Cloning and expression analysis of the chloroplast fructose-1,6-bisphosphatase gene from Pyropia haitanensis

Fructose-1,6-bisphosphatase（FBPase） is one of the key enzymes in Calvin circle and starch biosynthesis. In this study, the full-length of cpFBPase gene from Pyropia haitanensis was cloned by using rapid amplification of cDNA ends（RACE） technology. The nucleotide sequence of PhcpFBPase consists of 1 400 bp, including a 5′ untranslated region（UTR） of 92 bp, a 3′？UTR of 69 bp, and an open reading frame（ORF） of 1 236 bp, which can be translated into a 412-amino-acid putative peptides with a molecular weight of 44.3 kDa and a theoretical pI of 5.23. Multiple sequence alignment indicated that the protein belonged to the chloroplast FBPase enzyme. Phylogenetic analysis showed that the protein assembled with the cpFBPase of a thermal tolerant unicellular red micro-algae Galdieria sulphuraria. Expression patterns analyzed by qRT-PCR revealed that the expression of PhcpFBPase gene in the thallus phage was 7-fold higher than in the conchocelis phage, which suggested the different mechanisms of inorganic carbon utilization among the different life phages of P. haitanensis. And the different response modes of PhcpFBPase mRNA levels to high temperature and desiccation stress indicated that PhcpFBPase played an important role in responsing to abiotic stress.

Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis(Bangiales, Rhodophyta) by quantitative real-time PCR

Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these characteristics, gene expression has been investigated at the whole transcriptome level. Gene expression studies using quantitative real-time PCR should start by selecting an appropriate internal control gene; therefore, the absolute expression abundance of six housekeeping genes （18S rRNA （18S）, ubiquitin-conju-ating enzyme （UBC）, actin （ACT）, β-tubulin （TUB）, elongation factors 2 （EF2）, and glyceraldehyde-3-phos- phate dehydrogenase （GAPDH） examined by the quantitative real-time PCR in samples corresponding to different strains, life-cycle stages and abiotic stress treatments. Their expression stabilities were assessed by the comparative cycle threshold （Ct） method and by two different software packages： geNorm and NormFinder. The most stable housekeeping gene is UBC and the least stable housekeeping is GADPH. Thus, it is proposed that the most appropriate internal control gene for expression analyses in P. haitanensis is UBC. The results pave the way for further gene expression analyses of different aspects of P. haitanensis biology including different strains, life-history stages and abiotic stress responses.

Effects of elevated CO_2 on the photosynthesis and nitrate reductase activity of Pyropia haitanensis(Bangiales,Rhodophyta) grown at different nutrient levels

Pyropia haitanensis, a commercially important species, was cultured at two CO2 concentrations （390× 10^-6 and 700× 10^-6 （parts per million）） and at low and high nutrient levels, to explore the effect of elevated CO2 on the species under nutrient enrichment. Results show that in CO2-enriched thalli, relative growth rate （RGR） was enhanced under nutrient enrichment. Elevated CO2 decreased phycobiliprotein （PB） contents, but increased the contents of soluble carbohydrates. Nutrient enrichment increased the contents of chlorophyll a （Chl a） and PB, while soluble carbohydrate content decreased. CO2 enrichment enhanced the relative maximum electronic transport rate and light saturation point. In nutrient-enriched thalli the activity of nitrate reductase （NRA） increased under elevated CO2. An instantaneous pH change in seawater （from 8.1 to 9.6） resulted in reduction of NRA, and the thalli grown under both elevated CO2 and nutrient enrichment exhibited less pronounced reduction than in algae grown at the ambient CO2. The thermal optima of NRA under elevated CO2 and/or nutrient enrichment shifted to a lower temperature （10-15 ℃） compared to that in ambient conditions （20℃）. We propose that accelerated photosynthesis could result in growth increment. N assimilation remained high in acidified seawater and reflected increased temperature sensitivity in response to elevated CO_2 and eutrophication.

Identification of potential internal control genes for real-time PCR analysis during stress response in Pyropia haitanensis

Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde- 3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that a-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.

Identification and bioinformatics analysis of micro RNAs from the sporophyte and gametophyte of Pyropia haitanensis

Pyropia haitanensis(T.J.Chang et B.F.Zheng) N.Kikuchi et M.Miyata( Porphyra haitanensis) is an economically important genus that is cultured widely in China.P.haitanensis is cultured on a larger scale than Pyropia yezoensis,making up an important part of the total production of cultivated Pyropia in China.However,the majority of molecular mechanisms underlying the physiological processes of P.haitanensis remain unknown.P.haitanensis could utilize inorganic carbon and the sporophytes of P.haitanensis might possess a PCK-type C 4-like carbon-fixation pathway.To identify micro RNAs and their probable roles in sporophyte and gametophyte development,we constructed and sequenced small RNA libraries from sporophytes and gametophytes of P.haitanensis.Five micro RNAs were identified that shared no sequence homology with known micro RNAs.Our results indicated that P.haitanensis might posses a complex s RNA processing system in which the novel micro RNAs act as important regulators of the development of different generations of P.haitanensis.

Pyropia haitanensis polysaccharide extends lifespan by inhibiting protein aggregation in Caenorhabditis elegans

Pyropia haitanensis polysaccharide(LP)have been found for having many excellent functions such as anti-aging.Using Caenorhabditis elegans models,we evaluated the anti-aging activity of LP by observing the lifespan,reproduction,pharyngeal pumping,stress response,quantitative fluorescence of polyglutamic acid,and nuclear localization of DAF-16 of worms.The results reveal that LP could extend the adult lifespan of wild-type and polyQ nematodes,indicating a connection of its anti-aging benefit with the toxicity-suppressing effect.The number of polyglutamic acid aggregates in high concentration groups decreased by 24.39%(P<0.05)to the control.The high-dose group strongly induced DAF-16 nuclear translocation over intermediate and cytosolic localizations compared with the control(P<0.001).Therefore,we believe that LP could extend the lifespan and reduce the protein aggregation in C.elegans through nuclear DAF-16∷GFP expression.