广州地区番石榴根结线虫鉴定与14-3-3基因的克隆

【目的】摸清广州地区番石榴根结线虫的种类、分布及致病机制。【方法】观察番石榴根结线虫的雄虫、二龄幼虫、雌虫及其会阴花纹的形态学特征,根据根结线虫的通用引物#C2F3和#1108对线虫mt DNA的COⅡ和lr RNA基因间序列进行PCR扩增,获得750 bp的特异扩增产物,将扩增片段在Gen Bank上进行Blast比对。通过南方根结线虫与北方根结线虫的14-3-3序列设计简并引物,进一步扩增已鉴定的象耳豆根结线虫的14-3-3基因。【结果】广州地区番石榴根结线虫的形态与象耳豆根结线虫(Meloidogyne enterolobii)相似;其扩增片段与象耳豆根结线虫(M.enterolobii)比对,序列相似性在99%以上。扩增了象耳豆根结线虫的14-3-3基因,结果显示该基因的开放阅读框包含783 bp的片段,编码261个氨基酸,命名为Me-14-3-3。【结论】广州地区番石榴根结线虫为象耳豆根结线虫(M.enterolobii),14-3-3蛋白基因被成功克隆,扩增结果为进一步研究14-3-3蛋白基因在象耳豆根结线虫生长发育中的功能、摸清致病机制等方面奠定良好的基础。     

Amplification and sequence analysis of anti-D variable region gene with leader peptide sequence

AIM:To amplify from leader peptide region and obtain human monoclonal anti-D variable region gene with high specificity and affinity, and analyze the nucleotide and deduced amino acid sequences.METHODS:The total RNA was extracted from an Epstein-Barr-virus-transformed cell line secreting monoclonal anti- (rhesus D) antibody. The leader region primers containing a ribosome recognition site were designed. By using PCR method, the cDNA of human anti-(rhesus D) antibody (IgM κ) variable region gene was amplified. Cloning and subsequent sequence analysis of the variable region gene was performed. The deduced amino acid sequence was also compared and analyzed with previously published sequences.RESULTS:A band of approximate 440 and 410 base pairs were amplified using heavy chain primers and light chain primers, respectively. Sequence analysis indicated that the deduced amino acid sequence was in agreement with the characterization of the amino acid present in the human Ig variable region. CONCLUSION:The cloning and sequencing of a human anti- (Rhesus D) antibody variable region cDNA will make benefits for production of recombinant anti-(Rhesus D) antibody and prevention of Rh haemolytic disease in newborns.     

Association analysis of FcεRⅠβ gene with allergic asthma in Chinese

AIM: To investigate the linkage between the polymorphism of -109 and Glu237 in the high-affinity IgE receptorβ(FcεRⅠβ) gene and susceptibilty of allergic asthma in adults in a Chinese population. METHODS: Allergic asthma sample in adult and age-and sex-matched control were studied. A-109C/T and a coding variant Glu237Gly in FcεRⅠβ were detected with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: (1)The genotype frequencies were 0.415 for -109T/T, 0.491 for-109T/C and 0.094 for -109C/C in allergic asthma in adults. No significant difference in the distribution of-109C/T polymorphism was found between allergic asthma adult subjects and healthy control subjects. However, a homozygosity for the-109T allele was associated with increased total plasma IgE levels in subjects with allergic asthma (F=7.213, P<0.01). (2)The allele frequency of Gly237 in the patients and control was 0.245 and 0.142, respectively. There was a significant association between Gly/Gly genotype and allergic asthma in adults among allergic asthma patients. Gly237 was significantly associated with high IgE. CONCLUSIONS: It suggests that Gly237 vaviant of the FcεRⅠβ gene is involved in the development of allergic asthma. The-109C/T and Glu237Gly polymorphisms are two of the genetic factors identified, which affects total plasma IgE levels in adult allergic asthma in Han of Hubei province.     

Association of endothelial nitric oxide synthase gene rs7830 and rs3918188 polymorphisms with essential hypertension in Kazakh of Xinjiang region

AIM: To investigate the correlation between endothelial nitric oxide synthase (eNOS) rs7830 and rs3918188 locus polymorphisms and essential hypertension (EH) in the Kazakh of Xinjiang region. METHODS: Epidemiological case-control study was conducted. DNA was extracted by classic phenol-chloroform method, PCR amplification and purification. The rs7830 and rs3918188 of eNOS gene in 363 EH patients (EH group) and 370 normotensive controls (NT group) in the Kazakh of Xinjiang region were genotyped by the technique of SNaPshot single nucleotide polymorphism genotyping. The plasma levels of fasting blood glucose, uric acid, cholesterol and triglyceride were measured by biochemical methods. Determination of body mass index and waist-hip ratio was also conducted. RESULTS: Age (P<0.01), body mass index (P<0.01), triglyceride (P<0.01), low-density lipoprotein (P<0.05) and apolipoprotein A1/B (P<0.05) were the independent factors for EH in the Kazakh of Xinjiang region. No difference of eNOS gene rs7830 and rs3918188 loci in the genotype frequency and the allele frequency distribution between EH patients and normotensive controls in the Kazakh of Xinjiang region was observed (P>0.05). The frequency distribution of CA, CC, AC and AA haplotypes from eNOS gene rs7830 and rs3918188 loci between EH group and NT group also had no difference in the Kazakh of Xinjiang region (P>0.05). CONCLUSION: Age, body mass index and triglyceride are the independent risk factors, while low-density lipoprotein and apolipoprotein A1/B are the independent protective factors for EH in the Kazakh of Xinjiang region. The polymorphisms of eNOS gene rs3918188 and rs7830 loci are not related to EH in the Kazakh of Xinjiang region.     

Interaction of polymorphisms of ICAM-1 gene K469E and MCP-1 gene -2518A/G in invasion and metastasis of gastric carcinoma

AIM: To investigate the interaction of polymorphisms of intercellular adhesion molecule-1 (ICAM-1) gene K469E and monocyte chemoattractant protein-1 (MCP-1) gene -2518A/G in the invasion and metastasis of gastric carcinoma. METHODS: Based on TNM classification, 4 500 patients with confirmed gastric carcinoma from the First Affiliated Hospital of Xinxiang Medical University in China from December 2009 to November 2014 were divided into stageⅠ group, stage Ⅱgroup, stage Ⅲ group, stage Ⅳ group, and stage 0 group, with 900 cases in each group. No significant difference among the 5 groups in age, gender, ethnicity, birthplace and living habit was observed. The genetic polymorphisms of ICAM-1 gene K469E and MCP-1 gene -2518A/G were analyzed by the technique of polymorphism-polymerase chain reaction (PCR) in peripheral blood leukocytes of above-mentioned cases. RESULTS: Statistical tests showed signi-ficant differences in the frequencies of K469E (EE) and -2518A/G (GG) among each group (P<0.01). The risk of the invasion and metastasis of gastric carcinoma significantly increased in subjects with K469E (EE) genotype and in those with -2518A/G (GG) genotype. Combined analysis of the polymorphisms showed that distribution frequency of K469E (EE)/-2518A/G (GG) in stage Ⅰ group, stage Ⅱ group, stage Ⅲ group, stage Ⅳ group and stage 0 group was 39.22%, 53.22%, 59.22, 65.44% and 12.11%, respectively (P<0.01). The people who carried with K469E (EE)/-2518A/G (GG) had a high risk of the invasion and metastasis of gastric carcinoma, and statistical analysis suggested a positive interaction in a super-multiplicative model between K469E (EE) and -2518A/G (GG) in increasing the risk of the invasion and metastasis of gastric carcinoma. CONCLUSION: ICAM-1 gene K469E (EE) and MCP-1 gene -2518A/G (GG) are the risk factors in the invasion and metastasis of gastric carcinoma, and significant interactions between genetic polymorphisms of K469E and -2518A/G added the risk of the invasion and metastasis of gastric carcinoma.     

菠萝AcSERK15’上游区(-499/+258bp)转录活性的研究

【目的】探究Ac SERK1启动子的功能,有助于了解Ac SERK1的表达调控模式。【方法】以‘神湾’菠萝(Ananas comosus L.‘Shenwan’)为材料,将Ac SERK1启动子缺失序列与GUS融合,构建植物表达载体,并导入根瘤农杆菌GV3101中。利用农杆菌真空渗透法侵染烟草叶片,并检测GUS活性;利用浸染法转化菠萝胚性愈伤组织获得转基因植株,分析光照、2,4-D和4℃等处理后的GUS表达量。【结果】构建2个Ac SERK1启动子植物缺失表达载体,分别命名为p(-30/+258 bp)和p(-499/+258 bp)。烟草瞬时表达结果显示p(-499/+258 bp)表现出强的GUS活性,p(-30/+258 bp)表现出微弱的GUS活性。q RT-PCR结果表明,光照处理后,GUS表达量降低。相反的,2,4-D和4℃处理转基因菠萝植株后GUS表达量均显著增加。【结论】Ac SERK1启动子-499/-30 bp区段内含有光、生长素和低温响应元件。     

Effect of 5Aza-dc on demethylation of TIMP-3 gene promoter in carcinoma cells

AIM: To observe the effect of 5Aza-dc on demethylation of TIMP-3 gene promoter in carcinoma cells. METHODS: Both hepatocellular carcinoma cells (H2M) and epidermoid carcinoma cells (A431) with methylation of TIMP-3 promoter gene were treated with 5-Aza-2'-deoxycytidine (5Aza-dc). Invasion ability and motility of the cells were detected by Transwell experiments. Expressions of TIMP-3 protein and mRNA were detected by Western blotting and RT-PCR, respectively. TIMP-3 gene promoter methylation was detected by methylation-specific PCR (MSP). RESULTS: ① Invasion ability and motility of H2M and A431 cells were declined after treatment with 5Aza-dc; ② After treatment with 5Aza-dc, the expression of TIMP-3 protein and mRNA were increased in H2M and A431 cells; ③ After treatment with 5Aza-dc, methylation of TIMP-3 promoter gene was not detectable in the cell lines. CONCLUSIONS: 5Aza-dc induces demethylation in TIMP-3 promoter gene, restores TIMP-3 gene and protein expression, and inhibits invasion ability and motility of the carcinoma cells.     

Analysis on relationship of lung cancer with length polymorphisms of microsatellite D3S1234 and D3S1300 located on FHIT gene

AIM:To analyze the relationship between lung cancer and length polymorphisms of microsatellite D3S1234 and D3S1300 located on fragile histidine triad(FHIT) gene. METHODS:The case-control study was conducted among 54 subjects of cancer and 131 healthy subjects. The length polymorphism was detected with the methods of multiplex PCR and urea-polyacrylamide gel electrophoresis. Allele frequency was calculated and the difference of allele frequency between cancer subjects and healthy persons was analyzed by the method of Chi-square. The relationship between lung cancer and length polymorphism of D3S1234 and D3S1300 was studied by the method of Binary Logistic analysis.RESULTS:No obvious difference in allele frequency of D3S1234 was observed between cancer subjects and healthy persons，but significant difference in allele frequency of D3S1300 was found between two groups. The results of Binary Logistic analysis showed that smoking was related with an increased risk of lung cancer. When the effect of smoking was excluded,length polymorphisms of D3S1234 and D3S1300 were found to be associated with higher risk of lung cancer. Meanwhile,length polymorphisms of D3S1234 and D3S1300 and smoking were proved to have interactive effects on the risk of lung cancer. CONCLUSION:The results suggest that there are statistic associations between lung cancer and length polymorphisms of D3S1234 and D3S1300,which may be beneficial to diagnose lung cancer early and explore its pathogenesis.     

Interaction of polymorphisms in resistin gene promoter -420C/G,cytochromes P4501A1 gene MspI and cigarette smoking on nonalcoholic fatty liver disease

AIM: To investigate the interaction of polymorphisms of resistin gene promoter -420C/G, cytochromes P4501A1-MspI and cigarette smoking in nonalcoholic fatty liver disease (NAFLD). METHODS: The genetic polymorphisms in resistin gene promoter -420C/G and CYP1A1-MspI were analyzed by the technique of polymerase chain reaction (PCR) in peripheral blood leukocytes of 900 NAFLD cases and 900 healthy persons. RESULTS: The frequencies of -420C/G (GG) and CYP1A1-MspI (m2/m2) were 49.75% and 50.08% in NAFLD cases and 24.00% and 24.25% in healthy controls, respectively. Statistical tests showed a significant difference in the frequencies between the 2 groups (P<0.01). The risk of NAFLD with -420C/G (GG) was significantly higher than that of controls. Individuals who carried with CYP1A1-MspI (m2/m2) had a high risk of NAFLD. Combined analysis of the polymorphisms showed that the percentages of -420C/G (GG)/CYP1A1-MspI (m2/m2) in NAFLD and control groups were 39.83% and 12.83%, respectively (P<0.01). The people who carried with -420C/G (GG)/CYP1A1-MspI(m2/m2) had a high risk in NAFLD group. The cigarette smoking rate in NAFLD group was signi-ficantly higher than that in control group (P<0.01), and the statistic analysis suggested an interaction between cigarette smoking and -420C/G (GG) and CYP1A1-MspI (m2/m2), which increased the risk of NAFLD.CONCLUSION: -420C/G (GG), CYP1A1-MspI (m2/m2) and cigarette smoking are the risk factors in NAFLD. The interactions between genetic polymorphisms in -420C/G, CYP1A1- MspI (m2/m2) and cigarette smoking increase the risk of NAFLD.     

沙梨PpGAI3基因的分离与原核表达分析

【目的】分离沙梨DELLA蛋白编码基因PpGAI3,探究其蛋白在花芽休眠中的表达情况。【方法】从不同需冷量沙梨品种‘蜜雪梨’和‘黄花梨’中分离PpGAI3基因,重组到p EASY-Blunt E1表达载体中,转化BL21(DE3)宿主菌后经IPTG诱导表达,并对表达菌株进行SDS-PAGE和Western blotting检测。【结果】获得2种沙梨的PpGAI3基因,分别命名为PpGAI3mx和PpGAI3hh,Gen Bank登录号为KU954535和KX078215,其开放阅读框(ORF)为1 641 bp,编码546个氨基酸,预测其为亲水性不稳定蛋白。进化树分析表明PpGAI3mx和PpGAI3hh与白梨的亲缘关系最近;成功构建了p EASY-Blunt E1-PpGAI3原核表达载体,得到蛋白分子质量约60 ku的诱导表达产物。【结论】所得沙梨DELLA蛋白编码基因PpGAI3为植物DELLA蛋白家族中的成员,在梨花芽休眠过程中具有特异表达,且表达产物大部分以不溶性包涵体形式存在。     

Molecular cloning and functional analysis of a flavanone 3-hydroxylase gene from blueberry

Vaccinium corymbosum (blueberry) is touted as a superfood with numerous health benefits due to its high levels of flavonoids. Flavanone 3-hydroxylase (F3H) is a key regulatory enzyme of the flavonoid pathway. In this study, we cloned the full-length cDNA of F3H (designated VcF3H) from young blueberry leaves using rapid amplification of cDNA ends (RACE). The cDNA contained a 1080-bp open reading frame that encoded a 359-amino acid protein. The deduced VcF3H protein showed high similarities to other plant F3Hs. Conserved amino acid motifs required for ferrous iron binding (HXD) and 2-oxoglutarate binding (RXS) were identified in VcF3H, VcFLS (flavonol synthase), and VcANS (anthocyanidin synthase). Quantitative RT-PCR analysis demonstrated that VcF3H was expressed in all tissues tested, with particularly high expression in young leaves, fruits (pink and blue), and stems. Anthocyanins accumulated mainly in fruits, whereas flavonols were found mainly in leaves and stems. Furthermore, the expression pattern of VcF3H was similar to that of VcCHS, VcDFR, and VcANS in various tissues. Heterologous expression of VcF3H in Arabidopsis thaliana increased the anthocyanin content in leaves, but did not affect the flavonol content. Thus, VcF3H seems to be involved in anthocyanin synthesis in the flavonoid biosynthetic pathway when ectopically expressed in Arabidopsis.     

Effect of miR-7-5p targeting Itch gene on insulin resistance model of HepG2 cells

AIM:To preliminarily explore the relationship between microRNA-7-5p (miR-7-5p) and Itch gene and their relationship with insulin resistance by establishing insulin resistance model of HepG2 cells in vitro for detecting differential expression of miR-7-5p and its predicted target gene Itch in the state of insulin resistance. METHODS:The insulin resistance model of HepG2 cells was induced by suitable concentration of plamitic acid. The possible target genes and the associated signaling pathways of miR-7-5p were predicted based on bioinformatic analysis. The expression levels of miR-7-5p and Itch were detected by RT-qPCR and Western blot in the HepG2 cells with insulin resistance. RESULTS:The HepG2 cell model of insulin resistance was successfully induced by treatment with 0.25 mmol/L palmitic acid for 24 h. Compared with negative control group, the expression level of miR-7-5p detected by RT-qPCR in insulin resistance group was significantly decreased (P<0.01). Bioinformatic analysis showed that a considerable number of target genes of miR-7-5p were enriched in the ubiquitin proteasome system. Among them, E3 ubiquitin ligase Itch gene was the most relevant target gene to insulin resistance. The results of Western blot showed that the protein expression of Itch was up-regulated in the HepG2 cells under insulin resistance (P<0.01). CONCLUSION:miR-7-5p may be involved in the pathophysiological process of insulin resistance, which may directly or indirectly affect the normal transduction of insulin signaling pathway by targeting Itch gene.     

利用精密作图和DNA纤维荧光原位杂交把灯笼椒抗烟草花叶病毒属病毒L^3基因定位于含高度重复序列的类抗病基因簇的400kb区

我们用辣椒（Capsicum annuum）栽培种（已导入了灯笼椒Capsicum chinense L^3 基因）的种内F2代群体（2016株）和种间F2代群体（3391株）（由灯笼椒与Capsicum frutescence杂交产生）对灯笼椒抗烟草花叶病毒属病毒的L^3基因进行定位。通过L^3基因抗性紧密相关的AFLP分子标记的BAC文库的分析,揭示出番茄抗病同源基因12的存在。通过简并PCR技术,对来自35株不同辣椒的同源基因12的部分或全部编码序列进行克隆,且在种间组合中产生了17个遗传标记。图谱显示：L^3基因位于12同源基因标记IH1—04和BAC—end标记189D23M中间,L^3基因定位于包含两个不同BAC重叠群的区内,这两个不同的BAC重叠群分别由4个和1个无性系组成。DNA纤维荧光原位杂交揭示这两个重叠群被约30kb隔开。DNA纤维荧光原位杂交结果和BAC无性系的Southern杂交表明在高度重复序列中富集包含L^3基因位点的区。Southern杂交表明两个BAC重叠群包含多于十个的12同源基因拷贝体。相反,对于种间F2代群体,,重组后代没有结合位点,在种内F2代群体中,该结合位点存在于两个不同的BAC重叠群内,这两个不同的BAC重叠群分别由7个和2个无性系组成。而且,两个群体间结合位点分配的不同表明在含有L基因位点的区域连锁不平衡。     

利用精密作图和DNA纤维荧光原位杂交把灯笼椒抗烟草花叶病毒属病毒L^3基因定位于含高度重复序列的类抗病基因簇的400Kb区（连载）

结果 L^3基因的初步定位 我们首先利用NK群体定位了L^3基因,NK群体是辣椒栽培品种（C．annuumcultivar）和从灯笼椒（C．chinense）PI152225导入的含有L^3基因的衍生品种种内杂交的F2代群体。对抗PMMoV及敏感的F2后代的大量DNA进行三轮AFLP分析发现了应用7个组合的选择性PCR引物扩增到8个多态性DNA片段。从其中得到了两个SCAR标记。     

山核桃赤霉素氧化酶基因CcGA3ox的克隆和功能分析

[目的]赤霉素3-氧化酶(gibberellin 3-oxidases,GA3ox)是赤霉素(GAs)生物合成途径中关键酶之一,对山核桃(Carya cathayensis)中CcGA3ox基因进行克隆,并进行序列分析和功能验证.[方法]以山核桃体细胞胚为材料,提取RNA,采用PCR扩增技术,克隆山核桃CcGA3ox基...     

miR-485-5p inhibits viability and migration and invasion abilities of hepatocellular carcinoma Hep3B cells by targeting SOX5

AIM: To investigate the effects of microRNA-485-5p (miR-485-5p) on the viability, migration and invasion abilities of hepatocellular carcinoma cells and the underlying mechanism. METHODS: The expression levels of sex determining region Y-box 5 (SOX5) mRNA and miR-485-5p in the hepatocellular carcinoma Hep3B cells were detected by RT-qPCR with normal hepatocyte THLE-3 as control. Western blot was used to measure the expression levels of SOX5, proliferating cell nuclear antigen (PCNA), Ki67, cyclin D1 and matrix metalloproteinase-2 (MMP-2). The viability of Hep3B cells was measured by MTT assay. The migration and invasion abilities of the Hep3B cells were detected by Transwell assay. Dual-luciferase reporter assay system was applied to verify the relationship between miR-485-5p and SOX5. RESULTS: Compared with the control cells, the expression level of miR-485-5p was decreased in hepatocellular carcinoma cells Hep3B, Huh7 and HCCLM3 (P<0.05), while the expression of SOX5 at mRNA and protein levels were significantly increased (P<0.05). Over-expression of miR-485-5p inhibited the viability, migration and invasion of Hep3B cells. miR-485-5p targeted the 3′-UTR of SOX5 and negatively regulated the expression of SOX5. Knocking-down of SOX5 expression inhibited the viability, migration and invasion of Hep3B cells. Over-expression of SOX5 partially reversed the inhibitory effect of miR-485-5p over-expression on the viability, migration and invasion of Hep3B cells. CONCLUSION: miR-485-5p inhibits the viability, migration and invasion of Hep3B cells by targeting SOX5 gene. miR-485-5p is a potential molecular target for hepatocellular carcinoma.     

Effect of microtubule-destabilizing protein stathmin gene silencing on proliferation and apoptosis of nasopharyngeal carcinoma cell line 5-8F

AIM: To investigate the effects of stathmin gene silencing on nasopharyngeal carcinoma cell line 5-8F. METHODS: Double-strand siRNA targeting to stathmin gene was obtained by chemical synthesis and annealing, and was sub-cloned into the vector pGenesil-1.1. The plasmid was introduced into 5-8F cells by liposome-mediated transfection. The gene expression of stathmin, and the proliferation, morphology and apoptosis of the cells were analyzed by Western blotting, MTT assay and flow cytometry. RESULTS: The cell suppression rate in stathmin gene silencing group was (53.01?1.12)%, significantly higher than that in transfection reagent group and in negative control group. The cell apoptotic rate in stathmin gene silencing group was (8.75?0.67)%, also significantly higher than that in transfection reagent group and in negative control group (P<0.05). CONCLUSION: Silencing of stathmin gene in nasopharyngeal carcinoma cells inhibits the cell proliferation and induces cell apoptosis.