Leaf senescence is considered as one of important factors to decrease ornamental values of foliage plants. In the attempt to study and understand the molecular mechanism of leaf senescence, a senescent leaf cDNA library of Coleus blumei was constructed and a small EST library was obtained. According to the sequence of an EST fragment with a cystathionine beta synthase (CBS) domain, a novel leaf senescenceassociated gene (SAG) full-length cDNA encoding a CBS-domain-containing protein, denoted Cbcbs, was rapidly cloned using a strategy of RACE combined with cDNA library. The full length of the Cbcbs gene was 859 bp long (accession No. EF076754) and contained a 609 bp open reading frame (ORF) encoding a 202amino acid protein. One stop codon (TAA) was found in 5' UTR and one possible polyadenylation signal,AATAAA, and a pentanucleotide motif, ATTTA, were found in 3' UTR. The CbCBS contained a predicted mitochondrial targeting peptide in the N-terminal region, two conserved and intact CBS domains, four casein kinase Ⅱ (CK Ⅱ) phosphorylation sites, three protein kinase c (PKC) phosphorylation sites and one tyrosine sulfation (TS) site. Sequence comparisons and phylogenetic analysis showed that CbCBS was a novel senescence or stress-associated protein. The prediction analysis of secondary structure and three dimensional structure of CbCBS suggested that the chief function of the protein was decided by the CBS domain pair. The expression pattern of Cbcbs in leaves was analyzed by RT-PCR. It was demonstrated that Cbcbs gene was a senescence-associated gene (SAG) and expressed in all leaf stages, young stage (Y) being the lowest and terminal senescence stage (S3) being the highest, and was upregulated along with the leaf senescence.Function analysis showed that the mature CbCBS maybe acts as a sensor of cellular energy status and directly or indirectly regulates cellular energy levels to increase ATP content in mitochondria during periods of metabolic stress of senescent leaves.

Using RACE with a Fagopyrum dibotrys callus cDNA library, one clone, named FdMYBP1, encoding a putative R2R3 MYB protein was identified. FdMYBP1 appeared to be a full-length cDNA of 1159 bp encoding a protein of 265 amino acids. Through structure and property analysis of FdMYBPI with bioinformational methods, it was found that the amino acid sequence of FdMYBP1 showed great homology to other MYBP with the R2R3 repeat region in the N-terminus. Southern blot analysis indicated that FdMYBP1 belongs to a single copy gene in F. dibotrys genomes. The FdMYBP1 gene has the same classic characters with other MYBP and probably involved in the pathway of flavonoid metabolisms.
Cloning, Molecular ; Fagopyrum ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Plant Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-myb ; genetics ; metabolism

OBJECTIVETo establish a method for isolation of the total RNA from Fagopyrum cymosum callus.

METHODThe improved method combining that of CTAB extraction with the LiCl precipitation was used to isolate the total RNA from the four F. cymosum callus. The quality of the RNA was detected by UV spectrophotometric analysis, 0.8% non-denaturing agarose gel electrophoresis and RNA reverse transcription.

RESULTThe bands of 28S and 18S could be seen clearly by agarose gel electrophoresis, and the value of A260/A280 was between 1.9 and 2.0. The cDNA which was reverse-transcribed by the total RNA showed a wide length rage of 500 bp-5 kb.

CONCLUSIONThe RNA extracted by this method meets the requirement of reverse transcription-polymerase chain reaction (RT-PCR), construction of cDNA libraries, et al. This improved method can be used to isolate the total RNA from F. cymosum callus with the advantage of simpleness, efficiency and low cost.

Fagopyrum ; genetics ; growth & development ; RNA, Plant ; analysis ; isolation & purification

Aristolochic acid (AA), extracted from Aristolochiaceae plants, plays an essential role in traditional herbal medicines and is used for different diseases. However, AA has been found to be nephrotoxic and is known to cause aristolochic acid nephropathy (AAN).AA-induced acute kidney injury (AKI) is a syndrome in AAN with a high morbidity that manifests mitochondrial damage as a key part of its pathological progression. Melatonin primarily serves as a mitochondria-targeted antioxidant. However, its mitochondrial protective role in AA-induced AKI is barely reported. In this study, mice were administrated 2.5 mg/kg AA to induce AKI. Melatonin reduced the increase in Upro and Scr and attenuated the necrosis and atrophy of renal proximal tubules in mice exposed to AA. Melatonin suppressed ROS generation, MDA levels and iNOS expression and increased SOD activities in vivo and in vitro. Intriguingly, the in vivo study revealed that melatonin decreased mitochondrial fragmentation in renal proximal tubular cells and increased ATP levels in kidney tissues in response to AA. In vitro, melatonin restored the mitochondrial membrane potential (MMP) in NRK-52E and HK-2 cells and led to an elevation in ATP levels. Confocal immunofluorescence data showed that puncta containing Mito-tracker and GFP-LC3A/B were reduced, thereby impeding the mitophagy of tubular epithelial cells. Furthermore, melatonin decreased LC3A/B-II expression and increased p62 expression. The apoptosis of tubular epithelial cells induced by AA was decreased. Therefore, our findings revealed that melatonin could prevent AA-induced AKI by attenuating mitochondrial damage, which may provide a potential therapeutic method for renal AA toxicity.