Cloning And Expression Analysis Of The Genes Associated With Photosynthesis In Pyropia Haitanensis

Pyropia haitanensis is an economically important mariculture crop in south China and is also valuable in life science research.P.haitanensis displays a unique heteromorphic,digenetic life cycle that consists of a macroscopic haploid foliate gametophyte and a microscopic diploid filamentous sporophyte.These gametophytic and sporophytic phases show a number of morphological and physiologic differences,such as the difference of carbon fixation pathways in photosynthesis.In this study,the carbon fixation pathways in different life cycle of P.haitanensis was studied from the molecular level and enzyme activity level,and the the expressed level of genes and activity level of enzymes associated with Photosynthesis in P.haitanensis during desication stress were also analyzed by real-time fluorescent quantitative PCR?qPCR?.These results will provide valuable resources for the research of carbon fixation pathways and mechanisms of desiccation stress in P.haitanensis.The main results were as follows:1.Based on unigenes sequences,73 Unigenes associated with carbon fixation pathways in photosynthesis were found from P.haitanensis whole transcriptome data.2.Thirteen full-length genes associated with carbon fixation pathways in photosynthesis weresuccessfully cloned from P.haitanesis by using rapid amplification of cDNA ends?RACE?technology or direct PCR.The seven genes in C₃ pathway were named PhPGK,PhFBA,PhcFBPase,PhTKT,PhSBP,PhRPIA and PhRPE,respectively.They comprised 1871,1541,1406,2533,1209,1019 and 1446 nucleotides,respectively.And the cDNAs encoded proteins of551,265,366,733,321,234 and 274 amino acids,respectively.The six genes in C₄ pathway were named PhAST,PhMDH,PhNAD-ME,PhNADP-ME,PhPK and PhALT,respectively.They comprised 1923,1820,1649,1374,1927 and 1513 nucleotides,respectively.And the cDNAs encoded proteins of 434,346,501,236,609 and 495 amino acids,respectively.3.Based on the phylogenetic analysis of the genes associated with carbon fixation pathways in photosynthesis,we hypothesized that the evolution of photosynthesis in P.haitanensis were between macroalgae and microalgae,C₃ pathway was more likely close to the macroalgae and C₄pathway was more likely close to the unicellular algae.4.The expressions of the genes associated with carbon fixation pathways in photosynthesis during desiccation stress were measured by real-time quantitative PCR?qPCR?.The results showed that the expression levels of genes?except PhSBP and PhRPE?in C₃ pathway were not significantly affected by mild-to-moderate water loss?<60%?,But the expression levels of genes in C4 pathway?including PhDLDH,PhPEPCK,PhPEPC,PhAST,PhMDH,PhNAD-ME,PhALT and PhPK?were significantly up-regulated during mild-to-moderate water loss?<60%?.And,the expression levels of genes in C₃ and C₄ pathway were all significantly down-regulated under moderate and severe water loss.These results indicated that genes associated with carbon fixation pathways in P.haitanensis were positively affected by the desication stress.5.The activities of enzymes in C₃ pathway?Rubisco,GAPDH,cytoplasm FBP and chloroplast FBP enzyme?and C₄ pathway?ALT,MDH,NAD-ME,NADP-ME,PEPC,PK?were measured during different levels of deciation stress.The results showed that the enzyme activities?except NADP-ME and PK?were all firstly enhanced and then weakened.Compared with the expression levels of genes,the change of enzyme activities were presented a delayed state.This indicated that the expression of genes and the change of enzyme activities have positive correlations.6.The relative expression level of genes associated with carbon fixation pathways in thallus and conchocelis of P.haitanesis were also measured by qPCR.The results showed that the expression levels of genes in C₃ pathway?except RPTP and TIM?were significantly lower in the conchocelis than in the thallus,but the expression levels of genes in C₄ pathway?except AST?were significantly higher in the conchocelis than in the thallus.7.The activities of enzymes in C₃ pathway?Rubisco,cpFBPase,cFBPase,GAPDH?and in C₄ pathway?PEPC,MDH,NAD-ME,NADP-ME,PK,ALT?in thallus and conchocelis of P.haitanesis were also measured.The results showed that the antivities of enzymes in C₃ pathway were significantly lower in conchocelis than in thallus,but the antivities of enzymes in C₄pathway were significantly higher in conchocelis than in thallus.8.By comparing the expression levels of genes and activities of enzymes associated with carbon fixation pathways in thallus and conchocelis of P.haitanesis,we speculated that the carbon fixation pathway in the conchocelis and thallus were different,and the C₄-like pathway might play important roles in the conchocelis stage.