RESUMO
Sweet sorghum (Sorghum bicolor (Linn.) Moench) has promise as a bioenergy feedstock in China and other countries for its use in the production of ethanol as the result of its high fermentable sugar accumulation in stems. To boost biofuel production and extend its range, we seek to improve its stress tolerance. Proline acts as an osmolyte that accumulates when plants are subjected to abiotic stress. P5CS (Δ1-pyrroline-5-carboxylate synthetase) is a key regulatory enzyme that plays a crucial role in proline biosynthesis. We isolated two closely related P5CS genes from sweet sorghum, designated SbP5CS1 (GenBank accession number: GQ377719) and SbP5CS2 (GenBank accession number: GQ377720), which are located on chromosome 3 and 9 and encode 729 and 716 amino acid polypeptides, respectively. The homology between the two sweet sorghum P5CS genes was 76%. Promoter analysis of the two P5CS genes revealed that both sequences not only contained the expected cis regulatory regions such as TATA and CAAT boxes, but also had many stress response elements. Expression analysis revealed that SbP5CS1 and SbP5CS2 transcripts were up-regulated after treatment of 10-day-old seedlings of sweet sorghum with drought, salt (250mM NaCl) and MeJA (10µM). The expression levels of the both SbP5CS genes were significantly increased after 3-day drought stress. Under high salt treatment, peak SbP5CS1 expression was detected at 4h and 8h for SbP5CS2 in roots, while the trends of expression were nearly identical in leaves. In contrast, under drought and high salt stress, the up-regulated expression of SbP5CS1 was higher than that of SbP5CS2. When the seedlings were exposed to MeJA, rapid transcript induction of SbP5CS1 was detected at 2h in leaves, and the SbP5CS2 expression level increase was detected at 4h post-treatment. SbP5CS1 and SbP5CS2 also show different temporal and spatial expression patterns. SbP5CS2 gene was ubiquitously expressed whereas SbP5CS1 was mainly expressed in mature vegetative and reproductive organs. Proline concentration increased after stress application and was correlated with SbP5CS expression. Our results suggest that the SbP5CS1 and SbP5CS2 are stress inducible genes but might play non-redundant roles in plant development. The two genes could have the potential to be used in improving stress tolerance of sweet sorghum and other bioenergy feedstocks.
Assuntos
Ornitina-Oxo-Ácido Transaminase/genética , Sorghum/genética , Estresse Fisiológico , Acetatos , Sequência de Aminoácidos , Biocombustíveis , Ciclopentanos , Secas , Expressão Gênica , Dados de Sequência Molecular , Ornitina-Oxo-Ácido Transaminase/metabolismo , Oxilipinas , Filogenia , Reguladores de Crescimento de Plantas , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Prolina/metabolismo , Regiões Promotoras Genéticas , Salinidade , Plântula/metabolismo , Análise de Sequência de DNA , Sorghum/enzimologia , Transcriptoma , Regulação para CimaRESUMO
OBJECTIVE: To screen the antisense oligodeoxynucleotides (ASODN) that inhibit cultured hepatoblastoma HepG2 cell proliferation, and evaluate the antiproliferative potency of modified ASODN. METHODS: ASODN sequences were selected based on the secondary structure of human CDK7 mRNA predicted with RNAStructure 3.71 software. The binding thermodynamics of CDK7 mRNA to ASODN was analyzed by OligoWalk program. The sequences with the strongest effect against cultured HepG2 cell proliferation in vitro were selected, and the fragments complementary to 1-5 bases upstream or downstream to the complementary region were structurally modified and screened. RESULTS: The partial phosphorothioate ASODN complementary to 284-303 region of human CDK7 mRNA was the most powerful inhibitor, and the antiproliferative activity reached 40.4+/-12.6%; in the second round of screening, the antiproliferative activity of the full phosphorothioate ASODN complementary to the 287-306 region of the mRNA on HepG2 cells was 68.3+/-2.6%, with IC50 of 51.9+/-8.6 nmol/L. CONCLUSION: Proliferation of HepG2 cells can be significantly inhibited by the screened ASODN, which might be used as a lead compound in the development of specific CDK7 inhibitors.
Assuntos
Quinases Ciclina-Dependentes/genética , Inativação Gênica , Hepatoblastoma/genética , Neoplasias Hepáticas/genética , Oligodesoxirribonucleotídeos Antissenso/genética , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Hepatoblastoma/patologia , Humanos , Neoplasias Hepáticas/patologia , Fosforilação , RNA Mensageiro/genética , Transcrição Gênica/fisiologia , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
OBJECTIVE: To investigate the relationship of the transportation characteristics of Nolaterex, a new anti-cancer drug, with its sensitivity in tumor cells. METHODS: The sensitivities of 3 tumor cell lines (C6, SRS-82 and LoVo) to nolaterex were determined by growth inhibition test. After Nolaterex exposure, the intracellular drug concentration was measured by high-performance liquid chromatography. RESULTS: C6 was the most sensitive among the 3 cell lines, and the IC50 values of SRS-82 and LoVo cells were 6.8-fold and 13.8-fold higher, respectively, than that of C6 cells. In all the 3 cell lines, linear relationship between intracellular and extracellular drug concentrations was noted. The intracellular steady-state level achieved in C6 was significantly higher than the levels in the other two cell lines. CONCLUSIONS: The results indicate that nolaterex quickly enters the cells, and different cell lines may have different nolaterex-transporting capacities, thus partially accounting for different states of sensitivity of the tumor cells to nolaterex.
