| Carbonic anhydrase(CA)is found widely in animals,plants,microorganisms and algae,catalysis reversible inter-conversion of HC03-and C02.As one of the main protein of red blood cell components,there are numerous reports of animal carbonic anhydrase in biomedical.However,about carbonic anhydrase,especially the beta carbonic anhydrase,although studies have shown that it is involved in plant pH regulation,ion exchange,biosynthesis,photosynthesis,C02 transport and so on,the mechanism in higher plants still unclear,especially in maize.In addition,most studies of βCAs are confined to single mutants and double mutants,and these mutants had no phenotype under natural growth conditions.So,use the modle plant Arabidopsis thaliana as material,to explore βCAs mutants with obvious physiological phenotype,explore the mechanism of βCA in plants,and provide clues for the study of maize CA.First partwe built a random test set of maize proteins to evaluate the accuracy of six commonly used programs of subcellular locations:iLoc-Plant,Plant-mPLoc,CELLO,WoLF PSORT,SherLoc2,and Predotar.Our results showed that the accuracy of prediction varied greatly depending on the programs and subcellular locations involved.The programs using homology search methods(iLoc-Plant and Plant-mPLoc)performed better than those using feature search methods(CELLO,WoLF PSORT,SherLoc2,and Predotar).In particular,iLoc-Plant achieved an 84.9%accuracy for proteins whose subcellular locations have been experimentally determined and a 74.3%accuracy for all of the proteins in the test set.Regarding locations,the highest prediction accuracies for subcellular locations were obtained for the nucleus,followed by the cytoplasm,mitochondria,plastids,endoplasmic reticulum,and vacuoles,while the lowest were obtained for cell membrane,secreted,and multiplelocation.A total of seventeen full-length maize CA were predicted by the iLoc-Plant and Plant-mPLoc.For the program of iLoc-Plant,eleven of them in chloroplast orplastid,five of in cytoplasm,one in cell membrane;For the program of Plant-mPLoc,all of them in chloroplast.Second partIn this study,βcal βca2 βca4 Arabidopsis thaliana mutant plant were obtained for the first time with obvious physiological defects under natural growth conditions.According to their phenotypic characteristics,physiological and biochemical studies were carried out of growth and development,such as determination of photosynthetic index,sugar,pH and hormone on root growth,determination of carbohydrate.RNAseq was used for transcriptomic analysis,explore the molecular function of βCAs involved in plant.The main results were as follows:1.βcal βca2 βca3 and βcal βca2 βca4 triple mutants plant were obtained by hybridization.Compared with the wild type,βcal βca2 βca4 has obvious physiological defects,such as plant small and short,thick and green leaves,poor fertility,more lateral branches and long growth cycle and so on,indicating that βCAs participates in the growth and development of Arabidopsis.2.βCA1:βCA1,βCA2:βCA2 and βCA4:βCA4 functional transgenic vectors were constructed,transform into βcal βca2 βca4 triple mutant plants.Compared with the wild type and fical fica2 βca4 triple mutant plants,the functional phenotype of transgenic lines was restored,βCA1,βCA2 andβCA4 complemented the βal βca2 βca4 mutant phenotype,respectively.The results showed that theβCA gene resulted in the physiological defects of Arabidopsis plants.3.4 x 35S-βCA1:βCAl overexpressing vector was constructed,transform into wild type plants.Compared with the wild type,overexpression lines with leaf rosettes are compact and small,leaf round and green,flowering early and short growth cycle.In addition,there were two different fertility lines in the 4 x 35S-βCA1:βCA1 overexpression plants,one for the semi-sterile,the other for the whole sterile.Overexpression studies suggest that the βCA gene may be involved in promoting the processes of plant life.4.βca1 βca2 βca4 and 4 x 35S-βCA1:βCA1 plants were measured for their physiological indexes.βca1 βca2 βca4 homozygous triple mutant plant photosynthetic efficiency was lower than wild type,4x 35S-βCA1:βCA1 was higher than wild type,βcal βca2 βca4 heterozygote plant photosynthetic efficiency was same as wild type.βca1 βca2 βca4 triple mutant stoma index was similar with wild type,but the stomatal density per unit area was larger than wild type.These results indicate that PCA gene may be involved in plant photosynthesis.5.βCA gene effects on the root growth of Arabidopsis thalianaEffect of sugar on the root growth of βCA mutant in the initial stage of seed germination.At the sugar-free medium,the βca1 βca2 double mutant roots were slightly shorter than wild type,βca1 βca2βca4 triple mutant roots were hardly grow;at 1%sugar medium,the roots of βca1 βca2 βca4 triple mutant can growth,βca1 βca2 and βca1 βca2 βca4 were still shorter than wild type;with the increase of sugar concentration,the gap of root length between mutants and wild type were shortened.Effect of sugar on the root growth of βCA mutant of seedlings,the results showed that the root length of βca1βca2 and βca1 βca2 βca4 mutant were shorter than wild type in the medium with different sugar concentration.Whether it is seed or seedling,βca1 βca4 mutant was always similar with wild type.In the complementary plants study,the βCA1 complementary plant roots were shorter than wild type,andβCA2 complementary plant roots were shorter than wild type,it was suggested that the βca2 βca4 mutant root could also be sensitive to sugar.4 x 35S-βCA1:βCA1 overexpressing lines showed longer and more lateral roots.The results showed that βCA genes were involved in the roots growth of plant.In the study of the pH Effect of βca1βca2 mutant on the root growth,βca1βca2mutant was found to be more sensitive to pH than the wild type.The hormone study found that the roots of βcalca2 mutant were not sensitive to ABA,IAA and GA3,the results showed that the hormone had no effect on the roots growth ofβca1βca2 mutant.6.Using starch stain,transmission electron microscopy and starch content determination,it was found that the starch content in leaves of βca1 βca2 βca4 mutant was higher than wild type after 8 h dark.While the maltose,sucrose and glucose content were lower than wild type,indicating that βCAs were involved in starch degradation.However,βca1 βca2 βca4 mutant accumulation rate of starch was lower than wild type after Illumination begin.The results also indicated that the photosynthetic rate ofβca1/βca2 βca4 mutant was lower than wild type,which was in agreement with the results of photosynthetic indexes.Functional complementary transgenic plants were always same as the wild type.7.RNAseq study found that βca1 βca2 βca4 triple mutant plants have 986 up-regulated genes,627 down-regulated genes.GO analysis found that those genes mainly involved in metabolism and cell processes,binding and enzyme activity.Enrichment map analysis found that those genes mainly for signal,hormone response,photosynthesis,light-harvesting complex,brassinosteroid response,stress response,amino acid metabolism,hydrolase and transferase.Especially the down-regulation of Brassinolide-responsive genes,was consistent with the pHenotype of βca1 βca2 βca4 mutant and the previous studies.The main innovation was as follow:First time obtain βca1 βca2 βca4 Arabidopsis thaliana triple mutant with obvious physiological defects under natural growth conditions.New features of βCAs,such as the sensitivity of the root to sugars,involved in the degradation of starch and so on.First time analysis the βca1 βca2 βca4 triple mutant with RNAseq,the mechanism ofβCAs in plants was analyzed from the transcriptional level.It provides a theoretical basis for exploring the function of βCAs in higher plant growth and development,significance for the new function of βCAs gene in plant. |
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