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Articles by Haiqing Yu
Total Records ( 7 ) for Haiqing Yu
  Haiqing Yu , Chunbang Ding , Chun Zhang and Yonghong Zhou
  In order to obtain more cytological data, the karyotypes of Pseudoroegneria gracillima and P. kosaninii were investigated. Root tips of P. gracillima and P. kosaninii were pretreated in an ice bath, fixed in a mixture of 95% ethanol: glacial acetic acid and treated in 1 M HCl at 60°C in a water bath. Somatic cells were stained in Schiff at room temperature and the meristematic portions of the root tips were squashed in 45% acetic acid. The results show that: (1) P. gracillima is diploid with two pairs of satellites and P. kosaninii is octoploid with three pairs of satellites. The karyotypes of diploid P. gracillima and octoploid P. kosaninii are first reported, (2) the karyotype formulas of P. gracillima and P. kosaninii are 2n = 2x = 14 = 12 m (2sat)+2sm (2sat) and 2n = 8x = 56 = 42 m (6sat)+12sm+2st, respectively and (3) the karyotype of P. gracillima is 1A type, while P. kosaninii is 2B type. This demonstrated that there are great variations between the karyotypes of P. gracillima and P. kosaninii.
  Haiqing Yu , Chun Zhang , Zhiqiang Mei , Li Wang , Juan Li , Chenggang Duan , Xiaoyan Liu , Shu Gong , Lin Gan , Zhonghua Tao , Fuli Yao , Ye Yang , Chunyan Duan , Youping Liu , Chunyan Zhang , Chuanning Chen , Xiaoli Zheng , Hongxian Zhao , Jiyan Cheng , Xiaojun Tang , Yong Zhang , Yingxi Zhang and Junjiang Fu
  As a traditional medicinal herb in China, Penthorum chinense Push from Gulin county is regarded as genuine regional drug with better clinical effects. To discriminate the geographical origin of six P. chinense samples cultivated in three provinces, Randomly Amplified Polymorphic DNA (RAPD) analysis was carried out with an improved method to increase the resolution and production using 10 mer-random primers. Similarity index was ranged from 0.61 to 0.97, which demonstrated that samples from different localities displayed similar band patterns. However, based on the analysis of selected 13 primers, primers SBS-I9, SBS-I20 and SBS-Q9 produced distinguishable bands among Sichuan, Yunnan and Hubei P. chinense. T-test of mean S.I. values of six accessions demonstrated the significant differences between Luzhou or Sichuan samples and others. Cluster analysis indicated that cultivars with close geographic distributions were clustered together consequently. This suggested that there are RAPD site variations and local specialized genotypes among the six samples. The results indicated that RAPD analysis is effective in distinguishing the geographical origin of genuine regional drug P. chinense grown in Luhzou, Sichuan. The approach is a valuable tool to authenticate other morphologically similar herbal medicinal materials.
  Hong Yu , Haiqing Yu and Hui Liu
  Viola philippica is a widely used herb in Chinese traditional medicine. However, V. mandshurica is found as one of common substitutes or adulterants of V. philippica in the commercial market. In order to authenticate the two herbal species, an improved randomly amplified polymorphic DNA (RAPD) and Sequence-Characterized Amplified Region (SCAR) were performed to obtain species-specific DNA fragments. Three 10-mer random primers SBS-A8, SBS-I10 and SBS-Q6 were used for screening of specific RAPD markers and the former two primers demonstrated different amplification band patterns. Two candidate specific bands showed in two taxa by SBS-A8 primer were successfully cloned and sequenced respectively. Based on the RAPD marker sequences, four pairs of SCAR primers from V. philippica (VPF1/VPR1 and VPF2/VPR2) and V. mandshurica (VMF1/VMR1 and VMF2/VMR2) were designed. VPF1/VPR1 and VPF2/VPR2 primers yielded expected 694 bp and 403 bp amplicons with the V. philippica DNA only. DNA amplification using VMF1/VMR1 primers generated a single 574 bp band only in V. mandshurica, while VMF2/VMR2 primers produced 261 bp bands in two species. The results suggested that RAPD and SCAR analysis are effective in distinguishing genuine V. philippica. The methodology is a valuable tool to authenticate other morphologically similar herbal medicinal materials.
  Huailin Xiong , Bo Luo , Yujie Zheng , Yuanyuan Cui , Yingqian Zhang , Qin Wu , Xi Zeng and Haiqing Yu
  One improved randomly amplified polymorphic DNA (RAPD) technique has been used in animal phylogenetic relationships and fingerprints analysis by prolonging the ramp time. However, little was known whether the PCR ramp time and G/C content of primers effect on RAPD analysis of medicinal Gastrodia elata Bl. plants. The present study was conducted with the objectives to extract genomic DNA of G. elata, execute RAPD analysis with different PCR ramp time and discuss the relationship between the amplification efficiency and the G/C content of RAPD primers subsequently. The Cetyltrimethylammonium Bromide (CTAB) protocol was used in the genomic DNA extraction. Ten RAPD primers were randomly selected in PCR amplifications. The ramp time parameters from annealing to extension were used 0.3 and 3°C sec-1, respectively. The concentration of G. elata genomic DNA were about 50 ng μL-1 with a 1.93 purity. Obviously, the amplified band numbers and resolution were improved when using a 0.3°C sec-1 ramp time in the RAPD analysis. The band number increase is closely related to the G/(G+C) ratio of RAPD primers. Therefore, the extracted G. elata genomic DNA is suitable for PCR amplifications and the prolonged ramp time is helpful to improve the RAPD resolution and production. These are valuable references for molecular identification and biodiversity analysis of G. elata populations.
  Bo Luo , Huailin Xiong , Yingyu Mao and Haiqing Yu
  Currently, it is obscure whether Toll-like Receptor 2 (TLR2) is related to the pathogenesis and pathophysiology of Paederus Dermatitis (PD). This study focused on the analysis of TLR2 mRNA expression in spleen of PD mice. Pederin was extracted at -20°C by dipping six female Paederus fuscipes Curtis in 1 mL of 70% ethanol for one month. Mouse abdominal hairs were denuded gently using 4% Na2S. By evenly extending 20 μL pederin solutions in 8 mm diameter skin, PD were induced in mice. Erythema and edema symptoms were scored. At 44 h after induction, the expression of TLR2 mRNA in spleen was analyzed using a reverse transcription and semi-quantitative polymerase chain reaction (SQ-PCR) with GAPDH mRNA as a reference. Normal mice with unhaired abdominal skin were used as controls. In this study, erythema and edema symptoms were observable, especially at 20 h after pederin application. The relative quantity of TLR2/GAPDH mRNA was 1.32±0.213 and 0.896±0.036 in healthy group and PD group, respectively. The expression level of TLR2 mRNA in spleen was significantly decreased (p<0.05) after treatment with pederin. These results suggested that pederin is a Th1 (T helper cell 1) inducing agent and there are Th1 cytokines in PD mice in early stage after contact, however, down-regulation of TLR2 mRNA expression is most likely related to Th2 (T helper cell 2) cytokines. The pathogenesis and pathophysiology In PD mice are closely associated with Th1 and Th2 responses, especially with cytokines excretion. This hypothesis needs to be investigated further.
  Chenggang Duan , Zhiqiang Mei , Shu Gong and Haiqing Yu
  Penthorum sedoides L. and Penthorum chinense Push is closely related aneuploids in genus Penthorum L. based on the previous data. However, P. chinense are treated as variety or subspecies of P. sedoides successively by botanists and little was known about their relationships on molecular level. An improved randomly amplified polymorphic DNA (RAPD) and Sequence Characterized Amplified Region (SCAR) were performed to obtain their genetic characterizations and species-specific DNA fragments. Obviously, five 10-mer random primers SBS-A3, SBS-I3, SBS-I18, SBS-M6 and SBS-Q9 demonstrated different fingerprints. Six candidate specific bands (I3-PS, I3-PC, I18-PC, M6-PS, Q9-PS and Q9-PC) displayed in two taxa were successfully cloned and sequenced. Based on these sequences, six pairs of SCAR primers from P. sedoides (I3PSF/I3PSR, M6PSF/M6PSR and Q9PSF/Q9PSR) and P. chinense (I3PCF/I3PCR, I18PCF/I18PCR and Q9PCF/Q9PCR) were designed, respectively. However, four of six primer pairs yielded amplicons common in the two taxa. The DNA amplification using Q9PSF/Q9PSR primers generated a single 555 bp band only in P. sedoides and Q9PCF/Q9PCR primers produced a 760 bp fragment unique to P. chinense. The results suggested that P. sedoides and P. chinense are still closely related, although there are a lot of variations in RAPD genetic sites. The developed RAPD and SCAR techniques are effective and useful in revealing genetic characterizations of P. sedoides and P. chinense. Also, the SCAR analysis based on the improved RAPD method is powerful in authentication of species with close relationships.
  Ting He , Linfang Li , Jia Feng and Haiqing Yu
  Currently, little is known about a protocol in establishing the quantitative Real-Time PCR (qRT-PCR) system in gram-positive bacteria. Also, it is obscure whether the expression level of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (Cas9) in Listeria innocua is influenced by the pH. This study focused on providing a protocol for analyzing Cas9 gene expression in L. innocua at two pH conditions. The 37°C overnight culture was diluted 1:500 and grown in fresh Todd Hewitt Yeast extraction broth (THY) for 3 h. The collected pellet was washed two times in pH = 7.5 medium and was cultured for 4 h by using pH = 7.5 and pH = 6.0 medium, respectively. The culture was treated with RNA protection reagent for 5 min and the obtained pellet was suspended with mutanolysin for 30 min. Total RNA was extracted by using a kit and was incubated with DNase I, which was removed by adding phenol-chloroform-isoamyl alcohol thereafter. The purified RNA was precipitated via glycogen-ethanol treatment and was synthesized into cDNA. The 2‾ΔΔCt method was used to analyze the expression level of Cas9 gene with 16S rRNA gene as a reference. The gel electrophoresis showed that the quantity of 23S rRNA was about two-fold of 16S rRNA. The relative Cas9 expression levels of L. innocua in neutral and acidic medium were 1.08±0.49 and 1.66±0.54, respectively (p>0.05). Totally, the qRT-PCR system for analyzing Cas9 gene expression in L. innocua was successfully developed. The Cas9 expression level of L. innocua in acidic medium was similar to that in neutral medium.
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