| Research of this paper is focus on photosystem II subunits of Ginkgo biloba L., a primitive seed plant and the sole living member of the gymnosperm division Ginkgophyta. Total proteins and RNA were isolated from leaves during early phases of seedling growth, respectively; and psbA gene was cloned and sequenced. The whole work is concluded as follows.First, an improved protocol was developed to isolate total RNA in good yield and integrity from Ginkgo biloba leaves containing high levels of flavonoid glycosides, terpene lactones, carbohydrates and polyphenolic secondary metabolites. Polyvinylpolypyrrolidone at 2% and 4% β-mercaptoethanol were added to the standard CTAB extraction buffer and, after chloroform and phenol extraction, the pellet obtained by ethanol/acetate precipitation was washed and a second phenol/chloroform extraction was introduced to remove co-precipitated polysaccharides. Both A260/A230 and A260/A280 absorbancy ratios of isolated RNA were around 2 and the yield was about 0.4 mg/g fresh weight. At least seven distinct rRNA bands were detected by denaturing gel electrophoresis. Sharp hybridization signals were obtained from Northern blots with both nuclear and plastid gene probes. Two gene fragments: nuclear-encoded cab and chloroplast encoded rbcL were successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. The total RNA isolated by this protocol is of sufficient quality for subsequent molecularapplications.Second, changes in chlorophyll, protein content of photosystem II subunits and mRNA levels of genes encoding D1 and LHCII were investigated in the early phases of Ginkgo biloba seedling growth. During the early development, total chlorophyll levels increased quickly as the young leaves expanded while chlorophyll a/b ratio slightly decreased. As the results of western blot indicated, the accumulation of chlorophyll binding protein LHCII and CP43 increased as Ginkgo seedling developed, however, no significant changes of other Chlorophyll proteins, including D1, D2 and CP47, were detected. The mRNA levels of plastid gene psbA encoding PSII reaction center protein D1 and nuclear gene cab encoding chlorophyll a/b binding protein LHCII were analyzed in the same time. Observed on the northern blot results, psbA mRNA expression in 10d seedlings was about 1-fold higher than that of 5d seedlings, whereas nuclear encoded cab mRNA levels were approximately equivalent in those two samples. The results indicate that the structural and functional status of chloroplast during ginkgo seedling development may influence the expression of photosystem II subunits.Third, a cDNA fragment of psbA gene was cloned from Ginkgo biloba. Although psbA genes which encode photochemical reaction center core protein Dl have been cloned and sequenced in many other plants, there is no nucleotide information of the one from Ginkgo biloba in GenBank database. The new cloned cDNA was 1020 bp and had a high homology to other plant psbA genes. Analysis by BLAST showed that they were all from the ORF region and the translated polypeptide had 340 amino acid residues, including complete N-terminal end of D1 and missing only 13 residues in proprotein C-terminal extension which may be processed to form mature protein. Molecular modeling of this polypeptide indicated that its three dimensional structure strongly resembled that of cyanobacteria Thermosynechococcus elongates, which was elucidated recently from 3.5 A X-ray cystallization results (Ferreira 2004).
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