The wild resources of Paris polyphylla var. yunnanensis, a secondary endangered medicinal plant, are severely scarce. Introduction and cultivation can alleviate market demand. To screen phosphatolytic bacteria in the rhizosphere soil of P. polyphylla var. yunnanensis and provide data support for the development of high-efficiency microbial fertilizer, in this study, the dilution plate coating method was used to isolate and screen the phosphorus solubilizing bacteria with the ability of mineralizing organic phosphorus from the rhizosphere soil of wild and transplanted varieties of P. polyphylla var. yunnanensis in 10 different locations in Yunnan, Sichuan and Guizhou. After separation and purification, the phosphatolytic capacity was analyzed by qualitative and quantitative analysis. Combined with physiological and biochemical experiments, the strains were identified using 16 S rDNA sequencing analysis. Forty one strains were selected from the rhizosphere soil of P. polyphylla var. yunnanensis from 10 different habitats. Among them, 21 strains were obtained from the rhizosphere soil of the wild variety P. polyphylla var. yunnanensis and 20 strains were obtained from the rhizosphere soil of the transplanted variety. And significance analysis found that 41 organophosphate solubilizing strains had significant differences in their ability to solubilize phosphorus. The amount of phosphate solubilizing was 0.08-67.61 mg·L~(-1), the pH value was between 4.27 and 6.82. The phosphatolytic amount of strain Y3-5 was 67.61 mg·L~(-1), and the phosphorus increase amount was 57.57 mg·L~(-1). All 41 strains were identified as Gram-positive Bacillus. Combining physiological characteristic and phylogenetic trees, Bacillus mobilis Y3-5 was finally selected as the candidate rhizosphere phosphatolytic bacteria of P. polyphylla var. yunnanensis. The distribution of phosphorus solubilizing bacteria in the rhizosphere soil of P. polyphylla var. yunnanensis was different, and there were significant diffe-rences in phosphorus solubility. Organophosphate-dissolving strain Y3-5 is expected to be a candidate strain of P. polyphylla var. yunnanensis microbial fertilizer.
Bacillus ; Bacteria/genetics* ; China ; Liliaceae ; Phylogeny

The matK genes of 10 samples in Paris from Hunan, Yunnan and Jilin provinces were sequenced and compared. The phylogenetic tree was constructed based on the matK gene sequences and the ten pairs samples were divided into four groups. The results did not support the reality of four taxa named P. polyphylla var. pseudothibetica, P. polyphylla var. yunnanensis, P. polyphylla var. appendiculata and P. polyphylla var. chinenis. They are supposed to be treated as different forms of P. polyphylla var. polyphylla.
Genes, Plant ; genetics ; Liliaceae ; classification ; genetics ; Molecular Sequence Data ; Phylogeny

OBJECTIVETo study the morphological variations of Paris polyphylla var. yunnansensis in different population for genetic diversity and breeding.

METHODThe characters of roots, stalks, leave and flowers were observed. The results were analyzed by DPS software.

RESULT AND CONCLUSIONP. polyphylla var. yunnansensis showed plenty genetic diversity, there existed obvious differences in morphological characters of different population. Principal components analysis showed that the number of calyces, petal, carpels, stamens is main factor,which causes the morphological variations in different population. Cluster analysis shows that 26 populations are incorporates in two types as 45.08 Euclidean distance. Leaf area index is distinct different in this two types.

China ; Genetic Variation ; Liliaceae ; anatomy & histology ; classification ; genetics ; growth & development

OBJECTIVEStudies on DNA fingerprinting of eight species of Paris and application of restriction site amplification polymorphism (RSAP) to the identification of Paris.

METHODSequence-related amplified polymorphism (SRAP) molecular markers were used to detect the genetic diversity of 7 accessions of Paris collected from Tianquan and Baoxing in Sichuan, and one from Lijiang in Yunnan.

RESULTThe DNA fingerprinting of 8 species were generated by 18 primer combination screened from 45 primer combinations. Eight accessions were clustered into 4 groups by genetic distance.

CONCLUSIONBased on molecular biology methods of RSAP analysis, accurate molecular identification could be performed on traditional Chinese medicinal material plants in Paris, and provided molecular evidence for taxonomy and identification of different species in Paris.

DNA Fingerprinting ; Genetic Markers ; Genetic Variation ; Liliaceae ; genetics ; Polymorphism, Genetic

Phylogeography is a research hotspot in the field of the genetic diversity and core germplasm construction of endangered rare plants. Paris polyphylla var. yunnanensis is a rare plant species mainly distributed in China. Wild individuals have been overexploited for the last few decades because of increasing demand for such medicines. Therefore, it is great significance to study the phylogeography of P. poliphylla var. yunnanensis based on chloroplast gene trnL-trnF sequences. In this study, chloroplast genes trnL-trnF were used in the phylogeography analysis of 15 wild and 17 cultivated populations of P. polyphylla var. yunnanensis. This study revealed that based on the results of neutrality tests and mismatch analysis, the rapid expansion of wild population has not been detected in P. polyphylla var. yunnanensis. After aligning and sorting the obtained cpDNA sequences, a total of 15 haplotypes were detected in all 32 populations. One haplotype was unique to the wild population, and 5 haplotypes were unique to the cultivated population. It can be seen that the haplotype richness of cultivated population was higher than that of wild population. The wild populations of P. polyphylla var. yunnanensis were divided into two groups according to evolutionary relationship of haplotypes and distribution map of haplotypes. The haplotype of branch Ⅰ was mainly distributed in Guizhou, and the haplotype of branch Ⅱ was located in Yunnan and Huidong, Sichuan. Therefore, it's speculated that Guizhou and the west Yunnan region may be glacial refuge in the evolutionary history of wild populations of P. polyphylla var. yunnanensis, and in order to protect the wild resources more effectively, wild populations of P. polyphylla var. yunnanensis in these two areas should be included in the protection zone.
China ; Genes, Chloroplast ; Humans ; Liliaceae/genetics* ; Melanthiaceae ; Phylogeography

OBJECTIVETo clone the cDNA sequence of squalene synthase gene from Paris polyphylla, and characterize the biological features of the obtained SQS.

METHODUsing homology cloning and RACE technique, a full-length cDNA sequence of PpSQS gene was isolated from P. polyphylla. The obtained sequence was analyzed by bioinformatics softwares. A plasmid [named pET-30b (+)-PpSQS] was constructed for prokaryotic expression the recombinant PpSQS.

RESULTThe full-length cDNA of PpSQS gene is 1 498 bp, which contains a 1 212 bp ORF. Sequence analysis indicated that PpSQS encoded 403 amino acids residues with a calculated molecular weight (MW) of 46.36 kDa and an isoelectric point (pI) of 6.83. SDS-PAGE results showed that the recombinant PpSQS was expressed in Escherichia coli BL21 (DE3) by inducing with 1 mmol x L(-1) IPTG.

CONCLUSIONThe full-length cDNA sequence of PpSQS gene was obtained from P. polyphylla, and its molecular features were consisted with classic SQS in plant. The recombinant PpSQS was successfully expressed in E. coli.

Cloning, Molecular ; Escherichia coli ; genetics ; Farnesyl-Diphosphate Farnesyltransferase ; genetics ; Liliaceae ; enzymology ; Phylogeny ; Recombinant Proteins ; biosynthesis

In this study, the authors cloned a glycosyltransferase gene PpUGT2 from Paris polyphylla var. yunnanensis with the ORF length of 1 773 bp and encoding 590 amino acids. The phylogenetic tree revealed that PpUGT2 belonged to the UGT80A subfamily and was named as UGT80A49 by the UDP-glycosyltransferase(UGT) Nomenclature Committee. The expression vector pET28a-PpUGT2 was constructed, and enzyme catalytic reaction in vitro was conducted via inducing protein expression and extraction. With UDP-glucose as sugar donor and diosgenin and pennogenin as substrates, the protein was found with the ability to catalyze the C-3 hydroxyl β-glycosylation of diosgenin and pennogenin. To further explore its catalytic characteristic, 15 substrates including steroids and triterpenes were selected and PpUGT2 showed its activity towards the C-17 position of sterol testosterone with UDP-glucose as sugar donor. Homology modelling and molecule docking of PpUGT2 with substrates predicted the key residues interacting with ligands. The re-levant residues of PpUGT2-ligand binding model were scanned to calculate the corresponding mutants, and the optimized mutants were obtained according to the changes in binding affinity of the ligand with protein and the surrounding residues within 5.0 Å of ligands, which had reference value for design of the mutants. This study laid a foundation for further exploring the biosynthetic pathway of polyphyllin as well as the structure of sterol glycosyltransferases.
Ligands ; Glycosyltransferases/genetics* ; Sterols ; Phylogeny ; Ascomycota ; Liliaceae/chemistry* ; Melanthiaceae ; Diosgenin ; Sugars ; Glucose ; Uridine Diphosphate

The monocot mannose-binding lectin can inhibit HIV from infecting the target cells. The total RNA of Zephyranthes grandiflora was extracted and reversely transcribed into cDNA. Degenerate primers were designed based on the conserved regions of other monocot mannose-binding agglutinins by homology alignment. The 694bp full-length cDNA of Zephyranthes grandiflora agglutinin (ZGA) was cloned by RT-PCR, 3' and 5' RACE (rapid amplification of cDNA ends). The start codon and stop codon of ZGA were at 37-39bp and 529-531bp respectively. The NCBI Blast analysis result showed that ZGA gene encoded a protein precursor with signal peptide, mature protein and C-terminal cleavage sequence. The mature ZGA protein contained 106 amino acids residues and its molecular weight was 11.6KD. The percentages of identity of the deduced mature ZGA protein with those of Galanthus nivalis agglutinin, Narcissus hybrid cultivar agglutinin, Lycoris radiate agglutinin and Clivia miniata agglutinin were 71.8%, 81%, 81.8% and 84.5%, respectively. Blocks analysis revealed that ZGA had three functional domains and three mannose-binding boxes (QDNY).
Agglutinins ; genetics ; Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Liliaceae ; genetics ; Mannose-Binding Lectin ; genetics ; Molecular Sequence Data ; Sequence Analysis, DNA

The paper is aimed at studying the diversity of endophytic fungi community from Paris polyphylla var. yunnanensis, and to provide a scientific basis for the utilization value of the endophytic fungi as bioactive material resources. In the present study, endophytic fungi were isolated from roots, rhizomes and leaves of wild P. polyphylla var. yunnanensis collected from Baoshan, Heqing county and Songming city of Yunnan province, and identified and classified by morphological methods together with its ITS sequence analysis. Seven and forty-nine strains of endophytic fungi were isolated from P. polyphylla var. yunnanensis. They were identified belonging to 41 genus. In these 41 genus, 3 genus exist in root only, 12 genus only exist in rhizome and 8 genus only exist in leaf. There was difference in endophytic fungi isolated from different sample sites. Endophytic fungi diversity from rhizomes of Heqing site was the highest. Endophytic fungi similarity coefficient was low among different sites and tissues. Based on these results, it is reasonable to propose that endophytic fungi of P. polyphylla var. yannanensis from different tissue and different sample sites has a certain difference which is possibly relate to their different habitats, different structure and composition of each tissue.
Biodiversity ; Endophytes ; classification ; genetics ; isolation & purification ; Fungi ; classification ; genetics ; isolation & purification ; Liliaceae ; microbiology ; Molecular Sequence Data ; Phylogeny ; Plant Leaves ; microbiology ; Plant Roots ; microbiology ; Plant Stems ; microbiology

In this paper, the varying pattern of the amount of rhizospheric microorganisms, including bacteria, actinomycetes and fungus, was observed during the cultivation of Paris polyphylla var. yunnanensis. And the correlations between number of rhizospheric microorganisms and the quality of P. polyphylla var. yunnanensis were also studied. The results showed that the rhizospheric microorganism source of P. polyphylla var. yunnanensis was rich. The distribution of rhizospheric microorganisms (soil bacteria, fungus, actinomycetes, potassium-solubilizing bacteria, inorganic phosphorus-solubilizing bacteria, organic phosphorus-solubilizing bacteria) collected from different origin places existed significant difference (P < 0.05). The varying pattern for the amount of rhizospheric microorganisms was showed as following: the amount of bacteria > the amount of actinomycetes > the amount of fungus. The medicinal quality of P. polyphylla var. yunnanensis was influenced by their habits, and the increase of cultivation years caused the obvious decrease of the quality of P. polyphylla var. yunnanensis. Therefore, the increase of cultivation years will cause the variation of the soil micro-ecology flora, and decrease the nutrient absorption and the utilization of P. polyphylla var. yunnanensis, which will make the decrease of the medical quality of P. polyphylla var. yunnanensis.
Bacteria ; genetics ; growth & development ; isolation & purification ; Biodiversity ; China ; Fungi ; genetics ; growth & development ; isolation & purification ; Liliaceae ; chemistry ; microbiology ; Plant Extracts ; analysis ; Rhizome ; chemistry ; microbiology ; Rhizosphere ; Saponins ; analysis ; Soil Microbiology