Cloning And Expression Analysis Of Malonyl CoA-acyl Carrier Protein Geneand Fatty Acids Content Assay Under Environment Stress In Nannochloropsis Gaditana

Nannochloropsis ganada belongs to brown algae. Its oil content could be60%of thedry weight maximal. Fatty acid is given priority to C16and C18. Nannochloropsis ganada isrecognized as the most promising seawater algae for edible lipids. However few jobs havebeen focused on this. In order to understand fully the transcriptomic information anddifferential expressions of genes between low and high lipid producing phases, weinvestigated the Nannochloropsis ganada lipid metabolic pathways by sequencing thetranscriptome and determining quantitatively the abundances of gene transcripts ofNannochloropsis ganada at logarithm and stationary growth phases with Illumina Hiseq2000.The MCAT gene (NgMCAT) from Nannochloropsis ganada transcriptome library wasidentified and sequenced in our laboratory. Bioinformatic analysis was used to analysis theencoded protein sequence of NgMCAT. Also the changes of NgMCAT gene expression andfatty acid components of Nannochloropsis ganada under different temperature, concentrationof nitrates and sodium chloride were also investigateded by RT-qPCR and some that changedwith the expression of NgMCAT were also observed.A total of29,203unigenes and13,775unigenes with function annotations in NCBI weregenerated. About9,745unigenes were2-fold up in exponential growth phase and7305unigenes were2-fold up in stationary phase.12,153unigenes showed no difference. Theunigenes with COG annoations could be classified into25categories. Including Signaltransduction mechanisms, Energy production and conversion, Amino acid transport andmetabolism, etc.About4,135unigenes were2-fold up in exponential growth phase while5,015were2-fold up in stationary phase. All the unigenes were queried against the KEGGpathway database.818unigenes were2-fold up and238pathways were related to exponentialgrowth phase. While in stationary phase,1,148unigenes were2-fold up and248pathwayswere related.The full length of NgMCAT cDNA contained an ORF of1,059bp, a5′-UTR of158bp anda3′-UTR of452bp. The cDNA encoded a protein of352amino acids residues with deducedpI of6.06and MW of37.22kDa. The abundance of NgMCAT gene transcript at normaltemperature (25℃) was more stable than those at15℃and35℃. At15℃, the abundance ofNgMCAT transcript increased significantly (P<0.05), reaching the maximum at6h (4.25foldsof the control) and then decreasing continuously to an abundance lower than that of control.At35℃, the expression of NgMCAT increased a little in12h and then decreased slowly.When sodium nitrate content was limited, the expression of NgMCAT reached the maximumat12h (5.17folds of the control)(P<0.05), then decreased gradually. When sodium nitratecontent was excessive (150mg L1), the expression of NgMCAT was statistically constant(P<0.05) in24h, and then increased slightly but significantly (P<0.05). In comparison with the normal content (32g L1), the high content of sodium chloride (64g L-1) showed littleinfluence (P>0.05) on the expression of NgMCAT during culture except for72h at which theabundance of NgMCAT transcript decreased obviously (P<0.05).Under normal condition and when sodium chloride was excessive, the percentage of C14,C16:1, C20:4, C20:5increased with culturing time. However, the percentage of C16:0, C18:2, C18:1,C18:0displayed a contrary trend. The percentage of C14:0was fluctuant while at hightemperature, the percentage of C14:0increased at beginning and reached the maximum at24h.The percentage of C16:0decreased at both low and high temperature, but at low temperature,the decent rate of C16:0was faster. The percentage of C18:0also decreased at beginning butchanged little latterly at both low and high temperature. The percentage of C14:0increasedwhen nitrate was excessive, but it decreased when nitrate was limited. The percentage of C16:1increased significantly when nitrate was limited. The percentage of both C18:0and C20:4increased when nitrate was excessive and decreased when nitrate was limited. The percentageof C20:5decreased when nitrate was either limited or excessive; however, it decreased fasterwhen nitrate was limited.