Function Analysis Of Rabidopsis Thalinana Carbonic Anhydrase βCA6Gene

Agriculture is the indispensable base of human society.The nature and productivity of agriculture aredetermined by water and climate and largely directed by the products of agricultural research. With theincrease of the world population, we will need more and more food in future. And the deterioration ofenvironment and lack of the resources, will lead to the competition of the fuel and the food. So theimprovement of crop yields becomes the urgent problems of agricultural research. Fortunately,evolution has provided an example of a much more efficient photosynthetic system （C4） than thatpossessed by rice or wheat （C3）. C4plants have the higher water-use efficiency than C3plants, and useabout40%less nitrogen to achieve50%higher yields. Therefore, adding C4systems to C3ones will bethe most direct and effective method to improve food production through biotechnology.C4photosynthesis is a main kind of terrestrial plants photosynthesis. The kranz structure is the mosttypical morphological structure in C4plants, for the performance of two kinds of photosynthetic cellsaround the vascular. The RuBP has the dual role of oxidation/decarboxylation in C4photosynthesispathway, its activity depends on intracellular CO2/O2partial pressure. The CO2concentratingmechanism elevated CO2partial pressure in the vascular bundle sheath cells.So the C4photosynthesiscan reduce the loss of CO2in the process of photorespiration.Over a quarter of the carbon dioxide （CO2） fixed by photosynthesis in plant leaves is lost due tophotorespiration. Additionally, dark respiration consumes another30-40%of the fixed carbon in manyplant species. Reducing respiratory carbon loss has been proposed as a promising way to increase plantproductivity. However, respiration is also necessary for growth and respiration rates generally scalewith relative growth rates. Although early experiments suggested that the selection of genotypes withreduced dark respiration rates increased crop yields, a later study by Kraus and Lambers （2001）indicated that the increase in yield was not coupled to the respiration rate. Although attempts toincrease the amount of photorespiratory CO2that is refixed by photosynthesis have been preliminarilysuccessful, whether the manipulation of respiration rates by biotechnological means can positivelyaffect plant growth rates remains to be established.Carbonic anhydrase is a zinc metalloenzyme that catalyzes the reversible hydration of carbondioxide （CO2） and increases the inter-conversion between CO2and bicarbonate （HCO3-） in livingorganisms. Its widespread existence in all plants, animals, and microorganisms suggests that it has an important role in biological processes. In C4plants, a cytosolic CA is required for the hydration of CO2to provide HCO3-to phosphoenolpyruvate carboxylase （PEPCase） and the initial carboxylation reactionof the CO2-concentrating pathway. PEPCase activity is also present in C3plants and replenishes theKrebs cycle with intermediates （the anaplerotic pathway） necessary for amino acid production, whichimplies that some cytosolic or mitochondrial CA activity is also required to sustain this pathway.In spite of the C4photosynthesis is important in ecological and economic.But since C4photosynthesis pathway has been found, the mechanism of the genetic control of C4has been nobreakthrough.The genetic mechanism of C4kranz structure is unclear. So far transcription factors,kinase, phosphorylase or receptors closely associated with C4pathway have not been identified. of.Therefore, the studies of C4genes are important. CA play an important role in carbon fixation inphotosynthesis pathway, therefore, to research the mechanism of action of the CA will be of greatsignificance to the study of C4photosynthesis.There were three gene families of CA in Arabidopsis, and the AtβCA6located in mitochondria bypredict of bioinformatics. And the ATβCA6gene expression level increased in low CO2condition.Therefore we speculated the gene may related to the refixation of CO2released from the mitochondrialrespiration, and so we studied AtβCA6gene function. We bought the T-DNA insertion mutant（Salk₀65611） of AtβCA6to research the gene function.The main research results were shown as follows:1. A T-DNA insertion in the gene encoding βCA6（At1g58180） was verified in the insertion mutantline Salk₀65611using PCR analysis and sequencing. The amplified PCR fragments were sequencedto confirm that Salk₀65611had a T-DNA insertion in the seventh intron of At1g58180. We obtainedhomozygous mutants and confirmed that no AtβCA6transcript was detectable in these mutants.2. Transgenic Arabidopsis lines that expressed GUS under the control of AtβCA6promoter （1to1350bp upstream of the ATG of At1g58180） were used to identify the tissues and cell types in whichthe AtβCA6promoter was active. Our results indicate that the GUS gene was expressed in all parts ofArabidopsis seedlings, including in the roots and leaves.Analysis of the promoter by PlantCARE and PLACE software, we found that the gene promoter inaddition to the necessary elements such as TATA BOX、 CAAT enhancer, also contain multipleregulatory elements, such as light response element, salicylic acid response element, stress components,heat shock response of components and so on. And we also found a cis-acting element （GANTTNC） at 1105bp of promoter related to the CO2response in Chlamydomonas reinhardtii.For the study of CO2response element of AtβCA6genes, this article analyzes the CO2response element, the results indicatethat the gene’s promoter between464-726bp areas exist CO2response element and the gene’spromoter between726-1000bp areas exist a repressor3. In this research, we constructed the35S promoter-driven expression vector in which the gene wasin frame fused with the C terminal of GFP and the native promoter-driven expression vectors in whichthe gene was in frame fused with the N terminal or the C terminal of GFP, respectively and obtainedthe transgenic plants. And we found that the AtβCA6proteinlocated in arabidopsis mitochondria andthe gene didn’t express when the gene was fused with the N terminal of the GFP.4. A number of AtβCA6overexpressing lines （OE） were obtained by expressing AtβCA6under thecontrol of a35S promoter in a wild type background. In addition, we aimed to complement the T-DNAinsertion mutant by expressing AtβCA6under the control of the35S promoter in the homozygousSalk₀65611background （CM line）. Expression levels of AtβCA6in five OE lines were all highercompared to the wild type control. By comparison, expression levels of five CM lines were all similarto the WT.After one month of growth, the OE plants were significantly larger in size than the other lines,whereas the Salk₀65611line was the smallest. Compared to the WT, the overexpression of AtβCA6significantly increased the shoot fresh and dry mass, but these parameters were reduced in the AtβCA6knockout line （SALK₀65611）. The rosette leaf area and biomass of the CM lines were similar to thatof the WT.There was a trend for higher rates of net photosynthetic rates in AtβCA6OE lines, although thisdifference was not significantly different. The Salk₀65611knockout mutant had a significantly higherrespiration rate, whereas the OE lines showed a trend for lower respiration rates compared to the WT.We further analysed the relation between photosynthesis and the intracellular CO2concentration inthe leaves by CO2response curve. The CO2compensation points of the WT，Salk₀65611，OE and CMlines were75±8μmol mol-1,88±9μmol mol-1,74±5μmol mol-1and77±9μmol mol-1, respectively. Thisresult may help to explain why under low CO2conditions, the growth of the Salk₀65611knockoutmutant was inhibited. Nonetheless, the OE line showed an increased biomass accumulation whencompared to the WT.5. A normalized cDNA library from Arabidopsis thaliana wild type and Salk₀65611mutant lines was sequenced using an Illumina HiSeq2000. In total, around53million single-end100bp long readswere generated for each sample. After trimming adapters and filtering out low quality reads, weobtained more than47million reads （89.5%）, which were used for assembly. We mapped clean reads tothe Arabidopsis thaliana genome using TopHat.The assembled transcripts were annotated using gene ontology （GO） terms and MapMan annotations.GO labels for Arabidopsis thaliana from the Plant Gene Index website（http://compbio.dfci.harvard.edu/tgi/plant.html） were used in this study. All the reads were mapped to23826genes among which101genes where both the P value and FDR were lower than0.05wereselected for further analysis. These101genes included26up-regulated genes and75down-regulatedgenes. We used quantitative RT-PCR to validate these observed changes in the RNA-SEQ. Thefunctions of101identified genes were related to metabolism, ion transport, signal transduction, proteinmodification, transcription factors, phosphorylation, and stress responses.6. We analysed the metabonomics of the Arabidopsis wide type and knockout mutant Salk₀65611through proton nuclear magnetic resonance spectroscopy （1H NMR）. We exactly obtained38metabolites, including16kinds of amino acids and their derivatives,6kinds of sugars,7kinds oforganic acids,2kinds of Alcohols and others. It showed that the samples have high repeatability in theWT and Salk₀65611groups respectively by PCA and PLS-DA analysis. And we found that most ofthe sugars and organic acids significantly decreased in the Salk₀65611compared with WT. But mostof the amino acids were increased in Salk₀65611.The innovations of this thesis were shown as follows:It was the first systematic research to illustrate the gene function of Arabidopsis AtβCA6, such as thestudy of the function of the gene promoter element, subcellular localization, gene function,transcriptome and metabonomics analysis.It is the great significance to the study of C4photosynthesis.