficient was 0.99.2. Spraying zinc ion on leaves of oilrape with high CA activity was performed to investigate the effects of exogenous zinc on the photosynthetic characteristics and CA activity of oilrape in the field. It was revealed that spraying exogenous zinc ion enhanced net photosynthetic rate and CA activity, which helped to increase the yield of oilrape. Spraying exogenous zinc ion reduced transpiration rate and stomatal conductance simultaneously, which suggested the drought resistance of oilrape was improved. In order to testify the positive correlation between CA activity and drought resistance of oilrape, spraying zinc ion on leaves of potted oilrape was performed to investigate the effect of exogenous zinc on CA activity and drought resistance of oilrape. It was revealed that the CA activity, net photosynthetic rate, and proline content of zinc treated group were much higher than those of water treated group. However, the content of malondialdehyde of zinc treated group was lower than that of water treated group. The results indicated that the ability of resistance to drought was elevated after CA activity was increased by spraying zinc ion on leaves of oilrape. In a word, the two experiments about spraying zinc on leaves indicated that the oilrape with high CA activity has a characteristic of high yield and strong resistance to drought.3. We chose the oilrape with high CA activity as plant material. One pair of primers was designed based on the conserved sequences ofβ-CA gene in several Brassicaceae plants. A cDNA fragment of 996 bp was isolated by Polymerase Chain Reaction (PCR) using the primers. Utilizing the 996 bp fragment as a"core"sequence, the full-length cDNA sequence of CA from B. napus was determined by a RACE method with high-fidelity DNA polymerase. The full-length cDNA of B. napus CA was 1190 bp and was shown to comprise 47 bp of a 5'-untranslated region, 993 bp of an open reading frame, a termination codon, and 118 bp of a 3'-untranslated region. The open reading frame encodes a polypeptide of 331 amino acids. The amino acid sequence of CA from B. napus shared significant identity withβ-CA from A. thaliana (94% identity), which indicated that the CA gene from B. napus was attributed toβ-CA gene family. The CA gene from B. napus was designated as BnCA1 and had been deposited in the Genbank database under the accession number (GQ356780).4. Theβ-CA gene from B. napus was subcloned into the prokaryotic expression vector pET-32a(+), then the plasmid pET32-CA was transformed into E. coli BL21 (DE3). With the induction of IPTG (1 mM),β-CA gene was expressed in E. coli cells as a fusion protein at 28℃, with 6×His-tag at the N-terminus. A protein with an apparent molecular weight of 37.5 kDa was expressed after induction, which corresponded to the size of the fusion protein which had a calculated molecular weight of 37.5 kDa.5. The primary structure, secondary structure, three-dimensional structure and 3D structure alignment ofβ-CA from B. napus were predicted in PSORT, PSIPRED, Swiss-Model, and NCBI-VAST server, respectively. Theβ-CA from B. napus was located in chloroplast stroma. In the secondary structure,α-helix, random coil, andβ-sheet were 45.9%, 37.7%, and 16.4%, respectively. The three-dimensional structure ofβ-CA was constructed by homology modeling. The 3D alignment ofβ-CA from B. napus and a monomer ofβ-CA octamer from p. sativum (i.e. 1ekjG) showed that the 3D structure ofβ-CA from B. napus could match well with theβ-CA monomer from p. sativum. The alignment results implied thatβ-CA from B. napus was a monomer of theβ-CA octamer from B. napus.
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