Cloning and gene functional analysis study of dynamin-related protein GeDRP1E gene in Gastrodia elata / 药学学报

The gene GeDRP1E encoding dynamin-related protein 1E in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDRP1E gene was carried out by using ExPASy, ClustalW, MEGA, etc. Positive transgenic Arabidopsis plant and potato minituber were obtained with the genetic transformation system of Arabidopsis and potato. The plant height and seed setting rate of transgenic Arabidopsis, and agronomic characters, such as size, weight and starch content of potato minituber of transgenic potato were tested and analyzed. And GeDRP1E gene function was preliminarily investigated. The results showed that the open reading frame of GeDRP1E gene was 1 899 bp in length and 632 amino acids residues were encoded, with a relative molecular weight of 69.90 kDa and a molecular formula of C3079H4973N883O933S19. It was predicted that the theoretical isoelectric point was 7.27, the instability coefficient was 43.34, and the average hydrophilicity index was -0.259, which was indicative of an unstable hydrophilic protein. GeDRP1E has no transmembrane structure and signal peptide, and was localized in the cytoplasm. The phylogenetic tree showed that GeDRP1E was highly homologous with DRP1E proteins of other plant species, among which GeDRP1E had the highest homology with DcDRP1E (XP_020689662.1) in Dendrobium candidum, reaching 90.05%. GeDRP1E plant expression vector pCambia1300-35Spro-GeDRP1E was constructed by double digests, and Arabidopsis complementary mutant and potato overexpression strain of GeDRP1E gene were obtained by Agrobacterium-mediated gene transformation. Compared with the Arabidopsis AtDRP1E mutant, the height and seed setting rate of the GeDRP1E complementation mutant were rescued. The minituber of GeDRP1E overexpression potato had larger size, heavier weight and higher starch content, comparing to wild-type potato. It was preliminarily induced that GeDRP1E was involved in mitochondrial morphology regulation, which related to the growth and development of Arabidopsis plants and potato miniature. The research results laid a foundation for further elucidating the molecular mechanisms underlying the growth and development of G. elata tuber development.

Screening of an Armillaria gallica strain for Gastrodia elata cultivation / 中国中药杂志

This study aims to screen a strain from Armillaria for the cultivation of Gastrodia elata. Specifically, Armillaria strains were isolated from different producing areas of G. elata and identified. Based on the growth characteristics of the strains and the experiment on the cultivation of G. elata, an optimal A. gallica strain was screened out. The specific process is as follows. The fungus-gro-wing materials of G. elata were collected from four producing areas and the Armillaria strains were isolated(G,Y,S,H). The strains were then identified based on morphological observation and phylogeny analysis and the commonly used strains were determined. The sucrase genotypes of the strains were identified according to our previous research findings, and the growth characteristics of the strains, such as growth rate, diameter, dry weight, and polysaccharide content of the rhizomorphs, were measured. According to the biological characteristics and sucrase genotypes, two strains were selected for the cultivation of G. elata. The tuber yield and the content of gastrodin and p-hydroxybenzyl alcohol in the tuber of G. elata were measured to select the optimal strain. The results showed that the four strains were all A. gallica. The rhizomorphs of strains G and H of the same sucrase genotype had larger/higher length, growth rate, diameter, branch number, dry weight, and polysaccharide content than those of strains S and Y of the same sucrase genotype. The tuber yield and the total content of gastrodin and p-hydroxybenzyl alcohol in tuber of G. elata cultivated with strain H were 6.528 kg·m~(-2) and 0.566%, respectively, which were 4.58 and 1.30 folds those of G. elata cultivated with strain S. Strains H and S were screened out from four strains of A. gallica based on the growth characteristics and sucrase genotype. According to the tuber yield and content of total gastrodin and p-hydroxybenzyl alcohol in the tuber of G. elata, strain H was identified as the optimal one. The findings in this study are expected to lay a basis for cultivating G. elata with high yield and quality of tubers.

Cloning and gene function of dicarboxylate-tricarboxylate carrier protein in Gastrodia elata / 中国中药杂志

The gene GeDTC encoding the dicarboxylate-tricarboxylate carrier protein in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDTC gene was carried out by using ExPASY, ClustalW, MEGA, etc. Positive transgenic plants and potato minituber were obtained by virtue of the potato genetic transformation system. Agronomic characters, such as size, weight, organic acid content, and starch content, of potato minituber were tested and analyzed and GeDTC gene function was preliminarily investigated. The results showed that the open reading frame of GeDTC gene was 981 bp in length and 326 amino acid residues were encoded, with a relative molecular weight of 35.01 kDa. It was predicted that the theoretical isoelectric point of GeDTC protein was 9.83, the instability coefficient was 27.88, and the average index of hydrophilicity was 0.104, which was indicative of a stable hydrophilic protein. GeDTC protein had a transmembrane structure and no signal peptide and was located in the inner membrane of mitochondria. The phylogenetic tree showed that GeDTC was highly homologous with DTC proteins of other plant species, among which GeDTC had the highest homology with DcDTC(XP＿020675804.1) in Dendrobium candidum, reaching 85.89%. GeDTC overexpression vector pCambia1300-35Spro-GeDTC was constructed by double digests, and transgenic potato plants were obtained by Agrobacterium-mediated gene transformation. Compared with the wild-type plants, transgenic potato minituber harvested by transplanting had smaller size, lighter weight, lower organic acid content, and no significant difference in starch content. It is preliminarily induced that GeDTC is the efflux channel of tricarboxylate and related to the tuber development, which lays a foundation for further elucidating the molecular mechanism of G. elata tuber development.

Application of strigolactone analogs in storage of Gastrodia elata / 中国中药杂志

This study explored the preservation effect of strigolactone analogs on Gastrodia elata tubers and screened out the suitable preservation measures of G. elata to provide a safer and more effective method for its storage and preservation. Fresh G. elata tubers were treated with 7FGR24, 2,4-D isooctyl ester, and maleic hydrazide, respectively. The growth of flower buds, the activities of CAT, and MDA, and the content of gastrodin and p-hydroxybenzyl alcohol were measured to compare the effects of different compounds on the storage and preservation of G. elata. The effects of different storage temperatures on the preservation of 7FGR24 were compared and analyzed. The gibberellin signal transduction receptor gene GeGID1 was cloned, and the effect of 7FGR24 on the expression level of GeGID1 was analyzed by quantitative polymerase chain reaction(qPCR). The toxicity of the G. elata preservative 7FGR24 was analyzed by intragastric administration in mice to evaluate its safety. The results showed that compared with 2,4-D isooctyl ester and maleic hydrazide, 7FGR24 treatment had a significant inhibitory effect on the growth of G. elata flower buds, and the CAT enzyme activity of G. elata was the highest, indicating that its preservation effect was stronger. Different storage temperatures had different effects on the preservation of G. elata, and the preservation effect was the strongest at 5 ℃. The open reading frame(ORF) of GeGID1 gene was 936 bp in length, and its expression level was significantly down-regulated after 7FGR24 treatment, indicating that 7FGR24 may inhibit the growth of flower buds by inhibiting the gibberellin signal of G. elata, thereby exerting a fresh-keeping effect. Feeding preservative 7FGR24 had no significant effect on the behavior and physiology of mice, indicating that it had no obvious toxicity. This study explored the application of the strigolactone analog 7FGR24 in the storage and preservation of G. elata and preliminarily established a method for the storage and preservation of G. elata, laying a foundation for the molecular mechanism of 7FGR24 in the storage and preservation of G. elata.

A Soybean Straw Cultivation Medium for Armillaria gallica and Optimal Composition / 中国实验方剂学杂志

Objective:To explore the feasibility of replacing wood （or wood chips） with crop residues for culturing <italic>Armillaria gallica</italic> targeting the problems of forest resource destruction and increased cultivation cost caused by the extensive use of wood in <italic>Gastrodia elata</italic> cultivation， so as to reduce the cultivation cost of <italic>G. elata</italic>， promote the effective use of crop residues， and protect forest resources. Method:The growth situation of <italic>A. gallica</italic> in different media was observed， followed by the measurement of its growth rate using streaking method and the determination of total polysaccharide content of <italic>A. gallica</italic> by phenol-concentrated sulfuric acid colorimetric method. In order to further optimize the soybean straw cultivation medium， we carried out a four-factor three-level L₉（3⁴） orthogonal assay on the ratio of main ingredients， sucrose content， inorganic salt content， and water content. Result:The comparison of growing states of <italic>A. gallica</italic> cultured in different media revealed that <italic>A. gallica</italic> in soybean straw medium began to grow since the fourth day of inoculation， and the mycelium grew well， with the growth rate being 0.352 cm·d^-1， which was 1.48 times that in birch wood medium. The total polysaccharide content of <italic>A. gallica</italic> cultured in soybean straw medium was the highest， which was 39.260 mg·g^-1， much higher than 17.028 mg·g^-1 of that cultured in birch wood medium. This demonstrated the obvious advantage of soybean straw medium， whose main ingredients were soybean straw and wheat bran at the ratio of 8：2， with the sucrose and inorganic salt content accounting for 1% and 0.5% of the main ingredients， respectively. When the water content reached 50%， the growth rate of <italic>A. gallica</italic> was maintained at 0.392 cm·d^-1. Conclusion:This study has provided a basis for utilizing soybean straw instead of wood （or wood chips） as cultivation medium for <italic>A. gallica</italic>， thus better reducing the waste of forest resources and protecting the natural environment in the cultivation of <italic>G. elata</italic>.

Identification of four Armillaria strains and their effects on quality and yield of Gastrodia elata f. glauca / 中国中药杂志

In order to improve the quality and yield of Gastrodia elata f. glauca,determine the suitable Armillaria strains for the accompanying experiment in Xiaocaoba,Yiliang,four Armillaria strains were selected. They were used for G. elata cultivation,and the gene sequence,r DNA-ITS,β-tubulin and EF1-α of four Armillaria strains,were compared and analyzed. The yield was mesured in November which was based on previous laboratory research. The tubers were washed and steamed,then dried and powdered. The content of gastrodin and p-hydroxybenzyl alcohol was determined by UPLC,the polysaccharide was determined by phenol-concentrated sulfuric acid method. The results showed that the strains M1,M2,M3 and M4 were Armillaria gallica group but there were differences in the yield and active ingredient content when they were cultivated with the same G. elata. The yield of G. elata( Jian Ma) was the lowest when cultivated with Armillaria strain M3,but it was not the same when used M1,0. 981 kg·m-2,the highest yield in the four stains.The content of gastrodin was 0. 581%,the total content of gastrodin and p-hydroxybenzyl alcohol was 0. 595%,when accompanied with M1 strains. It was higher than other strains. The content of G. elata polysaccharide was 2. 132%,which was similar to the content of M3 strain,higher than that of M2 and M4 strain. Selecting phylogenesis of Armillaria strians,the content of active ingredient,and the yield as indicators,it was concluded concerned that the M1 strain was the best of four strains. The results will provide a theoretical basis and guidance for higher yield and quality in cultivation of G. elata in Yiliang.