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1.Application of SmartRoot in Phenotype Profile Analysis of Armillaria gallica

Xue-yan ZHANG ; Qi-tiao HU ; Cheng-wu FANG ; Yuan YUAN

Chinese Journal of Experimental Traditional Medical Formulae 2020;26(19):23-28

Objective:To establish a research platform for obtaining accurate phenotypic spectrum data is a technical difficulty that needs to be resolved in the research of traditional Chinese medicine resources. For example，the traditional phenotypic characterization method of Armillaria rhizomorph is mostly in a form of descriptive text，which is subjective and empirical. There is an urgent need for an objective and accurate method to characterize the phenotype of honey fungus rhizomorph. Method:Based on the image processing software Image J and the root identification plug-in SmartRoot combined with the Synbiosis ProtoCol 3 image analyzer，the growth picture of Armillaria spp. was analyzed，and the length，growth rate，branching situation，and angle of nascent rhizomorph of Armillaria gallica were measured to establish a measurement system for the phenotypic analysis of Armillaria rhizomorph. Result:Based on the method developed in this paper，the growth length，growth rate，number of branches，angle of nascent rhizomorph，and other phenotypic changes can be analyzed in a real-time manner without affecting the growth of Armillaria gallica. Armillaria spp. grew fastest at 9-12 days after generation，and the angle between the nascent rhizomorph and the parent rhizomorph was nearly vertical. This method had a certain correlation with the dry weight of traditional Armillaria biomass phenotypic parameters，with a high value in practical application. Conclusion:This study has established an objective，accurate，fast and real-time phenotypic analysis and measurement system for Armillaria rhizomorph，which expands the scope of application of SmartRoot and can be used for phenotypic analysis of traditional Chinese medicine resources under controlled experimental conditions.

2.Cloning and sequence analysis of codeine-O-demethylase gene from Papaver somniferum and Papaver rhoeas

Dan-dan HAO ; Qi-tiao HU ; Yuan YUAN ; Yong-fu GONG ; Fang CHEN ; Yu-jie WEI ; Lu-qi HUANG

Acta Pharmaceutica Sinica 2019;54(7):1312-1316

Codeine-O-demethylase (CODM) is a key enzyme in the biosynthesis of codeine and morphine. In this study, CODM gene sequences were cloned from Papaver somniferum and Papaver rhoeas, and were compared with each other by sequence alignment and bioinformatics analysis. The results showed that there were three genotypes of CODM in Papaver somniferum and five genotypes of CODM in Papaver rhoeas. Bioinformatics analysis showed that all CODM proteins had no signal peptide sequence, and these proteins were predicted to be non-secretory proteins, belonging to the Pcbc supergene family. Although the amino acid sequences of CODM in poppies are the same, the expression levels of CODM in different poppy resources are significantly different. It is speculated that the variation of transcription level of CODM may be related to its non-coding region sequence, which lays a foundation for further research on the synthesis and regulation mechanism of alkaloids in poppies.

3.Selection of Reference Genes for microRNA Real-time PCR Under Salt Stress in Armillaria gallica

Qi-rui YU ; Zhong-yi HUA ; Jun-hui ZHOU ; Qi-tiao HU ; Yuan YUAN ; Quan YANG ; Lu-qi HUANG

Chinese Journal of Experimental Traditional Medical Formulae 2020;26(19):35-42

Objiective： In the process of microRNA expression analysis by quantitative Real-time polymerase chain reaction（Real-time PCR），the selection of miRNA plays an important role in data standardization. Method:In this paper，13 Armillaria gallica.Candidate miRNAs were selected for bioinformatics analysis of their precursors，and the PMRD was used to predict similar sequences of their precursors，and the RNAfold was used to predict the secondary structure of the candidate miRNAs and their similar sequences. Real-time PCR was used to detect miRNAs expression in two genotypes of Armillaria gallica（genotype A，genotype B） before and after salt stress，and geNorm，NormFinder and BestKeeper were used to analyze the stability of miRNAs expression. Result:Secondary structure prediction and characterization of 9 candidate miRNA precursors showed that the miRNA predicted belonged to the miR family with typical stem-loop structure and the mature miRNAs were at the 5' or 3' end of the miRNA precursors.geNorm analysis showed that genotype A Armillaria gallica could select Novel-4* and Novel-9 as reference gene，genotype B could select Novel-9 and Novel-16 as its reference gene.NormFinder analysis showed that Novel-9 was stable in both genotype A and B Armillaria gallica.BestKeeper analysis showed that Novel-12* was stable in genotype A Armillaria gallica and Novel-2* was stable in genotype B Armillaria gallica. Conclusion:miRNA Novel-9 is the best stable reference gene，which lays a foundation for further research on the regulation mechanism of miRNA in Armillaria gallica.

4.Molecular identification of Armillaria gallica by PCR-RFLP analysis.

Yu-Ting LIANG ; Chao JIANG ; Jun-Hui ZHOU ; Qi-Tiao HU ; Yuan YUAN

China Journal of Chinese Materia Medica 2019;44(17):3622-3626

Armillaria gallica is a symbiotic fungus in the cultivation process of Gastrodia elata and Polyporus.The rhizomorph of A. gallica invades the stalk of the G. elata or the Sclerotium of the Polyporus,and is digested and utilized by the latter,becoming their important source of nutrition. Different nature of A. gallica affects the growth of G. elata and Polyporus. The authors collected A. gallica from 13 commercially available regions and screened two A. gallica,A and B,at the genetic and metabolic levels,in order to distinguish between the two A. gallica market. We have established convenient and effective DNA molecular identification method.By comparing the sequence differences between the A. gallica type A and type B invertase genes,PCR-RFLP primers were designed based on differential fragment. Primer ZTM.F/ZTM.R can amplified A. gallica type A and B,producing a band of about 304 bp in length. The restriction endonuclease EcoR V could recognize the difference sequence of A and B types of A. gallica. The type B was digested to form two fragments,thereby specifically identifying the A. gallica as type B. The established methods of PCR-RFLP is an accurate identification method for A. gallica. Therefore,in the cultivation process of G. elata and Polyporus,suitable strains can be selected according to different needs of variety,growth stage and ecological environment,and the yield and quality can be improved according to local conditions.
Armillaria ; classification ; Gastrodia ; microbiology ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Polyporus

1.Application of SmartRoot in Phenotype Profile Analysis of Armillaria gallica

2.Cloning and sequence analysis of codeine-O-demethylase gene from Papaver somniferum and Papaver rhoeas

3.Selection of Reference Genes for microRNA Real-time PCR Under Salt Stress in Armillaria gallica

4.Molecular identification of Armillaria gallica by PCR-RFLP analysis.

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