Cloning And Expression Analysis Of Carbonic Anhydrase Genes From Pyropia Haitanensis

Carbonic anhydrases（EC 4.2.1.1,CAs）are Zn-containing metalloenzymes that catalyse the reversible hydration of CO₂.They are widely present in plants and algae,which play important roles in the mechanisms of inorganic carbon utilization.In this study,based on the unigene sequences which obtained from whole transcriptome sequencing of Pyropia haitanesis,six full-length carbonic anhydrase genes were obtained by using rapid amplification of cDNA ends（RACE）technology;The expression level of the each CA gene in different CO₂ concentration,different life phages and during high temperature and desication stress also were analyzed by real-time fluorescent quantitative PCR（qPCR）;And then the pH compensation point of P.haitanesis and the CAs activity during stress were determined.The main results are as follows:1.Based on unigenes sequences,six full-length genes of carbonic anhydrase family were successfully cloned from P.haitanesis.The six CA genes were named PhαCA1,PhαCA2,PhβCA1,PhβCA2,PhβCA3 and PhγCA1,respectively.PhαCA1 and PhαCA2 belong to the family ofα-CA,PhβCA1,PhβCA2 and PhβCA3 belong toβ-CA family,and PhγCA1 belongs to LbeatH superfamily.2.The relative expressions of each PhCA gene in the conchocelis and thallus of P.haitanensis was measured,the results showed that the expressions of PhαCA1,PhαCA2,and PhγCA1 in the thallus phage were significantly higher than in the conchocelis phage,but the expressions of PhβCA1,PhβCA2,PhβCA3 in the thallus phage were significantly lower than in the conchocelis phage.These results further verified the different mechanisms of inorganic carbon utilization in the different life phages of P.haitanensis.3.The relative expression level of each PhCAs gene in thallus of P.haitanensis under different CO₂ concentration was measured,the results showed that the expressions of PhγCA1and PhβCA3 gene were not affected by CO₂ concentration,suggesting PhγCA1 and PhβCA3 are constitutive carbonic anhydrase;but the expressions of PhαCA1,PhαCA2,PhβCA1 and PhβCA2gene were significantly changed by CO₂ concentration,suggesting PhαCAl,PhαCA2,PhβCA1and PhβCA2 are inducible carbonic anhydrase.4.The relative expression level of each PhCA gene in thallus of P.haitanensis under different levels of desication and high temperature stress were measured,the results showed that except for PhαCA2 which was significantly down-regulated under the high temperature stress,PhαCAs and PhγCA1 genes were significantly up-regulated under the desiccation and high temperature stress.These results suggested that PhCAs genes played important roles in response to the desiccation and high temperature stress of P.haitanesis.5.The pH compensation points are different in the thallus and conchocelis of P.haitanensis each was 9.82 and 9.93 And under the culture condition extracellular carbonic anhydrase inhibitors（AZ）,the pH compensation point of thallus and conchocelis was 9.28 and 9.47,respectively.These results suggested that extracellular carbonic anhydrase was existed in P.haitanensis and active.6.Last,the activity of carbonic anhydrase in different stress was determined,the results showed that the change trends were same between the whole carbonic anhydrase and extracellular carbonic anhydrase during stress,but the response of extracellular carbonic anhydrase was more rapidly.These results suggested that the stress can significantly affect the utilization of inorganic carbon by restraining the activity of carbonic anhydrase.