RESUMEN
BACKGROUND: The phytohormone ethylene (ET) is a key signaling molecule for inducing the biosynthesis of shikonin and its derivatives, which are secondary metabolites in Lithospermum erythrorhizon. Although ETHYLENE INSENSITIVE3 (EIN3)/EIN3-like proteins (EILs) are crucial transcription factors in ET signal transduction pathway, the possible function of EIN3/EIL1 in shikonin biosynthesis remains unknown. In this study, by targeting LeEIL-1 (L. erythrorhizon EIN3-like protein gene 1) at the expression level, we revealed the positive regulatory effect of LeEIL-1 on shikonin formation. RESULTS: The mRNA level of LeEIL-1 was significantly up-regulated and down-regulated in the LeEIL-1-overexpressing hairy root lines and LeEIL-1-RNAi hairy root lines, respectively. Specifically, LeEIL-1 overexpression resulted in increased transcript levels of the downstream gene of ET signal transduction pathway (LeERF-1) and a subset of genes for shikonin formation, excretion and/or transportation (LePAL, LeC4H-2, Le4CL-1, HMGR, LePGT-1, LeDI-2, and LePS-2), which was consistent with the enhanced shikonin contents in the LeEIL-1-overexpressing hairy root lines. Conversely, LeEIL-1-RNAi dramatically repressed the expression of the above genes and significantly reduced shikonin production. CONCLUSIONS: The results revealed that LeEIL-1 is a positive regulator of the biosynthesis of shikonin and its derivatives in L. erythrorhizon hairy roots. Our findings gave new insights into the molecular regulatory mechanism of ET in shikonin biosynthesis. LeEIL-1 could be a crucial target gene for the genetic engineering of shikonin biosynthesis.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Lithospermum/genética , Naftoquinonas/metabolismo , Proteínas de Plantas/genética , Factores de Transcripción/genética , Lithospermum/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Interferencia de ARN , Factores de Transcripción/metabolismoRESUMEN
The phytohormone ethylene (ET) is a crucial signaling molecule that induces the biosynthesis of shikonin and its derivatives in Lithospermum erythrorhizon shoot cultures. However, the molecular mechanism and the positive regulators involved in this physiological process are largely unknown. In this study, the function of LeACS-1, a key gene encoding the 1-aminocyclopropane-1-carboxylic acid synthase for ET biosynthesis in L. erythrorhizon hairy roots, was characterized by using overexpression and RNA interference (RNAi) strategies. The results showed that overexpression of LeACS-1 significantly increased endogenous ET concentration and shikonin production, consistent with the up-regulated genes involved in ET biosynthesis and transduction, as well as the genes related to shikonin biosynthesis. Conversely, RNAi of LeACS-1 effectively decreased endogenous ET concentration and shikonin production and down-regulated the expression level of above genes. Correlation analysis showed a significant positive linear relationship between ET concentration and shikonin production. All these results suggest that LeACS-1 acts as a positive regulator of ethylene-induced shikonin biosynthesis in L. erythrorhizon hairy roots. Our work not only gives new insights into the understanding of the relationship between ET and shikonin biosynthesis, but also provides an efficient genetic engineering target gene for secondary metabolite production in non-model plant L. erythrorhizon.
Asunto(s)
Etilenos/farmacología , Regulación de la Expresión Génica de las Plantas/fisiología , Lithospermum/metabolismo , Liasas/metabolismo , Naftoquinonas/metabolismo , Raíces de Plantas/metabolismo , Clonación Molecular , Biología Computacional , ADN Complementario/genética , ADN Complementario/metabolismo , Liasas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Plantas Modificadas Genéticamente , Transducción de Señal/fisiologíaRESUMEN
Rpi-blb2, which is originally derived from Solanum bulbocastanum, is a broad-spectrum potato late blight resistance gene and belongs to the NBS-LRR family. Here, the LRR homologues of Rpi-blb2 were cloned with PCR method from 40 potato cultivars (including 20 resistant potato cultivars and 20 susceptible ones) and 7 wild potato populations. Then, the similarities of the sequences, polymorphic (segregating) sites, and nucleotide diversities were estimated by bioinformatic methods. The results showed that high nucleotide polymorphism and some hot-spot mutations existed in the LRR region of Rpi-blb2. The test of Ka/Ks ratio showed that the function of LRR was conserved because of the purifying selection, although different positions of the Rpi-blb2 LRR region were under different selection pressures. Moreover, the LRR region of Rpi-blb2 had no clear differentiation between the cultivated and wild potatoes.
Asunto(s)
Resistencia a la Enfermedad/genética , Evolución Molecular , Genes de Plantas , Enfermedades de las Plantas/genética , Polimorfismo Genético , Solanum tuberosum/genética , Secuencia de Aminoácidos , Datos de Secuencia MolecularRESUMEN
In the crystal structure of the title compound, C(21)H(25)NO(11), the hexopyranosyl ring adopts a chair conformation and the five substituents are in equatorial positions. An intra-molecular hydrogen bond between the amino group and a neighbouring carbonyl group is found. Two carbonyl groups are disordered and were refined using a split model.
