川滇高山栎QaGDU3基因的克隆及遗传转化

以川滇高山栎(Quercus aquifolioides Rehd. et Wils.)叶片为材料,分别采用不依赖于连接反应的克隆法、农杆菌花序侵染、实时荧光定量PCR、图像分析等方法克隆了QaGDU3基因,获得QaGDU3转基因拟南芥(Arabidopsis thaliana(L.)Heynh.)并分析转基因植物的表型变化,利用生物信息学对QaGDU3蛋白与其他物种的GDU3蛋白序列间的差异和进化关系进行了分析。结果显示,GDU3是种子植物特有的基因;QaGDU3属于阴离子渗透酶ArsB/NhaD超家族成员,可能具有氨基酸转运功能。QaGDU3基因在拟南芥中过表达能够激活水杨酸(SA)途径中AtPR1、AtACD6、AtCBP60g和AtPAD4基因的表达,且转基因拟南芥莲座大小随着QaGDU3基因表达量升高而变小。研究结果说明GDU3是种子植物所特有的氨基酸转运蛋白并参与植物生长发育的调控。     

葡萄蔗糖转化酶基因家族的生物信息学及响应逆境胁迫分析

蔗糖转化酶(invertase, INV)在植物生长发育和抵御胁迫中发挥着重要作用。研究从葡萄基因组数据库中鉴定出19个蔗糖转化酶基因,对基因结构和编码蛋白质的理化性质进行生物信息学分析,并利用qRT-PCR技术分析基因在不同激素和非生物胁迫条件下的表达特征,为进一步探索葡萄INV基因家族参与葡萄逆境响应提供了一定的理论依据。结果表明,(1)该基因家族编码蛋白的氨基酸长度在150～766 aa之间,理论等电点介于4.43～9.1之间,亚细胞定位预测发现其主要在细胞质中表达,此外液泡和细胞壁也存在部分基因表达;(2)共线性结果显示VvCINV与其他5个物种复制频率较高;(3)保守基序分析表明VvCwINV包含了所有的保守基序,且Glyco＿32和Glyco＿hydro＿100是VvINV基因主要结构域;(4)组织特异性表达分析发现多数基因在葡萄生长发育进程中都有表达;(5)qRT-PCR分析结果显示,VvINV基因家族在叶片中对激素处理和非生物胁迫的响应出现上调,VvCINV1在50 mg/L GA₃和10%PEG处理后上调表达极显著,VvCINV4在盐胁迫、ABA...     

实时荧光定量PCR分析中毛果杨内参基因的筛选和验证

实时荧光定量PCR(qRT-PCR)技术具有高灵敏性、高保真性和高特异性, 被广泛应用于基因表达的分析。在数据处理过程中, 选用稳定表达的基因作为内参基因对准确分析实验结果非常关键。以毛果杨(Populus trichocarpa)的不同组织以及锌胁迫下的组培苗为材料, 使用荧光定量PCR方法分析了TUA8、TUB6、ubiquitin、GAPDH、actin、18S rRNA和EF1α 7个看家基因的表达情况。通过geNorm、NormFinder和BestKeeper 3个程序的综合分析, 发现actin、ubiquitin、EF1α和18S rRNA的稳定性较好, 可用作毛果杨基因表达研究的内参基因; 而TUB6在不同组织中稳定性最差; GAPDH在锌胁迫下的组织中稳定性最差, 因此不适宜作为内参基因。毛果杨NAC基因的表达分析, 进一步验证了上述结果。该研究对采用qRT-PCR方法分析毛果杨基因表达过程中内参基因的选择具有指导作用, 同时对揭示NAC基因的功能也有一定的意义。     

苹果MAX1基因的克隆和表达及启动子分析

MAX1(MORE AXILLARY GROWTH1)是独脚金内酯(Strigolactones,SLs)合成途径中的关键基因,为了研究MAX1在苹果分枝调控中的功能,以苹果‘长富2号’(Malus domestica‘Nagafu 2’)腋芽为材料,采用PCR方法,克隆MdMAX1基因,进行生物信息学和表达水平分析;采用瞬时表达转化烟草,进行GUS染色,分析MdMAX1启动子活性。结果表明:(1)成功克隆得到苹果MAX1,其开放阅读框(ORF)1620 bp,编码539个氨基酸;系统进化和基序分析表明,MdMAX1和已知的A1型MAX1相似。(2)qRT-PCR分析表明,MAX1基因在‘长富2号’嫁接苗茎中高表达,并在腋芽本身有表达;RNA-seq分析表明,细胞分裂素(6-BA)处理苹果腋芽96 h后MAX1基因的表达水平显著降低。(3)成功克隆获得‘长富2号’MAX1启动子序列片段(1500 bp),顺式作用元件预测显示MdMAX1启动子序列中存在光响应元件,GUS活性分析表明光照处理能够减弱MAX1启动子的活性。该研究为进一步研究苹果MAX1参与SLs合成、调控苹果分枝的功能奠定了基础。     

The Expression of SIRT1 and DBC1 in Laryngeal and Hypopharyngeal Carcinomas

Rationale and Objective

Sirtuin 1 (SIRT1) plays an important role in tumorigenesis and is increased in many human tumors. DBC1 is a negative regulator of SIRT1 via promotion of p53-mediated apoptosis. It is necessary to investigate the expression of SIRT1 and DBC1 in laryngeal and hypopharyngeal squamous cell carcinomas (LSCC and HSCC) and its correlation with available clinical parameters.

Methods

The mRNA levels of SIRT1 and DBC1 were measured in 54 paired LSCC or HSCC tumors and corresponding adjacent noncancerous mucosae using quantitative RT-PCR (qRT-PCR). The protein levels of SIRT1 and DBC1 were also evaluated in 120 cases of patients with LSCC or HSCC using immunohistochemical staining. The correlation between SIRT1 and DBC1 expression and clinical parameters was analyzed with Pearson chi-square test.

Results

qRT-PCR assay showed that, compared with the paired adjacent noncancerous mucosae, SIRT1 mRNA was significantly decreased in tumors. The immunohistochemical results indicated that the SIRT1 protein was also downregulated in tumors compared with noncancerous mucosae. Moreover, decreased SIRT1 was significantly correlated with the tumor clinical stage and lymph node metastasis. Additionally, DBC1 mRNA was significantly increased in tumors compared with noncancerous mucosae. The immunohistochemical results indicated that the DBC1 protein was downregulated in tumors, which is inconsistent with the results obtained by qRT-PCR. Finally, decreased DBC1 protein was significantly correlated with tumor differentiation, lymph node metastasis, and p53 expression.

Conclusions

SIRT1 and DBC1 might be involved in the pathophysiology of laryngeal and hypopharyngeal squamous cell carcinomas and are associated with lymph node metastasis and p53 positive staining in LSCCs and HSCCs.     

MiR-422a as a Potential Cellular MicroRNA Biomarker for Postmenopausal Osteoporosis

Background

MicroRNAs (miRNAs) are a class of short non-coding RNA molecules that regulate gene expression by targeting mRNAs. Recently, miRNAs have been shown to play important roles in the etiology of various diseases. However, little is known about their roles in the development of osteoporosis. Circulating monocytes are osteoclast precursors that also produce various factors important for osteoclastogenesis. Previously, we have identified a potential biomarker miR-133a in circulating monocytes for postmenopausal osteoporosis. In this study, we aimed to further identify significant miRNA biomarkers in human circulating monocytes underlying postmenopausal osteoporosis.

Methodology/Principal Findings

We used ABI TaqMan miRNA array followed by qRT-PCR validation in human circulating monocytes from 10 high BMD and 10 low BMD postmenopausal Caucasian women to identify miRNA biomarkers. MiR-422a was up-regulated with marginal significance (P = 0.065) in the low compared with the high BMD group in the array analysis. However, a significant up-regulation of miR-422a was identified in the low BMD group by qRT-PCR analysis (P = 0.029). We also performed bioinformatic target gene analysis and found several potential target genes of miR-422a which are involved in osteoclastogenesis. Further qRT-PCR analyses of the target genes in the same study subjects showed that the expression of five of these genes (CBL, CD226, IGF1, PAG1, and TOB2) correlated negatively with miR-422a expression.

Conclusions/Significance

Our study suggests that miR-422a in human circulating monocytes (osteoclast precursors) is a potential miRNA biomarker underlying postmenopausal osteoporosis.     

苹果叶绿素合成关键酶基因MdHEMA1生物信息学和表达分析

HEMA1编码谷氨酰-tRNA还原酶(Glu TR)的合成,是叶绿素生物合成的关键酶基因。本研究利用RACE技术从苹果叶片中克隆Glu TR的编码基因,将其命名为MdHEMA1。生物信息学分析表明:MdHEMA1基因位于苹果8号染色体上,其CDS长1638 bp,编码545个氨基酸残基,蛋白分子量为59279.4 Da,等电点为8.45。蛋白序列及结构分析显示该蛋白包含保守的谷氨酰-tRNA还原酶的N端结构域、莽草酸/奎尼酸脱氢酶结构域及谷氨酰-tRNA还原酶二聚结构域。进化树分析显示MdHEMA1蛋白与白梨(Pyrus×bretschneideri)Pb HEMA1亲缘关系最近。qRT-PCR结果显示,MdHEMA1在根、茎、叶、花、果实各组织器官中均有表达,但光合组织(茎、叶、果实)中的表达水平较高;该基因在叶片和果实不同发育期表达存在差异,表达量与叶片和果实内叶绿素含量变化趋势一致;而且干旱胁迫能够诱导该基因表达。启动子分析显示MdHEMA1基因启动子区域含有多种非生物胁迫相关的顺式作用元件。     

Avoiding Pitfalls of Internal Controls: Validation of Reference Genes for Analysis by qRT-PCR and Western Blot throughout Rat Retinal Development

Background

Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development.

Methodology/Principal Findings

We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed.

Conclusion

Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.     

甜橙SPL基因家族的鉴定及其在成花诱导中的表达分析北大核心CSCD

SQUAMOSA promoter binding protein-like(SPL)基因家族在植物成花转变与花器发育中起重要调控作用。该研究基于甜橙(Citrus sinensis)基因组数据,对甜橙SPL家族成员及其启动子区进行生物信息学分析,并采用qRT-PCR检测其在甜橙不同花期、不同品种花芽和叶片的表达特异性,为深入探讨CsSPLs基因在甜橙成花诱导中的功能奠定基础。结果表明:(1)共鉴定出15个含有SBP保守结构域的甜橙SPL基因,分别命名为CsSPL1~CsSPL15,且分布在6条染色体上。(2)CsSPL1~CsSPL15基因编码的氨基酸长度为130~1075 aa,分子量为14.73~118.75 kD;系统发育分析将15个CsSPLs分成8个亚族,除CsSPL4外,其他CsSPLs均与拟南芥SPL家族成员聚类。(3)顺式作用元件分析表明,CsSPLs启动子中含有大量光响应元件,推测CsSPLs可能在甜橙响应光调控过程中发挥重要作用。(4)转录组数据分析显示,CsSPLs在甜橙愈伤组织、花、叶片和果实中均有表达,其中CsSPL3、CsSPL4、CsSPL7、CsSPL8、CsSPL12、CsSPL13、CsSPL14和CsSPL15在花和叶片中较高表达。(5)qRT-PCR检测显示,CsSPLs在甜橙不同花期、不同品种花芽和叶片组织中的表达均有显著上调趋势,且采样各时期下早花品种‘赣南早’花芽和叶片中CsSPLs的表达量均高于对照品种‘纽荷尔’。研究认为,甜橙成花组织内CsSPLs基因的高表达是其花期提前的主要因素。     

Differential expression of T-type calcium channels in P/Q-type calcium channel mutant mice with ataxia and absence epilepsy

Mutations in P/Q-type calcium channels generate common phenotypes in mice and humans, which are characterized by ataxia, paroxysmal dyskinesia, and absence seizures. Subsequent functional changes of T-type calcium channels in thalamus are observed in P/Q-type calcium channel mutant mice and these changes play important roles in generation of absence seizures. However, the changes in T-type calcium channel function and/or expression in the cerebellum, which may be related to movement disorders, are still unknown. The leaner mouse exhibits severe ataxia, paroxysmal dyskinesia, and absence epilepsy due to a P/Q-type calcium channel mutation. We investigated changes in T-type calcium channel expression in the leaner mouse thalamus and cerebellum using quantitative real-time polymerase chain reaction (qRT-PCR) and quantitative in situ hybridization histochemistry (ISHH). qRT-PCR analysis showed no change in T-type calcium channel alpha 1G subunit (Cav3.1) expression in the leaner thalamus, but a significant decrease in alpha 1G expression in the whole leaner mouse cerebellum. Interestingly, quantitative ISHH revealed differential changes in alpha 1G expression in the leaner cerebellum, where the granule cell layer showed decreased alpha 1G expression while Purkinje cells showed increased alpha 1G expression. To confirm these observations, the granule cell layer and the Purkinje cell layer were laser capture microdissected separately, then analyzed with qRT-PCR. Similar to the observation obtained by ISHH, the leaner granule cell layer showed decreased alpha 1G expression and the leaner Purkinje cell layer showed increased alpha 1G expression. These results suggest that differential expression of T-type calcium channels in the leaner cerebellum may be involved in the observed movement disorders.     

MYOZ2通过负调控TCAP的表达促进C3H10T1/2细胞成脂分化

本研究旨在明确钙调磷酸酶肌小节结合蛋白2(myozenin2,MYOZ2)的组织表达特性,阐明其对C3H10T1/2细胞成脂分化的影响及可能的作用机制.采集180日龄马身猪、60日龄ICR小鼠、35日龄罗斯肉鸡和12月龄小尾寒羊背最长肌、皮下脂肪和肝组织,检测MYOZ2基因mRNA表达.结果 显示,MYOZ2基因在所检...     

Prognostic Value and Targeted Inhibition of Survivin Expression in Esophageal Adenocarcinoma and Cancer-Adjacent Squamous Epithelium

Background

Survivin is an inhibitor of apoptosis and its over expression is associated with poor prognosis in several malignancies. While several studies have analyzed survivin expression in esophageal squamous cell carcinoma, few have focused on esophageal adenocarcinoma (EAC) and/or cancer-adjacent squamous epithelium (CASE). The purpose of this study was 1) to determine the degree of survivin up regulation in samples of EAC and CASE, 2) to evaluate if survivin expression in EAC and CASE correlates with recurrence and/or death, and 3) to examine the effect of survivin inhibition on apoptosis in EAC cells.

Methods

Fresh frozen samples of EAC and CASE from the same patient were used for qRT-PCR and Western blot analysis, and formalin-fixed, paraffin-embedded tissue was used for immunohistochemistry. EAC cell lines, OE19 and OE33, were transfected with small interfering RNAs (siRNAs) to knockdown survivin expression. This was confirmed by qRT-PCR for survivin expression and Western blot analysis of cleaved PARP, cleaved caspase 3 and survivin. Survivin expression data was correlated with clinical outcome.

Results

Survivin expression was significantly higher in EAC tumor samples compared to the CASE from the same patient. Patients with high expression of survivin in EAC tumor had an increased risk of death. Survivin expression was also noted in CASE and correlated with increased risk of distant recurrence. Cell line evaluation demonstrated that inhibition of survivin resulted in an increase in apoptosis.

Conclusion

Higher expression of survivin in tumor tissue was associated with increased risk of death; while survivin expression in CASE was a superior predictor of recurrence. Inhibition of survivin in EAC cell lines further showed increased apoptosis, supporting the potential benefits of therapeutic strategies targeted to this marker.