Extraction and bioactivity analysis of total protein of K326 tobacco leaves with high expression of Nicotiala alata defensin 1 gene / 中国生物制品学杂志

@#Objective To extract the total protein of K326 tobacco leaves with high expression of Nicotiana alata defensin 1(NaD1) gene and analyze its bioactivity.Methods Total proteins were extracted from Nicotiana alata flowers,wild type(WT) and K326 tobacco leaves(transgenic) with high expression of NaD1 gene,and determined for the concentrations by Bardford method,while for the antibacterial activity against fungi by filter paper method,and for the inhibition activity on cancer cells(HeLa cells) by CCK-8.Results The total protein concentrations of Nicotiana alata flowers,WT and transgenic K326 tobacco leaves were 11.25,10.33 and 10.14 mg/mL,respectively.The antibacterial activity of total protein from transgenic K326 tobacco leaves against Candida albicans was(85.68±3.08)%,which was 1.33 and 1.14 times that of total protein from WT K326 tobacco leaves and Nicotiana alata flowers,respectively(F=15 339,P <0.05);The antibacterial activity against Fusarium oxysporum was(148.48±2.47)%,which was 1.09 and 1.08 times that of total protein from WT K326tobacco leaves and Nicotiana alata flowers,respectively(F=4.927,P <0.05).The IC_(50) value of transgenic K326 tobacco leaf protein on HeLa cells was the smallest(6.11 mg/mL),and the inhibitory activity was 1.56 and 1.21 times that of total protein of WT K326 tobacco leaves and Nicotiana alata flowers,respectively(F=89.748,P <0.05).Conclusion The total protein of K326 tobacco with high expression of NaD1 gene has good antibacterial and anticancer bioactivities,which provides an experimental basis for producing antibacterial and anticancer biological agents with tobacco as bioreactor.

Interspecies Phylogenetic Analysis of / 生物医学与环境科学（英文）

Objective@#This study aims to investigate the infection of @*Method@#Infection of the definitive human host and intermediate fish host by @*Results@#In 2016-2020, the average population infection rate of Hunan was 1.38%, while in Tongdao County the rate was up to 26.90%, and the highest fish infection rate was detected in Qiyang County (99.44% in the dorsal fin of @*Conclusion@#The systematically study of

Identity of Spirometra theileri from a Leopard (Panthera pardus) and Spotted Hyena (Crocuta crocuta) in Tanzania

In the present study, a Spirometra species of Tanzania origin obtained from an African leopard (Panthera pardus) and spotted hyena (Crocuta crocuta) was identified based on molecular analysis of cytochrome c oxidase I (cox1) and NADH dehydrogenase subunit I (nad1) as well as by morphological observations of an adult tapeworm. One strobila and several segments of a Spirometra species were obtained from the intestine of an African male leopard (Panthera pardus) and spotted hyena (Crocuta crocuta) in the Maswa Game Reserve of Tanzania. The morphological characteristics of S. theileri observed comprised 3 uterine loops on one side and 4 on the other side of the mid-line, a uterine pore situated posterior to the vagina and alternating irregularly either to the right or left of the latter, and vesicular seminis that were much smaller than other Spirometra species. Sequence differences in the cox1 and nad1 genes between S. theileri (Tanzania origin) and S. erinaceieuropaei were 10.1% (cox1) and 12.0% (nad1), while those of S. decipiens and S. ranarum were 9.6%, 9.8% (cox1) and 13.0%, 12.6% (nad1), respectively. The morphological features of the Tanzania-origin Spirometra specimens coincided with those of S. theileri, and the molecular data was also consistent with that of S. theileri, thereby demonstrating the distribution of S. theileri in Tanzania. This places the leopard (Panthera pardus) and spotted hyena (Crocuta crocuta) as new definitive hosts of this spirometrid tapeworm.

The identification of Sect. Cruciata (Gentiana) species using mtDNA nad1/b-c and nad5/d-e fragments / 药学学报

italic>Gentiana section Cruciata (Gentianaceae) is a medicinally important section of herbs, including Chinese traditional medicine Gentianae Macrophyllae Radix and Tibetan herb Jieji. Here, we assess the taxonomic significance using mtDNA nad1/b-c and nad5/d-e sequence data. A total of 144 nad1/b-c and nad5/d-e sequences from 11 species within Gentianaceae were obtained, including 138 sequences from 10 species within Gentiana section Cruciata and 6 sequences from Halenia elliptica (outgroup). The results showed that mtDNA nad1/b-c has species- level resolution within the section of Cruciata, i.e. the variable in the position 45 “C” could be used as a stable marker locus to distinguish G. robusta from other taxa; the variable in the position 352 and 353 “GA” could distinguish G. crassicaulis and G. tibetica from other taxa within the section. Intraspecies genotype variability was detected in nad1/b-c sequences of G. officinalis and G. siphonantha, respectively. These genotypes could be used as potential DNA barcode. In addition, intraspecies genotype variability was detected in nad5/d-e sequences of G. macrophylla, G. officinalis and G. siphonantha, respectively. Based on the stable marker locus, a species-specific PCR protocol was developed using the primer PF to identifying G. robusta in the section. This study could expand the understanding of the diversity of mtDNA nad1/b-c and nad5/d-e in the genus Gentiana, and provide the essence for the species identification within Gentiana section Cruciata.

Molecular and Morphologic Identification of Spirometra ranarum Found in the Stool of African Lion, Panthera leo in the Serengeti Plain of Tanzania

The present study was performed with morphological and molecular analysis (cox1 and nad1 mitochondrial genes) to identify the proglottids of spirometrid tapeworm found in the stool of an African lion, Panthera leo, in the Serengeti plain of Tanzania. A strand of tapeworm strobila, about 75 cm in length, was obtained in the stool of a male African lion in the Serengeti National Park (34° 50′ E, 02° 30′ S), Tanzania, in February 2012. The morphological features of the adult worm examined exhibited 3 uterine coils with a bow tie appearance and adopted a diagonal direction in the second turn. The posterior uterine coils are larger than terminal uterine ball and the feature of uteri are swirling rather than spirally coiling. The sequence difference between the Spirometra species (Tanzania origin) and S. erinaceieuropaei (GenBank no. KJ599680) was 9.4% while those of S. decipiens (GenBank no. KJ599679) differed by 2.1% in the cox1 and nad1 genes. Phylogenetic tree topologies generated using the 2 analytic methods were identical and presented high level of confidence values for the 3 major branches of the 3 Spirometra species in the cox1 gene. The morphological and molecular findings obtained in this study were nearly coincided with those of S. ranarum. Therefore, we can know for the first time that the African lion, Panthera leo, is to the definitive host of this tapeworm.

Genetic and Morphologic Identification of Spirometra ranarum in Myanmar

In the present study, we identified a Spirometra species of Myanmar origin (plerocercoid) by molecular analysis using mitochondrial cox1 and nad1 genes, as well as by morphological observations of an adult tapeworm. Spargana specimens were collected from a paddy-field in Taik Kyi Township Tarkwa Village, Yangon, Myanmar in December 2017. A total of 5 spargana were obtained from 20 frogs Hoplobatrachus rugulosus; syn: Rana rugulosa (Wiegmann, 1834) or R. tigrina (Steindachner, 1867). The plerocercoids were used for experimental infection of a dog. After 4 weeks of infection, an adult tapeworm was recovered from the intestine of the dog. Morphologically, the distinct features of Spirometra sp. (Myanmar origin) relative to S. erinaceieuropaei and S. decipiens include a uterine morphology comprising posterior uterine coils that larger than the terminal uterine ball and coiling of the uteri diagonally (swirling) rather than spirally. The cox1 sequences (1,566 bp) of the Myanmar-origin Spirometra species showed 97.9% similarity to a reference sequence of S. decipiens (GenBank no. KJ599679) and 90.5% similarity to a reference sequence of S. erinaceieuropaei (GenBank no. KJ599680). Phylogenetic tree topologies were identical and presented high confidence level of values for the 3 major branches of the 3 Spirometra species in cox1 and nad1 genes. These results indicated that Myanmar-origin Spirometra species coincided with those of S. ranarum and may be considered as a valid species.

In silico cloning and comparative analysis of NAD1 gene in three common human parasites / 中国血吸虫病防治杂志

Objective To in silico clone the NAD1 gene of three common parasites and analyze their bioinformatics,so as to lay the foundation for further research on the NAD gene. Methods By using the in silico cloning method,the full length cDNA (s)of NAD 1 genes of Clonorchis sinensis,Ascaris lumbricoides and Schistosoma japonicum were got,then their physical and chemical properties,compositions of amino acids,subcellular localizations,binary and ternary structures were contrastively an-alyzed.Results The three kinds of NAD1 proteins were similar in the relative molecular weight,subcellular localization,and physical and chemical properties.The NAD1 proteins were highly similar in binary and ternary structures of A.lumbricoides and S.japonicum.The phylogenetic analysis showed that C.sinensis,A.lumbricoides and S.japonicum belonged to the different evolu-tionary branches with a certain of genetic distance. Conclusion The three NAD1 genes got from C.sinensis,A.lumbricoides and S.japonicum by in silico cloning belong to the same gene of different species,which can be widely used in the researches of heritable variation of parasites.

Genotyping and gene nucleotide polymorphism of epidemic strain of Echino-coccus granulosus metacestode from humans and sheep in Tianjun region, Qinghai Province / 中国血吸虫病防治杂志

Objective To understand the genotypes and nucleotide polymorphisms of Echinococcus granulosus metacestode from humans and sheep in Tianjun region,Qinghai Province. Methods The specific primers were designed according to the cox1 and nad1 genes of E.granulosus mitochondrial genome sequences accessed by GenBank.The primers were used to detect the cyst samples from 16 sheep and 2 humans infected with E.granulosus in Tianjun region of Qinghai Province by PCR,then the PCR amplification products were sequenced,the genotypes and nucleotide polymorphisms of the cox1 and nad1 genes were analyzed.Results The 18 isolated samples all belonged to E.granulosus G1 genotype.Among all the isolates,9 haplotypes ex-isted in the cox1 gene with 16 nucleotide mutation sites,and there were 0 to 5 nucleotide differences with the highest variation rate of 0.31%,whereas 7 haplotypes occurred with 15 nucleotide mutation sites,and there were 1 to 8 nucleotide differences with the highest variation rate of 0.89% for the nad1 gene.Conclusions The epidemic genotype of E.granulosus is G1 in hu-mans and sheep in Tianjun region of Qinghai Province,and the nucleotide polymorphisms of the cox1 gene were more abundant than those of the nad1 gene,and the resolution of the nucleotide polymorphisms of cox1 gene is higher than that of the nad1 gene used in E.granulosus isolates.

Practical Algorisms for PCR-RFLP-Based Genotyping of Echinococcus granulosus Sensu Lato

Echinococcus granulosus sensu lato (s.l.) is a causative agent of cystic echinococcosis or cystic hydatid disease in humans and domestic and wild animals. The disease is a serious health problem in countries associated with poverty and poor hygiene practices, particularly in livestock raising. We introduced a practical algorism for genotyping the parasite, which may be useful to many developing countries. To evaluate the efficiency of the algorism, we genotyped 3 unknown strains isolated from human patients. We found that unknowns 1 and 3 were included in G1, G2, and G3 genotypes group and unknown 2 was included in G4 genotype (Echinococcus equinus) according to the algorisms. We confirmed these results by sequencing the 3 unknown isolates cox1 and nad1 PCR products. In conclusion, these new algorisms are very fast genotype identification tools that are suitable for evaluating E. granulosus s.l. isolated from livestock or livestock holders, particularly in developing countries.

Application of mitochondrial nad 1 intron 2 sequences to molecular identification of some species of Dendrobium Sw / 中草药

Objective Application of a new molecular marker to the identification of Dendrobium (Orchidaceae) species. Methods Complete sequences of the mitochondrial nad 1 intron 2 for nine species of Dendrobium Sw. were amplified and determined. Results Seventeen variable sites were found in the aligned 872 bp of nad 1 intron 2 sequences. Eight of the nine Dendrobium species except D. loddigesii could be identified by the nad 1 intron 2 sequences. Conclusion The mitochondrial nad 1 intron 2 sequences could be used as a new molecular marker for the identification of Dendrobium species.