Expression of TMEM16A as a calcium-activated chloride channel in Fischer rat thyroid follicular epithelial cells and its electrophysiologic pro-perties

AIM：To investigate the expression of transmembrane protein 16A(TMEM16A) in Fischer rat thyroid follicular epithelial (FRT) cells and its electrophysiologic properties. METHODS：The eukaryotic expression vector of pUB6/V5-TMEM16A was constructed and transfected into FRT cells by liposome-mediated transfection. In order to obtain the high efficiency of gene transfection and expression, the quantity and ratio of lipid/DNA complexes were optimized. The FRT cells stably expressing TMEM16A were gained by the selection with blasticidin and confirmed by the techniques of RT-PCR and immunofluorescence. The expression and location of TMEM16A in the FRT cells were observed under an inverted fluorescence microscope. TMEM16A protein was associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent protein and patch-clamp technique. RESULTS：The results of double digestion and sequencing indicated that TMEM16A was cloned into pUB6/V5. The results of RT-PCR and immunofluorescence confirmed that TMEM16A was expressed in the FRT cells after transfection with TMEM16A. The classical calcium-activated chloride channel currents were recorded in the FRT cells stably expressing TMEM16A by the technique of patch-clamp and halide-sensitive fluorescent protein YFP-H148Q/I152L. CONCLUSION：The protein expression of TMEM16A in the FRT cells was observed. TMEM16A is the molecular identity of calcium-activated chloride channels.     

Construction and identification of FRT cell line stably expressing UT-A2

AIM: To obtain an FRT cell line that can stably express urea transporter A2 (UT-A2) and provide a cell model for screening UT-A2 inhibitors. METHODS: FRT cells stably expressing aquaporins 1 (AQP1) and YFP were transfected with the recombinant plasmid pUB6/V5-UT-A2 by eukaryotic expression plasmid-lipoplast mediating pathway. The stable UT-A2-FRT cell line was cloned by selection with BSD and confirmed by Western blotting and immunofluorescence staining. The urea permeability across the plasma membrane was detected by a 2 mol/L urea lysis assay. RESULTS: We have obtained a stable UT-A2-FRT cell line. Western blotting analysis showed that UT-A2 protein was expressed stably in this cell line. The immunofluorescence staining detection indicated UT-A2 expression in the plasma membrane. It was found that there was significant urea permeability in this cell line by 2 mol/L urea lysis assay. CONCLUSION: We constructed an FRT cell line that could stably express UT-A2 in plasma membrane in the non-renal epithelia cell. The cell line will be used to screen UT-A2 inhibitors.     

乙酰苯胺和2-氯苯磺酰胺诱导IN2-2启动子在芥菜中的表达

分别用不同浓度(50、100、200mg·L-1)的除草剂安全剂类似化合物乙酰苯胺和2-氯苯磺酰胺处理IN2-2::GUS转基因芥菜。结果显示:乙酰苯胺和2-氯苯磺酰胺处理后,IN2-2启动子在芥菜根、叶、花器官的花萼、花瓣、雄蕊及花粉中表达,但不在胚珠中表达。100mg·L-1乙酰苯胺以及50mg·L-12-氯苯磺酰胺适合用于在芥菜中调控IN2-2启动子的表达,乙酰苯胺较2-氯苯磺酰胺诱导表达所需时间更短。高浓度的2-氯苯磺酰胺影响芥菜种子发芽及生长发育,并且明显抑制IN2-2启动子的表达活性。4℃低温胁迫诱导IN2-2启动子在幼苗叶片中表达,IN2-2启动子轻微受150mmol·L-1Na Cl胁迫表达,但不受重金属Gu~(2+)离子的诱导表达。     

Expression and identification of ANO1 in mouse cardiomyocytes

AIM: To explore the expression of anoctamin 1 (ANO1), one of calcium-activated chloride channels (CaCCs), in mouse cardiomyocytes and its functional properties. METHODS: The cardiomyocytes from the myocardial tissues of C57BL/6 mice were isolated with enzyme and purified by the differential adherent method. The cells were stained with monoclonal anti-sarcomeric actin and Cy3 to evaluate the purity of the myocardial cells. RT-PCR was used to detect the mRNA expression of ANO1 in the mouse cardiomyocytes. The protein expression of ANO1 in the mouse cardiomyocytes was determined by Western blotting analysis. The fluorescence quenching kinetics experiment was used to identify the ion transport properties of ANO1 in the mouse cardiomyocytes. RESULTS: The results of RT-PCR confirmed that ANO1 was expressed in freshly isolated myocardial cells. The results of Western blotting clearly demonstrated the protein expression of ANO1 in primarily cultured myocardial cells. Fluorescence quenching kinetics experiment on freshly isolated single myocardial cell revealed a pronounced outward rectifying property of the ANO1. The functional properties were similar to the classic CaCCs. CONCLUSION: ANO1 expression was identified in the mouse myocardial cells. The function of CaCCs was generated by ANO1, suggesting that ANO1 is the molecular basis of CaCCs.     

契斯曼尼番茄果实中可溶性固形物和β–胡萝卜素含量相关基因QTL 分析

 以野生番茄契斯曼尼LA0317与栽培番茄自交系9706为亲本，构建了包括152个株系的BC₂S₆群体。利用155个SSR标记、21个CAPS标记和88个Indel标记构建了契斯曼尼番茄的遗传连锁图谱，总图距1 134.0 cM，标记间平均距离4.29 cM。采用复合QTL区间作图法对番茄果实中的可溶性固形物含量和β–胡萝卜素含量进行QTL分析。定位到10个与可溶性固形物含量相关的QTLs，分别位于第3、4、5、6、9、10、11号染色体上，其中有5个QTL效应值在12%以上。定位到6个与β–胡萝卜素含量相关的QTLs,分别位于第1、2、6、9号染色体上。其中,位于6号染色体上的qBC6b效应值达到54.7%，为一主效QTL。此外，还获得了可用作育种材料的5个高可溶性固形物含量株系和8个高β–胡萝卜素含量的株系。     

Expression and mechanisms of hsa_circ_087631 in patients with primary biliary cholangitis

AIM To explore the expression and mechanisms of circular RNA hsa_circ_087631 in the patients with primary biliary cholangitis （PBC）. METHODS RT-qPCR was used to detect the expression of hsa_circ_087631 in PBC patients and healthy controls. Hsa-miR-346-overexpressing lentiviral vector pLenti-EF1a-EGFP-F2A-Puro-CMV-MCS was constructed and transfected into human acute T cell leukemia Jurkat cells， and then the expression levels of hsa_circ_087631， Bcl-6 mRNA and interleukin-21 （IL-21） mRNA were detected by RT-qPCR. Dual-luciferase reporter assay was performed to identify the interactions between hsa_circ_087631 and hsa-miR-346. RESULTS The expression of hsa_circ_087631 in the PBC patients was significantly increased compared with the healthy subjects. Hsa-miR-346-overexpressing lentiviral vector pLenti-EF1a-EGFP-F2A-Puro-CMV-MCS was successfully constructed. The expression of hsa-miR-346 was significantly increased after the hsa-miR-346-overexpressing lentiviral vector was transfected into the Jurkat cells， while the expression levels of hsa_circ_0087631， Bcl-6 mRNA and IL-21 mRNA were significantly decreased. After wild-type or mutant hsa_circ_087631 vector and hsa-miR-346 mimics were transfected into 293T cells， the results of dual-luciferase reporter assay showed that hsa-miR-346 significantly decreased the luciferase activity of wild-type hsa_circ_087631 （P<0.01）， but the regulation did not change significantly after mutation of the predicted binding site. CONCLUSION Peripheral blood hsa_circ_087631 level is elevated in the PBC patients. The hsa_circ_087631/hsa-miR-346/Bcl-6 signaling may take effect in human T cells. Hsa-miR-346 significantly reduces the expression of hsa_circ_087631， but it may not be regulated by predicted binding sites.     

低温胁迫下北海道黄杨叶肉细胞Ca2+和Ca2+-ATPase的变化

 利用焦锑酸钙沉淀和硝酸铅沉淀的电镜细胞化学方法,以室温生长的北海道黄杨植株为对照,研究了人工4 ℃低温胁迫过程中北海道黄杨（Euonymus japonicus‘Cuzhi’）叶肉细胞Ca²⁺和Ca²⁺-ATPase的动态变化。在4 ℃低温胁迫的初期（3 ~ 12 h）,北海道黄杨叶肉细胞间隙和液泡内的Ca²⁺沉淀颗粒减少,而细胞质和细胞核内的Ca²⁺水平升高,但Ca²⁺-ATPase在细胞的分布几乎没有变化,主要分布在质膜和液泡膜上,有较高的活性;低温胁迫24 h,细胞质和细胞核内增加的Ca²⁺开始回到细胞间隙和液泡中,Ca²⁺-ATPase在质膜和液泡膜上活性增强;在低温胁迫48 ~ 96 h,细胞内的Ca²⁺又回到低温胁迫前的低水平,但Ca²⁺-ATPase在质膜和液泡膜上仍有很高的活性。叶肉细胞内Ca²⁺稳态平衡和Ca²⁺-ATPase的活性变化与植物的抗寒性存在一定的相关性。     

CDA-2 induces differentiation and inhibits proliferation of human SWO-38 glioma cells

AIM: To investigate the differentiation-inducing effect of cell differentiation agent-2 (CDA-2) in human SWO-38 glioma cell line in vitro.METHODS: The inhibitory effect of CDA-2 on cell proliferation was assessed by MTT assay and colony formation assay.Cell morphology was determinded by light microscopy observation,and the expression of GFAP (glial fibrillary acidic protein) was detected by immunohistochemistry and Western blotting.Western blotting was also applied to explore the expression of PPARγ and COX-2.RESULTS: The data showed that CDA-2 inhibited proliferation and induced differentiation of SWO-38 cells.The inhibition efficiency was time-dependent and dose-dependent .The IC₅₀ of CDA-2 was (2.33±0.37) g/L and (0.51±0.01) g/L,respectively when cells were treated for 72 h and 10 days.CDA-2 caused differentiation of human glioma cells as indicated by outgrowth of long processes and expression of astrocyte marker GFAP.Simultaneously,the expression of PPARγ increased after 3 h of CDA-2 treatment，while the expression of COX-2 decreased after 48 h of CDA-2 treatment.CONCLUSION: CDA-2 inhibits proliferation and induces differentiation of SWO-38 cells.These effects may be through increasing cellular GFAP,PPARγ level and decreasing COX-2 expression induced by CDA-2.     

Overexpression of Smad7 inhibits TGF-β-induced Smad2 mRNA and protein expression in peritoneal mesothelial cells

AIM: To investigate the role of Smad7 in the Smad2 expression induced by transforming growth factor-β₁ (TGF-β₁) in rat peritoneal mesothelial cells (PMCs).METHODS: Rat PMCs were cultured at different doses of TGF-β₁ (0，1.25，2.5，10 μg/L) for different time (0，5，15，30，60，120 min).PCDNA3-Smad7 was then transfected into cultured rat PMCs by lipofectamine, and the cells were stimulated like the above.Endogenous Smad2 and Smad7 expression was evaluated by RT-PCR and Western blotting.RESULTS: TGF-β₁ induced increase in Smad2 mRNA and protein expression at 5 min, peaked at 30 min, and declined to baseline levels at 120 min, which was in a time-dependent manner.TGF-β₁ also induced Smad7 mRNA expression at 5 min, and then declined, down to the lowest at 30 min, but at 60 min it increased again.Smad2, Smad7 mRNA and protein expression induced by TGF-β₁ were also dose-dependent.After transfection, overexpressions of Smad7 mRNA and protein in rat PMCs were observed, which did not decline with time.The expression of Smad2 mRNA significantly decreased by 33%, 56%, 67%, 71%, 63% and 57% (P<0.05), the expression of Smad2 protein declined by 78%，89%，89%，88% and 76% (P<0.05) respectively at 0, 5, 15, 30, 60 and 120 min.CONCLUSION: Overexpression of Smad7 inhibits Smad2 gene and protein expression in peritoneal mesothelial cells.Smad7 may be a negative regulator of TGF-β₁ signaling.     

牡丹休眠相关基因PsGRASs筛选、克隆和表达特性分析

通过筛选、克隆牡丹PsGRASs基因并进行表达模式分析,初步解析其是否参与牡丹休眠解除,以期为牡丹反季节催花提供候选基因。利用HMM(Hidden Markov Model)模型,基于GRAS基因家族的种子(Pfam号为PF03514)序列,利用BioEdit软件对牡丹花芽休眠的454测序转录组数据进行本地检索,筛选到2个具有GRAS结构域的序列。通过RACE扩增得到了其全长cDNA,其中PsGRAS1序列为2 308 bp,开放阅读框(open reading frame,ORF)为1 848 bp,编码615个氨基酸;PsGRAS2为2 034bp,ORF为1560bp,编码518个氨基酸,两个编码PsGRASs蛋白的同源性为19.87%。进化树结果表明,PsGRAS1蛋白与拟南芥GRAS家族中的DELLA亚家族的GAI和RGLs聚到一个大的分支,PsGRAS2蛋白与拟南芥HAMs(AtSCL6/15/22/26/27)并为一支,说明PsGRAS1和PsGRAS2来自GRAS家族的两个不同分支。组织表达结果显示PsGRAS1和PsGRAS2在牡丹初花期不同组织中表达水平不同,PsGRAS1在叶中表达水平最高,在雄蕊中最低;而PsGRAS2在苞片中表达水平最高,在花瓣中最低。在牡丹花芽休眠解除过程中,PsGRAS1呈下调趋势,低温28 d表达水平最低,而PsGRAS2呈先上调后下调趋势,低温14 d表达水平最高。在外源GA3处理7 d的牡丹花芽中,PsGRAS1的表达显著下降,而PsGRAS2显著提高。这表明PsGRAS1和PsGRAS2都参与了休眠过程,但功能不同。     

与黄瓜抗白粉病相关基因连锁的AFLP标记的获得

 以黄瓜抗白粉病母本Q9和感白粉病父本Q10及组合(津春3号)的F 分离群体为试材，采用BSA法建立了对白粉病的抗病组和感病组，AFLP引物组合P18M47在两组间表现多态，且呈共显性。经F₂单株分析，在高抗个体和高感个体中分别仅扩增出约238 bp和236 bp的特异片段，而在中间类型个体中同时扩增出了该两个特异片段(238 bp／236 bp)，经连锁值测定，表明该特异带与白粉病抗病相关基因紧密连锁，连锁距离为5.56 cM。测序结果显示，目标片段的大小分别为238 bp和236 bp，且两个片段的差异仅为两个“T”碱基的插入或缺失。BLAST查询表明该两个片段为新发现的黄瓜DNA序列。     

大白菜渗透压应激活化蛋白激酶基因SRK2F的克隆与表达

以不耐盐、不耐旱的大白菜自交系SY-14-06为试材,提取根部总RNA,反转录为c DNA。根据大白菜SRK2F基因设计引物,PCR扩增SRK2F基因CDS序列1044bp。SRK2F编码347个氨基酸,预测分子量为39.3k D,理论等电点为4.88。利用p EASY-E1原核表达载体构建原核表达质粒p EASY-E1-SRK2F,转化表达菌株Transetta(DE3),通过SDS-PAGE检测该蛋白的表达。经Smart-embl预测其具有丝/苏氨酸激酶特有结构域,位于第4~260位氨基酸处。经Clustal X2比对,其与拟南芥同源性最高。最后利用镍离子金属螯合亲和层析介质对该蛋白进行纯化,得到了纯化的融合蛋白。     

Construction of shRNA targeting AGR2 gene and effect of AGR2 on biological function of nasopharyngeal carcinoma cells

AIM: To construct the shRNA targeting anterior gradient protein 2 (AGR2) gene for exploring the effect of AGR2 on the biological behavior of nasopharyngeal carcinoma (NPC) cells.METHODS: The expression of AGR2 at mRNA and protein levels in NPC cell lines 6-10B and 5-8F was detected by real-time PCR and Western blot. The pSR-GFP/Neo-AGR2-shRNA expression vector targeting AGR2 was constructed. Based on the interference targeting AGR2, the cell migration and motility were determined by Transwell migration and motility assays.RESULTS: The expression of AGR2 was increased in NPC cell line 5-8F compared with NPC cell line 6-10B (P<0.05). When the AGR2 expression in 5-8F cells was interfered, the cell migration, invasion and tumorigenicity were weakened.CONCLUSION: The expression of AGR2 is up-regulated in NPC cell line 5-8F. pSR-GFP/Neo-CLU-shRNA successfully inhibits the expression of AGR2 in NPC cell line 5-8F. AGR2 inhibits the migration, invasion and tumorigenicity of 5-8F cells in vivo.     

Identification of HLA-A2 restricted cytotoxic T lymphocyte epitopes from tumor antigen PIWIL2

AIM: To identify the human leucocyte antigen A2 (HLA-A2) restricted cytotoxic T lymphocyte (CTL) epitopes from tumor antigen PIWIL2. METHODS: RT-PCR and Western blot was used to determine the expression of PIWIL2 in cancer cell lines MCF-7, SW480 and HT-29. HLA-A2 epitopes from PIWIL2 protein were predicted by the software of BIMAS, RankPep, NetMHC, NetCTL1.2 and IEDB. The peptides were synthesized by standard solid-phase methods. The binding affinity of the peptides to HLA-A2 molecules was evaluated by T2 cells binding assay. ELISPOT assay was used to investigate the levels of IFN-γ. The cytotoxicity assay in vitro was also used to determine the ability of inducing T cell response by the peptides. RESULTS: The expression of PIWIL2 was observed in MCF-7, SW480 and HT-29. The candidate peptide P485, P493 and P965 showed moderate affinity toward HLA-A2 molecule. ELISPOT assay showed P485 and P965 induced CTLs of IFN-γ release form CTLs. The CTLs induced by P485 and P965 lysed the MCF-7 cells. CONCLUSION: The peptides P485 and P965 are excellent HLA-A2 restricted cytotoxic T lymphocyte epitopes from the tumor antigen PIWIL2, which could serve as new candidates towards antitumor peptide vaccines.     

Effect of human xeroderma pigmentosum D on expression of Mdm2 and Mdm4 in HepG2 cells

AIM：To investigate the effects of human xeroderma pigmentosum D (XPD) on the expression of murine double minute 2 (Mdm2) and murine double minute 4 (Mdm4) in human hepatoma cells. METHODS：Recombinant plasmid pEGFP-N2/XPD and vacant plasmid pEGFP-N2 were transfected into HepG2 cells using liposome, and the cells were divided into blank control group, pEGFP-N2 group and pEGFP-N2/XPD group. The cell growth was detected by MTT assay. The cell cycle and apoptotic rate were examined by flow cytometry. The mRNA and protein expression levels of XPD, Mdm2, Mdm4 and P53 were determined by RT-PCR and Western blotting. RESULTS：The results of MTT assay showed that the cell growth was inhibited by the transfection of pEGFP-N2/XPD. The results of flow cytometry showed that the transfection of pEGFP-N2/XPD increased the cell number in G 1 phase, decreased the cell number in S phase and increased the apoptotic rate of HepG2 cells. The results of RT-PCR and Western blotting showed that the transfection of pEGFP-N2/XPD increased the expression of XPD, decreased the expression of Mdm2 and Mdm4, and increased the expression of P53. CONCLUSION：XPD down-regulates Mdm2 and Mdm4 expression and up-regulates P53 expression in hepatoma cells. Moreover, the proliferation of hepatoma cells can be inhibited and the apoptosis can be induced by XPD.     

蝴蝶兰AP2/ERF家族基因的克隆及在低温下表达特性分析

用RT-PCR方法从‘大辣椒’蝴蝶兰叶片中克隆得到4个AP2/ERF家族基因，命名为PhAP2/ERF1 ~ PhAP2/ERF4。蛋白结构域和序列比对发现PhAP2/ERF1 ~ PhAP2/ERF4均含有1个AP2结构域， PhAP2/ERF2还含有1个B3结构域。进化树分析表明4个蛋白与兰科AP2/ERF家族成员亲缘关系最近，PhAP2/ERF1属于AP2/ERF家族DREB亚家族中的A1类，PhAP2/ERF2属于RAV亚家族，PhAP2/ERF3属于ERF亚家族的B4类，PhAP2/ERF4属于DREB亚家族中的A2类。用实时荧光定量PCR方法检测低温驯化和低温胁迫条件下PhAP2/ERF1 ~ PhAP2/ERF4在‘大辣椒’和‘富乐夕阳’蝴蝶兰叶片中的表达特性，结果表明：4个基因在‘大辣椒’叶片中的表达趋势一致，表达量在低温胁迫早期达到最高，在胁迫中晚期有所下降；而4个基因在‘富乐夕阳’叶片中的表达趋势有较大差异，PhAP2/ERF1和PhAP2/ERF2的表达在低温处理整个过程中没有被诱导或仅有较小幅度增加，PhAP2/ERF3对低温的响应时间显著晚于‘大辣椒’，PhAP2/ERF4的表达趋势与‘大辣椒’差异较小。蝴蝶兰AP2/ERF在抗冷品种‘大辣椒’和不抗冷品种‘富乐夕阳’叶片中的表达差异暗示AP2/ERF基因家族在蝴蝶兰低温胁迫响应中起重要调控作用。     

miR-222 enhances HBx-HepG2 cell growth via regulation of BCL2L13 gene

AIM: To investigate the regulation of miR-222 on BCL2L13 gene and its effect on the growth and apoptosis of HBx-HepG2 cells, and to explore the underlying molecular mechanisms. METHODS: The expression level of miR-222 was detected by RT-qPCR. The HBx-HepG2 cell growth was examined by MTT and colony formation assays. The cell cycle and apoptosis were analyzed by flow cytometry. The recombination vector pmirGLO-BCL2L13 was constructed, and dual-luciferase reporter experiment was performed to validate the target of miR-222. RESULTS: The expression level of miR-222 in the HBx-HepG2 cells was significantly higher than that in the L02 cells (P<0.05). Over-expression of miR-222 enhanced HBx-HepG2 cell growth, changed cell cycle, and inhibited apoptosis (P<0.05). Knockdown of miR-222 reduced HBx-HepG2 cell growth, changed cell cycle, and increased cell apoptotic rate (P<0.05). BCL2L13 was down-regulated in the HBx-HepG2 cells as compared with L02 cells (P<0.05), and knockdown of miR-222 in the HBx-HepG2 cells increased the expression level of BCL2L13 (P<0.05). The results of dual-luciferase reporter assay and restore experiment showed that miR-222 negatively regulated the expression of BCL2L13 via targeting 3'UTR of BCL2L13, resulting in the promotion of HBx-HepG2 cell growth. CONCLUSION: miR-222 enhances HBx-HepG2 cell growth via down-regulation of BCL2L13.     

植物中Gibberellin 2-oxidases基因研究进展

赤霉素2–氧化酶(Gibberellin 2-oxidases,GA2oxs)是赤霉素代谢过程中的关键酶,根据底物特异性其可分为C20-GA2oxs和C19-GA2oxs两类,其中C20-GA2oxs直接作用于GA合成前体(GA12,GA53),C19-GA2oxs负责分解活性GA(GA1,GA4)及其前体(GA20,GA9)。GA2oxs由多基因编码,参与维持植物生长发育多个过程,受外源环境与内源因素严格而精密调控。研究表明GA2oxs通过调节植物体内赤霉素水平影响植物的生长发育,如株高、种子萌发、开花和结实等。主要综述了GA2oxs基因时空表达模式,生理功能,转录调控等方面的研究进展,为了解其对植物生长发育的调控机制提供参考,同时也有助于在生产实践中更好地利用其开展基因工程研究,定向培育植物新品种。