Cloning and Sequence Analysis of rbcS Gene of Wild Barley (Hordeum brevisubulatum) under Salt Stress

[Objective] The aim was to study the cloning and sequence analysis of rbcS gene of wild barley under salt stress. [Method] The tender leaf blade of wild barley under salt stress was taken as the experimental material. The primers were designed according to the homology of rbcS gene sequences of wheat and barely in Genbank; then PCR amplification,recovery,ligation,transformation and sequencing of rbcS gene were carried out. [Result] Two rbcS genes including rbcS1 and rbcS2 with the length of 1 252 and 908 bp respectively were cloned from the barely genome. rbcS1 and rbcS2 were both composed by two exons and one intron. The exons length of the two genes was the same of 525 bp,encoding 174 amino acids,and the homology between them was 96%; however,the intron length of rbcS1 and rbcS2 was 448 and 107 bp respectively.

NUCLEOTIDE SEQUENCE OF RIBULOSE 1,5 BISPHOSPHATE CARBOXYLASE/OXYGENASE LARGE SUBUNIT GENE FROM MILLET(SETARIA ITALICA) *

The 3.0 kb BglⅡ+XbaⅠrestriction fragment of millet(Setaria italica) chloroplast genome containing rbcL gene had been cloned into pBluescript SK (-) vector, then the restriction map and the 1990 bp complete nucleotide sequence was determined. The 1431 bp coding region of the gene consists of 476 amino acid residues with a predicted molecular weight of 52679 D. The 389 bp 5′ upstream region has the putative -10 box, -35 box and SD sequence, similar to that of procaryotes. The 170 bp 3′ downstream region contains three stem loop structures. Comparison of the rbcL gene sequences between C 4 plants and several C 3 plants reveals no difference in the coding region, promoter and 3′ downstream region. It might be concluded that the rbcL gene sequence has no relation with its cell specific expression.

Characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase and transcriptional analysis of its related genes in Saccharina japonica(Laminariales,Phaeophyta)

Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments, and consequently may have highly efficient ribulose-1, 5-bisphosphate carboxylase/ oxygenase （Rubisco） activity for carbon assimilation. In our study, we cloned the full-length Rubisco gene from S.japonica （SJ-rbc）. It contained an open reading frame for a large subunit gene （SJ-rbcL） of 1 467 bp, a small subunit gene （SJ-rbcS） of 420 bp, and a SJ-rbcL/S intergenie spacer of 269 bp. The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa, 5.81 and 15,84 kDa, 4.71, respectively. After induction with 1 mmol/L isopropyl-β-D- thiogalactopyranoside for 5 h and purification by Ni2＋ affinity chromatography, electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein. Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations： increased expression during the day but suppressed expression at night. This observation implied that Rubisco played a role in normal gametophytic growth and development. In juvenile sporophytes, mRNA levels of SJ-rbcL, carbonic anhydrase, Calvin-Benson- Bassham cycle-related enzyme, and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance. Similarly, expression of these genes was up-regulated under blue light irradiance at 350 umol/（m2.s）. Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements.

Subcloning and Sequencing of the Form II Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Rhodopseudomonas palustris

Rhodopseudomonas palustris, one of purple nonsulfur photosynthetic bacteria, fixes carbon dioxide via Calvin-Benson cycle and has been shown previously to express form I and form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). The gene cbbM, which encodes the form II enzyme from Rhodopseudomonas palustris, has been subcloned and sequenced. The deduced amino acid sequence is highly with the form II RubisCO from photosynthetic bacteria, including Rhodospirillum rubrum (PDB ID: 9rub), but appears to be more distantly related to the large subunit of the form I RubisCO found in photosynthetic bacteria, chemoautotrophic bacteria and higher plants. Several regions highly conserved among L 8S 8 and L x enzymes correspond with regions previously implicated in catalytic activity and subunit interactions.