Algal strain, cultivation conditions, and biomass assay
C. reinhardtii CC425 (mt) was purchased from the Chlamydomonas Genetics Center at Duke University. Cells were grown on a Tris-acetate-phosphate (TAP) agar plate, inoculated into 100 mL Erlenmeyer flasks containing 50 mL of HSM and N-deficient HSM (HSM-N) media . The HSM medium was composed of NH4Cl (0.500 g L-1), MgSO4 · 7H2O (0.020 g L-1), CaCl2 · 2H2O (0.010 g L-1), K2HPO4 (1.440 g L-1), KH2PO4 (0.720 g L-1), NaAc (2.000 g L-1), H3BO3 (0.001 g L-1), MnCl2 · 4H2O (0.005 g L-1), ZnSO4 · 7H2O (0.022 g L-1), FeSO4 · 7H2O (0.005 g L-1), CoCl2 · 6H2O (0.002 g L-1), Na2Mo7O24 · 4H2O (0.002 g L-1), and Na2 · EDTA (0.050 g L-1). The HSM-N medium contained the same components but with NH4Cl instead of NaCl. All cultures were maintained in an incubator shaker (230 rpm at 25°C) and then exposed to continuous illumination at a light intensity of 150 μmol · m-2 · s-1.
Biomass concentration (g/L) was determined by measuring the optical density of the samples at 490 nm (OD490) as described in a previous study . To generate the standard curve, a series of C. reinhardtii CC425 samples with different biomass concentrations were collected. The OD490 and cell dry weight were gravimetrically determined using dried cells to plot the standard curve of OD490 versus biomass concentration (g/L). Samples were diluted to appropriate ratios to ensure that the measured OD490 values ranged from 0.15 to 0.75, if applicable. Biomass concentration was then calculated using the following formula: cell dry weight (g/L) = 0.7444 × OD490 – 0.0132 (Additional file 1: Figure S1).
Lipid content analysis
The Nile Red fluorescence method and GC/MS were used to determine lipid and TAG levels [24, 25]. Algal cells were directly stained with 0.1 mg/mL Nile Red for 10 min, and then fluorescence was measured at excitation and emission wavelengths of 470 and 570 nm, respectively. The fluorescence value was calculated using the Equation FD (470/570) = (A
2 – A
1), where A2 is the fluorescence value of algal cells after staining with Nile Red, and A
1 is that of algal cells before staining (Additional file 1: Figure S2). Total lipid extraction was carried out according to a modified method. Logarithmic-phase algal cells were collected by centrifugation and extracted using an extraction buffer (methanol : chloroform : methanoic acid, 2 : 1 : 0.1), 1 M KCl, and 0.2 M H3PO4. The lipids were obtained by centrifugation at 13780 g for 3 min. For TAG separation, we used Si60 silica TLC plates for thin-layer chromatography. The TLC plates were immersed in 0.15 M (NH4)2SO4 for 30 s and stored in an airtight container for two days. These plates were then placed in an oven at 120°C for 2.5 h and cooled at room temperature. Samples were then placed under N2 flow, and TAGs were observed on TLC plates through iodine staining. Lipid analysis was conducted as previously described. Fatty acid methyl esters derived from TAG were analyzed through GC/MS . For the microscopic assay, images were acquired using a Nikon 80i Fluorescence Microscope after cells were stained with Nile Red. Nile Red signals were captured using an excitation wavelength of 480 nm, and emission was collected between 560 nm and 600 nm [27–29]. Thirty cell lipid droplets from each algal strain were examined to determine the difference between lipid contents.
Total RNA was prepared as described by Li et al. with some modifications . Cells from 10 mL of cultivated algae were collected by centrifugation at 10,000 × g for 1 min. After a series of phenol–chloroform extractions, nucleic acids were precipitated with two volumes of absolute ethanol and then washed with 75% ethanol. Finally, the air-dried pellet was dissolved in 40 μL of RNase-free water. RNA concentration was determined through spectrophotometry, and the integrity was examined through agarose gel electrophoresis.
Cloning of CrCIS gene
First-strand cDNA was synthesized using SuperScriptTM III Reverse Transcriptase (Invitrogen, USA) according to the instructions of the manufacturer. A fragment of the CrCIS gene was amplified by PCR using the primers CrCISL: 5′-TACTTGGCCCGTGCCTGTAT-3′ and CrCISR: 5′-CCTCCCTCCTTCATGTGTGT-3′. PCR reactions were performed in a final volume of 25 μL containing 1× PCR reaction buffer, 2 mM MgCl2, 0.4 μmol of each primer, 0.25 mM dNTPs, 1 μL DMSO, 0.5 M Betain, and 0.5 U Taq DNA polymerase (Promega, USA) according to the following program: 4 min at 95°C; 35 cycles of denaturation for 40 s at 95°C, annealing for 40 s at 58°C, and elongation for 20 s at 72°C; and 10 min at 72°C. After purification using the EZ-10 Spin Column DNA Gel Extraction Kit (BBI, Canada), DNA was inserted into vector pMD18-T following the instructions of the manufacturer (TaKaRa, Japan). The resulting plasmid was designated as pMD18T-CrCIS. The sequences of the cloned CrCIS gene were verified through double-stranded sequence analysis (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd).
Construction of the RNAi vector against CrCIS gene
To construct the RNAi vector against CrCIS gene, a fragment of C. reinhardtii 18S gene was amplified with primers 5′-CGAACTTCTGCGAAAGCAT-3′ and 5′-TCAGCCTTGCGACCATACT-3′ and then inserted into pMD18-T producing pMD18T-18S. The fragment of CrCIS and its reverse complementary sequences were amplified through PCR using pMD18T-CrCIS as the template and the primers CISRNAiL: 5′-GACGCGCACAGCGGCGTGCT-3′ and CISRNAiR: 5′-CCTCCCTCCTTCATGTGTGT-3′. The PCR fragment was then digested with KpnI/BamHI and HindIII/SalI, after which the fragment was inserted into the corresponding cloning sites of pMD18T-18S. The fragment yielded pMD18-CrCIS F-18S-CrCIS R, which contained an inverted repeat sequence of CrCIS (CrCIS IR). pMD18-CrCIS F-18S-CrCIS R was double digested with KpnI and HindIII to obtain CrCIS IR. Finally, CrCIS IR was inserted as a blunt-end fragment into EcoRI-digested pMaa7/XIR to produce pMaa7IR/CrCIS IR.
Construction of the overexpression vector of CrCIS gene for Chlamydomonas
To construct the overexpression vector of CrCIS gene, the coding sequence of CrCIS was amplified through PCR using pMD18T-CrCIS and primers 5′-TTCAAGATCTGATGCTGGCCACGGCC-3′ and 5′-TAACACTAGTTTACGCGGACTGGCC-3′ as templates. The fragment was digested with NcoI/SpeI and inserted into similarly digested pCAMBIA1302 to yield pCAMBIACIS facilitating the overexpression of CrCIS.
Transformation of Chlamydomonas
Transformation of C. reinhardtii strain CC425 was performed as described by Kindle . C. reinhardtii cells were grown in TAP medium until having a cell density of 1–2 × 106 cells/mL. Cells were collected by centrifugation, washed twice, and then resuspended in TAP medium until having a cell density of approximately 1 × 108 cells/mL. Plasmid DNA was introduced into the cells through the glass bead procedure. In each case, 2 μg of plasmid DNA was added to a mixture containing 400 μl of cells, 100 μl of 20% polyethylene glycol, and 300 mg of sterile glass beads. The reaction was mixed for 15 s on a benchtop vortex. To enable induction of RNAi or gene expression, cells were allowed to recover for one day before plating onto selective media. RNAi transformants were selected on TAP medium containing 1.5 mM l-tryptophan, 5 μg/mL paromomycin, and 5 μM 5-FI. Meanwhile, pCAMBIACIS transformants were selected on TAP medium containing 50 μg/mL hygromycin. Plates were incubated under dim light (approximately 50 μmol · m-2 · s-1 photosynthetically active radiation), and isolated transgenic strains were kept under constant selective pressure.
Expression of CrCIS in Escherichia coli BL21
To express CrCIS in E. coli BL21, the coding region was amplified from pMD18T-CrCIS with primer pairs GEXCIS-F: 5′-TAAAGGATCCATGCTGGCCACGGCC-3′ and GEXCIS-R: 5′-TAAGCTCGAGTTACGCGGACTGGCC-3′. The amplified fragments were digested with BamHI/SalI and then inserted into similar digested pGEX-6p-1 to administer pGEXCIS. The transformation of E. coli BL21 and subsequent foreign protein detection by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and purification of the glutathione S-transferase (GST)-fusion protein were conducted as described by Sambrook and Russell .
Quantitative real-time PCR
Samples for real-time PCR analysis were performed as described by Fei et al. and Deng et al. [10, 33]. RNA was extracted using TRIzol Reagent (Shanghai Sangon Biological Engineering Technology & Service Co.). Single-strand cDNA was synthesized using an Invitrogen SuperScriptTM III cDNA synthesis kit with 100 ng of RNA, and random primers performed at 65°C for 5 min, 25°C for 5 min, and 42°C for 50 min. Real-time PCR was performed on a BioRad iCycler iQ Real-Time PCR Detection System using SYBR Green as the fluorescent dye. Each reaction was performed with a final volume of 25 μl with the following components: 0.2 pmol of each primer, 1 μl of cDNA, and 12.5 μl of SYBR Green Mix (Invitrogen SYBR GreenER qPCR). Water was used to adjust the volume to 25 μl. The iCycler protocol was performed as follows: denaturing at 95°C, 5 min, and 40 cycles of denaturing at 95°C for 30 s; annealing at 54°C for 30 s; and amplification at 72°C for 15 s. The specificity of PCR amplification was examined with a melting curve program (55°C to 100°C at a heating rate of 0.5°C/s). 18S rRNA was used as the control with primers 18SrRNAF (5′-TCAACTTTCGATGGTAGGATAGTG-3′) and 18SrRNAR (5′-CCGTGTCAGGATTGGGTAATTT-3′). The expression of this control gene was measured and concluded to be constant under all conditions used in this study. The gene-specific primers listed in Table S2 (Additional file 1) were used to evaluate the quantity of target cDNA. The amplification rate of each transcript (Ct) was calculated through the PCR baseline-subtracted method performed with iCycler software at a constant fluorescence level. Ct values were determined over three repeats. Relative fold differences were calculated based on the relative quantification analytical method (2-ΔΔCT) using 18 s rRNA amplification as an internal standard .
Detection of CIS activities
Transgenic algal samples cultivated in the log phase were collected and centrifuged (3000 rpm for 5 min). Algal cell pellets were then collected and washed with phosphate buffer. Cells were sonicated in 3 mL of extract buffer and then centrifuged (3000 rpm for 5 min) to collect the supernatant totaling a volume of 6 mL through adding extract buffer. The enzyme extract to be detected was obtained by dialyzing with extract buffer. CIS activity was measured as described by Sienkiewicz et al. with some modifications . The dialyzed enzyme sample was mixed with 40 mmol/L Tris–HCl buffer (pH 9.0), 0.04 mmol/L DTNB, 0.08 mmol/L acetyl–CoA, and 0.04 mmol/L OAA. The absorbance of the samples requiring 15 s per scan was measured, and the total scan time was 3 min.