朱顶红实时荧光定量PCR中不同组织器官内参基因的筛选

以朱顶红（Hippeastrum vittatum）不同组织器官为试验材料,通过实时荧光定量PCR技术检测肌动蛋白基因（ACT）、亲环蛋白基因（CYP）、转录延伸因子基因（EF-1α）、甘油醛–3–磷酸脱氢酶基因（GAPDH、GAPDH2）、α微管蛋白基因（TUA）和β微管蛋白基因（TUB）这7个常用的看家基因的表达水平,并利用geNorm、NormFinder和BestKeeper综合评价其表达稳定性。结果表明,EF-1α和GAPDH2的表达水平较高,而且相对稳定;其次为TUB、TUA和GAPDH。geNorm和NormFinder软件分析结果显示,在不同组织器官中CYP和GAPDH2的表达稳定性最高,其次是EF-1α;进一步分析表明,CYP的表达稳定性虽然较高,但是其表达丰度较低,故不适合作为内参基因。BestKeeper分析表明,EF-1α和GAPDH2在不同组织器官中的表达稳定性较好。综合分析表明,EF-1α和GAPDH2在所有样品中表达量较高,稳定性较好。以筛选出来的EF-1α、GAPDH2以及EF-1α + GAPDH2作为内参基因检测朱顶红花器官调控基因PI的表达水平,结果表明,以上述两个基因及其组合为内参基因得到的PI表达模式相同,而且在花器官中的表达高于茎盘和根,基本符合PI调控花模型的机理。因此,EF-1α、GAPDH2或EF-1α + GAPDH2可作为朱顶红的内参基因进行相关基因的表达调控研究。     

苹果实时荧光定量PCR分析中内参基因的筛选

【目的】为筛选苹果实时荧光定量PCR实验中最适内参基因,【方法】应用实时荧光定量PCR技术,分析5个传统内参基因18SrRNA、ACTB、GAPDH、UBQ、TUB在苹果不同基因型、不同组织、果实不同发育时期的mRNA表达差异情况。供试的6个不同基因型苹果分别为:新疆野生苹果、八棱海棠、丽江山荆子、‘津轻’、‘国庆’、‘金冠’;6种不同组织为果皮、果肉、叶片、愈伤组织、花瓣、种子;5个果实发育不同时期为花后28、50、74、95、115 d。【结果】经geNorm程序分析发现5种内参基因的表达稳定性各异,UBQ在果实不同基因型和不同发育时期的基因表达分析中最稳定;ACTB和UBQ在6种不同组织中表达均稳定。【结论】UBQ在参试样品中表达均比较稳定,是研究苹果基因表达分析中理想的内参基因。     

钝裂银莲花花色素合成相关基因qRT-PCR内参基因的筛选

为了筛选适合于钝裂银莲花类黄酮/花青素合成途径中相关基因qRT-PCR表达分析时的内参基因,根据钝裂银莲花蓝/白不同花色花器官组织的转录组测序结果,选取了多聚泛素酶基因(polyubiquitin,UBQ)、微管蛋白基因(β-tubulin,-TUB)、水通道蛋白基因(aquaporin,AQP)、肌动蛋白基因(act...     

Effect of betaine on liver injuries in aging db/db mice induced by lipids

AIM: To evaluate the effect of betaine on lipid metabolism disorder in inherited db/db mice with long-term nonalcoholic fatty liver disease (NAFLD).METHODS: Experimental NAFLD models were established by feeding the db/db mice with high-fat diet. Fifty 7-month-old db/db mice were randomly divided into 5 groups: the mice in low, medium and high dose groups were given betaine by intragastric administration at doses of 200 mg/kg, 400 mg/kg and 800 mg/kg for 6 weeks, respectively,while the mice in saline control group and positive drug group were given normal saline and positive control drug,respctively. All the animals were killed, and serum alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL) and glucose tolerance were detected. The pathological changes of the liver tissues were also observed.RESULTS: Betaine significantly decreased the levels of ALT, TC and LDL (P<0.05 or P<0.01). The pathological changes of the liver tissues indicated that the content of lipid in the hepatocytes of betaine treatment groups was less than that in saline control group.CONCLUSION: Betaine significantly improves the lipid metabolism and the liver function in the aging db/db mice, and reduces the accumulation of lipid in the hepatocytes.     

石蒜属植物实时荧光定量PCR内参基因的选择

以石蒜属植物换锦花（Lycoris sprengeri）、石蒜（Lycoris radiata）和中国石蒜（Lycoris chinensis Traub）不同组织器官、不同花发育时期以及不同杂交种的幼小鳞茎为研究材料,利用qRT-PCR技术检测Actin、EF-1α、GAPDH、5S rRNA、Ubiquitin和β-Tubulin等6个内参基因的mRNA表达情况,利用geNorm、NormFinder、BestKeeper和Reffinder软件综合评价6个内参基因的表达稳定性。结果表明,石蒜属植物种间和种内内参基因表达差异明显,在分析换锦花不同组织基因表达时可选用β-Tubulin、Actin和GAPDH作为内参基因,分析不同花期的基因表达时宜选用5S rRNA、EF-1α、Actin和β-Tubulin作内参基因。中国石蒜不同组织最合适的内参基因是5S rRNA、Ubiquitin、EF-1α和β-Tubulin,而不同花期最合适的内参基因则是Ubiquitin、β-Tubulin。分析石蒜不同组织间基因表达的适宜内参基因是β-Tubulin和5S rRNA,不同花期为Ubiquitin、β-Tubulin以及5S rRNA。不同杂交种鳞茎适宜内参基因为β-Tubulin和Actin。     

药用蒲公英低温和高温胁迫下内参基因筛选与相关基因表达分析

为研究不同温度胁迫下药用蒲公英最适内参基因与温度响应相关基因的表达,分别以低温(4℃)和高温(38℃)胁迫0、3、6、12、24和48 h共12个处理的叶片为材料,选取10个候选内参基因18S、EF1α、TUB、40S、GAPDH、β-actin、ACT11、TUA、60S和SKIP,利用RT-qPCR技术以及geNorm、NormFinder和BestKeeper软件评价其稳定性。利用筛选出的最适内参基因对药用蒲公英12个温度响应基因(AP2/ERF、Dof、ICE1、MYB、b ZIP、NTL6、HSF、Gols、HSP、NAC、XCT和WRKY)的表达水平进行定量分析。结果显示:温度胁迫下药用蒲公英最适内参基因为18S和GAPDH;药用蒲公英AP2/ERF、HSF、Glos、HSP、NAC、XCT基因在低温和高温胁迫下均为先上调后下调表达,bZIP、NTL6在温度胁迫下波动变化,Dof、ICE1、MYB在低温胁迫下先上调后下调表达,高温胁迫下持续下调表达,WRKY在高温胁迫下表达量远远高于低温胁迫。依据基因表达量推测,药用蒲公英低温和高温胁迫的响应机制存在差异,AP2/ERF、D...     

‘琯溪蜜柚’荧光定量PCR内参基因的筛选

【目的】为了克隆‘琯溪蜜柚’的管家基因,并筛选合适的管家基因作为内参,【方法】以‘琯溪蜜柚’盛花期后5个不同时期的果实汁胞,以及根﹑茎﹑叶为材料,采用实时荧光定量PCR技术,分析actin1﹑β-tubulin﹑18S rRNA﹑EF1-α和Ubiquitin 5个管家基因在不同材料中的表达情况,并结合geNorm、NormFinder﹑comparative Delta-CT和BestKeeper 4种评估方法进行稳定性分析。【结果】结果表明,actin1和β-tubulin是‘琯溪蜜柚’果实发育进程中较为稳定的内参基因,而EF1-α和β-tubulin则是研究不同组织特定靶基因表达中稳定的内参基因。【结论】筛选出了‘琯溪蜜柚’最佳的内参基因β-tubulin,为今后目的基因的表达分析奠定了基础。     

Identification of reference genes for real-time quantitative polymerase chain reaction based gene expression studies on various Olive (Olea europaea L.) tissues

Reference genes are essential for the normalisation of the expression data of quantitative real-time PCR for the purposes of validation. Although several reference genes have been validated for the olive, comprehensive analyses including an excessive number of candidate reference genes still require study in various olive tissue samples. In this work, a total of 40 candidate reference genes were tested for their stability in 8 different olive tissues (root, apical bud, lateral bud, pedicel, young leaf, mature leaf, fruit mesocarp, and seed) with the utilisation of the most popular software programs including GeNorm, NormFinder, BestKeeper, and ΔC_t. The analyses of expression stability of candidate reference genes using quantitative real-time PCR demonstrated ubiquitin-conjugating enzyme (UBC1) as the most stable reference gene for the studied tissues of the olive. The GeNorm software also calculated the optimum reference gene combinations as two which consist of UBC1 and the Clathrin adaptor complex medium subunit (CLATHRIN) genes. This study provides the most stable reference gene combination for normalisation of target genes for quantitative real-time PCR gene expression studies on the olive.     

Role of chlorogenic acid in endothelial dysfunction in db/db mice

AIM: To explore the effects of chlorogenic acid (CGA) on endothelial dysfunction in db/db mice and the possible mechanism. METHODS: Male db/db mice (n=12) were divided into control group and CGA group, with 6 mice in each group. The mice in CGA group were treated with diet containing 0.02% CGA, while the mice in control group were given normal diet only. The observation period was 12 weeks. Fasting blood glucose level, tail blood pressure and the body weight were analyzed each week. At the end of the 12th week, the mice were anesthetized and blood was taken from carotid artery. The plasma levels of heme oxygenase-1 (HO-1), catalase (CAT), NAD(P)H dehydrogenase quinone 1 (NQO1) and glutathione peroxidase-1 (GPx-1) were measured by ELISA. The mouse aortas were isolated, and the superoxide anion and nitric oxide (NO) levels were measured by DHE and DAF-2 DA staining, respectively. Wire Myograph System was used to detect the vasorelaxation of db/db mouse aorta. The protein levels of peroxisome proliferator-activated receptor α (PPARα), nuclear factor E2-related factor 2 (Nrf2), phosphorylated AMP-activated protein kinase (p-AMPK), phosphorylated endothelial NO synthase (p-eNOS), P22^phox and P47^phox were determined by Western blot. RESULTS: Dietary CGA decreased fasting blood glucose and body weight in db/db mice as compared with control group (P<0.01 or P<0.05). The plasma levels of HO-1, CAT, NQO1 and GPx-1 in CGA group were higher than those in control group (P<0.01 or P<0.05). Administration of CGA for 12 weeks attenuated superoxide anion level, increased NO level in the mouse endothelium and improved endothelium-dependent relaxation of the db/db mouse aorta. CGA also increased the protein levels of PPARα, Nrf2, p-AMPK and p-eNOS, and decreased P22^phox and P47^phox levels (P<0.01). CONCLUSION: Dietary CGA improves db/db mouse endothelium-dependent relaxation. This effect may be related to the increases in the levels of antioxidant molecules PPARα, Nrf2 and p-AMPK, and the up-regulation of antioxidant capacity, thus decreasing the oxidative stress, promoting eNOS phosphorylation, and increasing NO level.     

Latent membrane protein 1 regulates expression of p27 and RPLP0 in nasopharyngeal epithelial and nasopharyngeal carcinoma tissues

AIM: To observe the protein expression of p27 and ribosomal phosphoprotein large P0(RPLP0) regulated by latent membrane protein 1 (LMP1) in nasopharyngeal epithelial and nasopharyngeal carcinoma tissues. METHODS: The protein levels of p27 and RPLP0 and the relationship with LMP1 were analyzed by Western blotting. The protein expression of LMP1, p27 and RPLP0 was also detected by the method of immunohistochemistry in 30 nasopharyngeal epithelial and 60 nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues. Meanwhile, the significance of clinical pathology was evaluated. RESULTS: The positive rate of LMP1 protein was 73.3% and 90.0% in nasopharyngeal epithelial and nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues, respectively. Compared with the LMP1-negative tissues, the protein levels of RPLP0 were low in the nasopharyngeal epithelial and nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues with LMP1-positive expression, but the levels of RPLP0 protein were overexpressed. The protein expression of RPLP0 and RPLP0 was related to the age of nasopharyngeal carcinoma patients, the protein level of LMP1, the metastasis of lymph nodes and the TNM classification. The positive expression of p27 protein at high level was usually observed in the patients with young age, or had the characteristics of LMP1 (-), non-metastasis of lymph nodes, and in I or II stage of TNM classification. However, the protein expression of RPLP0 was low (P<0.05). CONCLUSION: LMP1 down-regulates p27 and up-regulates RPLP0 in nasopharyngeal epithelial and nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues.     

Irbesartan alleviates hepatic steatosis in db/db mice by inducing auto-phagy

AIM: To investigate the effect of irbesartan on the fatty liver of db/db mice and whether autophagy is involved in the process. METHODS: Male db/db mice (n=24) were randomly divided into model group and irbesartan group, and 12 db/m mice with similar age and weight were selected as normal control group. After 16 weeks of intervention respectively, the fatty liver-related parameters including body weight, liver index, blood lipid, liver function and pathological changes in the liver were observed. The protein levels of p-PI3K, p-Akt, and p-mTOR, as well as Atg-7, beclin-1 and LC3B in the liver tissues were detected by Western blot, and the autophagosomes in the liver were observed under electron microscope. RESULTS: Compared with the model group, the body weight, liver index, blood lipids, alanine and aspartate aminotransferase were decreased in irbesartan group (P<0.05). Moreover, the pathological changes in the liver were significantly ameliorated in irbesartan group than that of model group. Importantly, the protein levels of p-PI3K, p-Akt and p-mTOR were decreased with irbesartan administration, while the expression of Atg-7, beclin-1 and LC3B-Ⅱ was increased(P<0.05), which resulted in a distinct increase in autophagosomes. CONCLUSION: Irbesartan alleviates hepatic steatosis in db/db mice by inhibiting the PI3K/Akt/mTOR signaling pathway and upregulating the protein expression of Atg-7, beclin-1 and LC3B-Ⅱ, thereby inducing autophagy in hepatocytes.     

喀西茄内参基因实时荧光定量PCR表达稳定性评价

为了筛选在喀西茄（Solanum aculeatissimum）中表达稳定的内参基因,选取7个常用内参基因GAPDH、ACTIN、18sRNA、UBQ、TUA、EF1和CYP,利用实时荧光定量PCR技术,结合GeNorm、NormFinder和RefFinder软件对其在喀西茄不同组织、不同植物生长调节剂处理、非生物和生物胁迫下的表达稳定性进行评价。结果表明,TUA和18sRNA在喀西茄不同组织中的表达稳定性最好,18sRNA和ACTIN在不同植物生长调节剂处理条件下表达水平最稳定,EF1和GAPDH在干旱和盐条件下表达稳定性最好,TUA和EF1在所有喀西茄测试样品组中（包括不同组织、不同植物生长调节剂处理、非生物及生物胁迫）的表达稳定性最高,而CYP和ACTIN在喀西茄不同试验条件下的表达稳定性较差。     

高温胁迫下茄子qRT-PCR内参基因筛选及稳定性分析

为筛选茄子(Solanum melongena L.)高温胁迫下稳定表达的内参基因,以不同茄子品系(种)为研究对象,利用实时荧光定量PCR技术对来自茄子高温胁迫转录组数据库8个候选内参基因(SmEF1a、SmEF2、Sm40s RPS29、Sm60s RPL24、SmTRX、SmCK I、SmDAHPS I、SmUCP)和SmActin在不同试验情况下进行表达检测,并结合GeNorm、Norm Finder、Best Keeper和ReFinder软件综合评价9个内参基因的表达稳定性。结果表明,在茄子热敏品系05-1和耐热品系05-4经高温处理不同时间的样品和不同组织样品以及10个耐热性不同的茄子品系(种)中,9个内参基因的表达丰度及稳定性存在差异;茄子热敏品系05-1和耐热品系05-4高温胁迫处理不同时间样品中表达稳定性最好的是SmEF1a和SmUCP;其不同组织中表达水平最稳定的是SmEF1a和SmTRX;高温胁迫下不同茄子品系(种)中以SmTRX和SmEF2的表达稳定性最好。综合来看,SmEF1a和SmTRX在所有茄子试验样品中的表达稳定性最好,而SmActin和SmCK I的表达稳定性较差。     

桦褐孔菌实时荧光定量PCR内参基因的筛选

笔者采用软件GeNorm分析7个内参基因在桦褐孔菌(Inonotus obliquus)中表达的稳定性.结果表明,在不同pH培养基处理下,最适内参基因数为2,act和gapdh为最适内参基因;高温刺激下,最适内参基因数为2,act和gapdh为适宜内参基因;在菌核发育阶段,最适内参基因数为2,gapdh和cyp为最适内参基因;在菌丝体发育阶段,最适内参基因数为3,act,tub和ubq为适宜内参基因;在整体试验样本中,最适内参基因数为2,act和gapdh为最稳定的内参基因.     

Effects of Yigu capsule on the level of cbf α-1 mRNA in bone of ovariectomized osteoporosis rats

AIM: To analyze the regulatory effect of Yigu capsule on core binding factor alpha 1 (cbf α-1) gene expression in bone of ovariectomized osteoporosis (OP) rats. METHODS: Thirty-six 10-month old Sprague-Dawley female rats were randomized into three groups: sham-operated group, model group and drug group. After intervention by the corresponding methods, the femurs were collected, SYBR green Ⅰ fluorescence quantitative PCR technique was applied with the internal control of GAPDH according to the relative quantitative formula (2-ΔΔCt), the differentially expressed multiples between the model group or drug group and sham-operated group were calculated. RESULTS: Quantitative formula analysis showed that the level of cbf α-1 gene expression in bone of model groups was decreased than that in sham-operated rats ［compared with sham-operated group, P<0.01, it was (9.9×105)-(1.6×104) times］. While in drug groups the level of cbf α-1 gene expression was 0.19 to 0.92 times than that in the sham-operated group, no significant difference was observed (P>0.05). CONCLUSION: The results of this study show that cbf α-1 gene expression in bone tissue of ovariectomized OP rat is decreased, and Yigu capsule increases the level of cbf α-1 gene expression in bone of OP, indicating that Yigu capsule induces bone marrow mesenchymal stem cells into osteoblasts.     

Role of nitric oxide in reactivity of isolated lymphatics to substance P in shock rats

AIM: To observe the role of nitric oxide (NO) in the reactivity of isolated lymphatics to substance P (SP),which presents a biphasic change, in the hemorrhagic shock (HS) rats with the technique of lymphatic perfusion in vitro. METHODS: Male Wistar rats were randomly divided into control group (surgical procedure only) and shock group (the rats were further divided into shock 0.5 h and shock 2 h groups after the HS model was established). A segment of lymphatics was pressed and perfused in vitro at transmural pressure of 3 cmH₂O after thoracic ducts were separated from the rats at the corresponding time points in each group. The lymphatics of shock 0.5 h and shock 2 h were incubated with different drugs for changing the activity of No and nitric oxide synthase (NOS), respectively. The end-systolic diameter, end-diastolic diameter, contraction frequency (CF) and passive diameter of isolated lymphatics were measured, while the contraction amplitude (CA), tonic index (TI) and fractional pump flow (FPF) were calculated after stimulated with gradient SP. Different values between pre-and post-administration of SP in CF, CA, TI and FPF were calculated and expressed as ΔCF, ΔTI, ΔCA and ΔFPF for further assessing the reactivity of lymphatics. RESULTS: NO donor L-Arg reduced ΔCF, ΔTI and ΔFPF of 0.5 h-shocked lymphatics treated with different concentrations of SP. The effect of L-Arg was obviously suppressed by a soluble guanylate cyclase inhibitor ODQ. ΔCF, ΔTI and ΔFPF increased strikingly compared with shock 0.5 h+L-Arg group in the presence of SP at certain concentration, and ΔCF and ΔFPF increased remarkably compared with control group. NOS inhibitor L-NAME elevated ΔCF, ΔTI and ΔFPF of 2 h-shocked lymphatics treated with different concentrations of SP and the manifestation of lymphatics exceeded the values of control levels. In the experiment of 2 h-shocked lymphatics treated with L-NAME+phosphodiesterase inhibitor aminophylline (AP), the effect of L-NAME was suppressed significantly, which manifested by the decrease in ΔCF, ΔTI and ΔFPF as compared with the values of shock 2 h+L-NAME group in the presence of SP at the concentrations of 1×10^-8 mol/L and 3×10^-8 mol/L. CONCLUSION: These data indicate that NO involves in the biphasic modulation of shocked lymphatics and the effect might be involved in the action of cyclic guanosine monophosphate.     

牡丹泛素延伸蛋白基因ubiquitin克隆及其作为内参基因的研究

以牡丹品种‘洛阳红’（Paeonia suffruticosa‘Luoyanghong’）为试验材料,采用RT-PCR方法获得一个牡丹泛素延伸蛋白基因Psubiquitin（PsUBI）,其cDNA开放阅读框（ORF）长度为447 bp,编码148个氨基酸,GenBank登录号为KP742952。序列比对发现,该基因与其他23种植物泛素延伸蛋白核苷酸序列的相似性均在81%以上,氨基酸序列的相似性达96%。进化分析表明,牡丹泛素延伸蛋白与棉花泛素延伸蛋白的亲缘关系最近。实时荧光定量PCR结果显示,在牡丹不同组织器官中,相对于其他5个常用看家基因（GAPDH、GAPDH1、tubulin、tubulin2、18S rRNA）,ubiquitin的PCR扩增曲线的CT值最为恒定,尤其是在不同花器官中的CT值完全一致。进一步分析发现该基因在牡丹的根、茎、叶片、花、雄蕊和心皮组织中均恒定表达,特别是花器官中的表达量几乎完全一致。以ubiquitin作为内参基因探讨控制花器官发育的基因PsAG的表达情况,结果显示PsAG的表达模式与其作用位点相吻合,ubiquitin更适宜作为牡丹花器官研究的内参基因。