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The WRKY family genes, which play an important role in plant morphogenesis and stress response, were selected based on the data of the full-length transcriptome of Asarum heterotropoides. Using AtWRKY33, which regulates the synthesis of the camalexin in the model plant Arabidopsis to compare homologous genes in A. heterotropoides, primers were designed to amplify the open reading frame(ORF) fragment of AhWRKY33 gene by RT-PCR using total RNA of A. heterotropoides leaves as template. Real-time PCR results showed that there was a significant difference between the aerial part and the underground part of A. heterotropoides, the toxic aristolochic acid content is highly expressed in the leaves higher than the root. After verification, the WRKY33 gene of A. heterotropoides is ORF long 1 686 bp, encoding 561 amino acids.AhWRKY33 had two conserved WRKYGQK domains. According to the classical classification, it belongs to group Ⅰ WRKY transcription factor. A. heterotropoides WRKY33 had some homology with amino acids of other species. The study successfully constructed the plant eukaryotic expression vector PHG-AhWRKY33 and transformed Arabidopsis thaliana, the transgenic Arabidopsis was obtained by PCR detection and hygromycin resistant plate screening. It found that the germination of transgenic Arabidopsis seeds was accelerated and the stress resistance was increased. It laid a foundation for further analysis of WRKY transcription factor in the growth and development of A. heterotropoides and the synthesis of secondary metabolites.
Assuntos
Arabidopsis , Genética , Proteínas de Arabidopsis , Genética , Asarum , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Folhas de Planta , Proteínas de Plantas , Genética , Fatores de Transcrição , Transformação GenéticaRESUMO
Chlorophyll content,leaf mass to per area,net photosynthetic rate and bioactive ingredients of Asarum heterotropoides var. mandshuricum,a skiophyte grown in four levels of solar irradiance were measured and analyzed in order to investigate the response of photosynthetic capability to light irradiance and other environmental factors. It suggested that the leaf mass to per area of plant was greatest value of four kinds of light irradiance and decreasing intensity of solar irradiance resulted in the decrease of leaf mass to per area at every phenological stage. At expanding leaf stage,the rate of Chla and Chlb was 3. 11 when A. heterotropoides var. mandshuricum grew in full light irradiance which is similar to the rate of heliophytes,however,the rate of Chla and Chlb was below to 3. 0 when they grew in shading environment. The content of Chla,Chlb and Chl( a+b) was the greatest value of four kinds of light irradiance and decreasing intensity of solar irradiance resulted in its decreasing remarkably( P<0. 05). The rate of Chla and Chlb decreased but the content of Chla,Chlb and Chl( a+b) increased gradually with continued shading. The maximum value of photosynthetically active radiation appeared at 10: 00-12: 00 am in a day. The maximum value of net photosynthetic rate appeared at 8: 30-9: 00 am and the minimum value appeared at 14: 00-14: 30 pm at each phenological stage if plants grew in full sunlight. However,when plants grew in shading,the maximum value of net photosynthetic rate appeared at about 10: 30 am and the minimum value appeared at 12: 20-12: 50 pm at each phenological stage. At expanding leaf stage and flowering stage,the average of net photosynthetic rate of leaves in full sunlight was remarkably higher than those in shading and it decreased greatly with decreasing of irradiance gradually( P < 0. 05). However,at fruiting stage,the average of net photosynthetic rate of leaves in full sunlight was lower than those in 50% and 28% full sunlight but higher than those in 12% full sunlight. All photosynthetic diurnal variation parameters of plants measured in four kinds of different irradiance at three stages were used in correlation analysis. The results suggested that no significant correlation was observed between net photosynthetic rate and photosynthetically active radiation,and significant negative correlation was observed between net photosynthetic rate and environmental temperature as well as vapor pressure deficit expect for 12% full sunlight. Positive correlation was observed between net photosynthestic rate and relative humidity expect for 12% full sunlight. Significant positive correlation was observed between net photosynthetic rate and stomatal conductance in the four light treatments. Only,in 12% full sunlight,the net photosynthetic rate was significantly related to photosynthetically active radiation rather than related to environmental temperature,vapor pressure deficit and relative humidity. In each light treatment,a significant positive correlation was observed between environmental temperature and vapor pressure deficit,relative humidity as well as stomatal conductance. Volatile oil content was 1. 46%,2. 16%,1. 56%,1. 30% respectively. ethanol extracts was 23. 44%,22. 45%,22. 18%,21. 12% respectively. Asarinin content was 0. 281%,0. 291%,0. 279% and 0. 252% respectively. The characteristic components of Asarum volatile oil of plant in different light treatments did not change significantly among different groups.
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Asarum , Fisiologia , Efeitos da Radiação , Clorofila , Fotossíntese , Folhas de Planta , Efeitos da Radiação , Luz SolarRESUMO
Objective: To analyze the change rule of main chemical components in Asarum heterotropoidesvar.mandshuricum seedling during the growing process.Method:Whole seedling samples (one week and two weeks) and the mature plant (three months) of A.heterotropoidesvar.mandshuricum were collected and every sample was divided to aerial part (stems and leaves) and underground part (roots).The secondary metabolites were qualitatively identified by HPLC-TOF-MS and the quantitative identification was carried out at the same time with asarinin as index component.Result: A total of 6 known compounds were identified from the underground part of A.heterotropoidesvar.mandshuricum as α-asarone (1),N-isobutyl-2,4,8,10-dodecatetraenamide (2),9-methoxyaristolactam Ⅳ(3),asarinin (4),caulesnarinside (6) and chalcononaringenin 2',4'-di-O-β-D-glucopyranoside (7),respectively,the peak area values showed that the contents of these compounds increased gradually with the growth time.A total of 4 known compounds were identified from the aerial part of this herb as N-isobutyl-2,4,8,10-dodecatetraenamide (2),caulesauroneside (5),caulesnarinside (6) or chalcononaringenin 2',4'-di-O-β-D-glucopyranoside (7) and peonidin 3-caffeoylgentiobioside (8).Asarinin was identified only in the underground part of mature plant,its content was 155.4 μg·g-1.Conclusion: The species and contents of secondary metabolites are quite different in the aerial and underground parts of A.heterotropoidesvar.mandshuricum.At different growth stages of A.heterotropoides var.mandshuricum seedling,the types and contents of secondary metabolites in the same site are also different,while the contents of main components show an increasing trend with the growth time.
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Objective: To clone the enzyme genes related with methyl eugenol synthesis and characterize the corresponding sequence information based on the transcriptome sequencing of Asarum heterotropoides. Methods: RT-PCR was performed to obtain the full length cDNA of the phenylalanine lyase (PAL) gene, cinnamic acid-4-hydroxylase (C4H) gene, 4-hydroxycinnamoyl-CoA ligase (4CL), and cinnamyl alcohol dehydrogenase (CAD) genes by using young leaves as materials. The acquired enzyme genes were analyzed by bioinformatics. Results: The ORF lengths of AhPAL, AhC4H, Ah4CL, and AhCAD were 2 157, 1 278, 1 623 and 1 071 bp, which respectively encoded 718, 425, 540, and 356 amino acids. Four proteins had respective conserved domains. The amino acid sequences of AhPAL, Ah4CL, and AhCAD were similar to those of other reported species except for AhC4H. Conclusion: Four enzyme genes related with methyl-eugenol synthesis in A. heterotropoides were separated and analyzed using bioinformatics method. These results would lay the important foundation for functional analysis of corresponding genes and for elucidating the regulation mechanism of methyl-eugenol biosynthesis.
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Objective: To screen the optimum conditions of proliferation, regenerated bud differentiation, and adventitious roots induction of Asarum heterotropoides petiole-derived callus so as to establish the effective system for callus proliferation and regeneration. Methods: Petiole-derived calli were subcultured in the proliferation medium supplemented with different concentration of 6-BA and NAA. Then green and compact calli were chosen to culture in differentiation medium with different exogenous hormones for the induction of regeneration bud and adventitious roots. After being acclimated, regenerated plants were transplanted. Results: The effective callus proliferation medium was 1/2 MS supplemented with 0.40 mg/L 6-BA and 0.10 mg/L NAA, then 1/2 MS medium supplemented with 0.4 mg/L 6-BA and 0.05 mg/L NAA was suitable for regenerated bud differentiation while regenerated buds grew better in 1/2 MS medium without any hormone. The medium of 1/4 MS supplemented with 0.25 mg/L IBA was best for the induction of adventitious roots. Conclusion: The optimum culture conditions of callus proliferation and differentiation in A. heterotropoides are determined to establish the regeneration system and obtain the regenerated plantlet successfully.
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Objective: To optimize the proper condition for callus induction of Asarum heterotropoides var. mandshuricum. Methods: The effects of different explants, medium types, and plant growth regulators on the callus induction of A. heterotropoides var. mandshuricum were studied by using the petioles and leaves as explants. Also we aimed to discover the morphology characteristics of callus in different periods through paraffin method. Results: The efficient callus induction medium was 1/2 MS + 0.6 mg/L 6-BA + 0.15 mg/L NAA while using petioles and leaves during young period as explants. Callus induction from young leaves lasted so long time. Furthermore it was found that the induced calli were embryogenic for they were composed of compact, small and irregular arrangement cells. Subsequently these calli could be differentiated to develop the regeneration plantlet. Conclusion: Young petiole is the optimal explant for callus induction of A. heterotropoides var. mandshuricum, and the efficient callus induction condition we optimized would provide a basis for establishing the subsequent regeneration system.
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Objective To approach the toxicity of Danggui Sini plus Wuzhuyu Shengjiang Tang and Asarum heterotropoides Fr.var. mandshuricum Kitag. Method To observe the Ca2+, Na+-K+-ATPase from the mice renalis tissue in order to prove the medicine curative effect. Result The Danggui Sini plus Wuzhuyu Shengjiang Tang and Asarum heterotropoides Fr.var. mandshuricum Kitag. can obviously increase the Ca2+ and decrease the Na+-K+-ATPase of the mice. Conclusion The Danggui Sini plus Wuzhuyu Shengjiang Tang and Asarum heterotropoides Fr.var. mandshuricum Kitag. has very obviously toxicity to mice by influence the Ca2+ and Na+-K+-ATPase of the tissue homogenate.