Dissertation > Biological Sciences > Botany > Plant Cell Genetics > Plant Genetic Engineering

Cloning and Characterization of a Rice (Oryza Sativa L.) Gene Encoding a Temperature-dependent Chloroplast ω-3 Fatty Acid Desaturase

Author WangJingWen
Tutor ShenDaLeng
School Fudan University
Course Genetics
Keywords fatty acid in situ hybridization 0sfad8 Oryza sativa L. ω-3 fatty acid desaturase transgenic tobacco flavonoid-3’, 5’ -hydroxylase homology modeling molecular docking algorithm Phalaenopsis
CLC Q943.2
Type PhD thesis
Year 2006
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1A cDNA, designated 0sfad8, encoding a chloroplast ω-3 fatty acid desaturase responsible for trienoic fatty acid formation, was isolated from the leaves of Oryza sativa L. by RT-PCR. Southern hybrization analysis indicated that a small gene family composed of two copies or closely linked genes exist. RT-PCR and RNA in situ hybridization showed that the accumulation of 0sfad8 mRNA was abundant in leaves but hardly detectable in roots. The 0sfad8 transcript level in leaves was much higher at 15 ℃ than that at normal temperature (25 ℃). In situ hybridization also showed particularly prominent expression of 0sfad8 in the palisade layer and spongy parenchyma cells of leaves when exposed to 15 C conditions for 5 days and 10 days. Two transgenic lines (8S-52 and 8S-101) harboring the 0sfad8 ORF in sense orientation under the control of the CaMV 35S promoter, contained increased levels of hexadecatrienoic (16:3) and linolenic (18:3) fatty acids. When exposed to 2 ℃ for 7 days, the damage observed to the control plants was significantly alleviated in the 8S-52 and 8S-101 lines. The amounts of trienoic fatty acids in an 0sfad8 antisense line (8A-35) declined 40.2% compared to the control plants. The 8A-35 plants survived after growth at 44 ℃ for 3 days while the control plants died. These data suggest that 0sfad8 encodes a temperature-dependent chloroplast ω-3 fatty acid desaturase.2 A novel cDNA for the flavonoid-3’ , 5’ -hydroxylase (F35H) gene was cloned from petals of Phalaenopsis, and designed to be Phf35h (accession number DQ148458 in GenBank/EMBL/DDBJ). The genomic clone of Phf35h was isolated by a PCR-based strategy. Nucleotide sequence analysis revealedthat its genomic clone contains one intron and an open reading frame encoding a polypeptide of 507 amino acid residues. Southern hybridization analysis indicated the presence of a single gene coding for Phf35h. RT-PCR analysis showed that the Phf35h mRNA is transcribed in late phase of petal development, which is concomitant with the appearance of anthocyanins in petal tissue. The transcript is abundant in the purple petals but not in leaves or roots. The three-dimensional model of PhF3’ 5’ H protein is classified into an a -domain which contains most of the α -helixes with three small β -sheets, a β -domain that contains the larger β -sheets with three small α -helixes by homology modeling. The substrate binding site for dihydrokaempferol on PhF3’ 5’ H protein was determined by molecular docking algorithm. A highly conserved HPPTPLSLPH sequence was predicted to contact the aromatic ring of dihydrokaempferol.

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