Cloning of Cotton Delta-12 Oleate Desaturase Gene FAD2-1 and Construction of Its ihpRNA and amiRNA Interference Vectors

Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cotton delta-12 oleate desat- urase gene GhFAD2-1 containing an open reading frame of 1 158 bp was cloned for constructing RNAi vector. A 515 bp long specific fragment of this gene was se- lected for constructing ihpRNA vector under the control of a seed-specific promoter NAPIN, named pFGC1008-NAPIN-FAD2-1; meanwhile miRNA gene-silencing vector pCAMBIA1302-amiRNA-FAD2-1 targeting GhFAD2-1 was also constructed.

ENHANCEMENT OF RADIATION-INDUCED APOPTOSIS IN RAJI CELL LINE BY BC1-2 ANTISENSE OLIGODEOXYNUCLEOTIDE

Objective: To investigate whether the Bc1-2 antisense oligonucleotide(ASODN) may enhance radiation-induced apoptosis in Raji cell line. Methods: Cell surviving fraction was determined using the trypan blue dye exclusion assay. The expression level of bc1-2 protein was assayed by immunofluorescence using fluoresce isothiocyanate label. Apoptosis was detected by Giemsa staining and flow cytomertric cell cycle analysis. Results: It was found that Bc1-2 ASODN combined with radiation had significantly reduced the number of viable cells (P<0.05). There was no difference on cell survival between mismatch Bc1-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone. Bc1-2 ASODN combined with radiation could significantly inhibit expression of Bc1-2 protein in Raji cells (P<0.05). Cells treated with Bc1-2 ASODN combined with radiation at 72 h displayed classic apoptotic changes. Apoptosis rates of Raji cells treated with Bc1-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone, respectively. Conclusion: Bc1-2 antisense oligonucleotide can enhance radiation-induced apoptosis in Raji cell line.

根癌农杆菌介导反义fad2-1基因转化大豆的研究

通过根癌农杆菌介导法将反义fad2-1基因导入大豆子叶节,研究了农杆菌菌液浓度、预培养时间、共培养时间、恢复培养时间、乙酰丁香酮浓度和pH值对大豆子叶节形成丛生芽的影响,建立了有效的转化体系,获得了转基因大豆再生植株。PCR、PCR-Southern blotting检测结果表明,反义fad2-1基因成功转入并整合于大豆基因组。GC检测结果表明,转反义fad2-1基因大豆种子油酸含量比非转基因大豆种子的油酸含量提高了14.44%,而亚油酸含量降低了20.27%。

同步抑制棉籽FAD21-与FatB基因表达的RNAi载体构建

采用PCR技术,分别扩增获得靶标基因FAD21-与FatB的功能区域片段426与501bp,通过PCR引物引入特定的酶切位点,采用DNA标准重组技术与Gateway技术相结合的方法,以表达载体pBI121及RNA干扰载体pANDA35HK为基础,利用种子特异性启动子,成功构建出棉籽特异抑制双基因表达的RNAi载体。

油用向日葵脂肪酸脱氢酶基因FAD2-1的克隆与表达分析

为了解FAD2-1基因在油用向日葵品质形成过程中的作用,以油用向日葵为材料,利用RT-PCR方法对高含油率保持系材料改HA89(含油率为46%)和低含油率保持系材料86-1(含油率为38%)的脂肪酸脱氢酶基因FAD2-1进行克隆与表达分析。结果表明:获得了脂肪酸脱氢酶基因FAD2-1全长cDNA编码序列,全长为1 137bp,编码378个氨基酸。核苷酸序列和蛋白序列比对结果表明,该基因在这2份材料中第44位和第102位核苷酸存在差异,并且都导致了氨基酸的改变。种子发育不同阶段油酸和亚油酸积累动态分析和qRT-PCR研究结果表明,FAD2-1基因表达量变化趋势与亚油酸含量变化趋势基本一致,而与油酸含量变化趋势基本相反。

大豆FAD2-1b基因沉默载体的构建

旨在培育生物安全的转基因大豆。设计引物、采用RT-PCR从高蛋白大豆品种‘黑农35’中克隆大豆种子特异表达启动子GY1;从高油大豆‘黑农37’中克隆FAD2-1b基因内含子1;从5个转基因受体大豆品种中克隆FAD2-1b基因片段,作为正向臂和反向臂。连接这些目的片段,构建筛选标记基因为bar,启动子为GY1,内含子为FAD2-1b基因内含子1的双T-DNA载体。将双T-DNA载体转化大肠杆菌DH5α,扩增得到的重组质粒经酶切和序列分析鉴定正确,命名为pDT-FAD2I。本研究构建了针对大豆FAD2-1b基因的沉默载体,为进一步获得FAD2-1b基因沉默大豆,培育转基因安全的高品质大豆奠定了基础。

油用向日葵FAD2-1基因RT-PCR体系的建立与优化

为了建立向日葵FAD2-1基因高效特异的PCR扩增体系,以油用向日葵86-1为材料提取总RNA,并反转录合成cDNA,以cDNA第一条链为模板对FAD2-1基因全长编码序列的PCR体系进行了优化。结果表明:根据已公布的FAD2-1序列设计3对特异性引物,每对引物设计了5个退火温度,筛选出适于该基因PCR扩增的引物及其退火温度,并将PCR扩增条件中循环数缩短为30次,延伸时间缩短为7min,此改进提高了PCR反应的效率。

棉花FAD2-1基因的RNAi载体构建及高油酸新种质的获得

【目的】在棉花纤维产量和品质不受影响的前提下,创造高油酸、低亚油酸棉花新种质。【方法】棉花FAD2-1基因是催化单不饱和脂肪酸的油酸形成多不饱和脂肪酸的亚油酸的关键基因,采用信息分析学方法分析其蛋白质性质、结构、功能;克隆GhFAD2-1的保守片段381 bp,构建RNA干涉载体,采用农杆菌介导的下胚轴侵染方法将干涉载体转入棉花。利用气相色谱-质谱联用仪(Gas chromatography-mass spectrometer,GC-MS)测定转基因T2~T4植株种子的脂肪酸成分及含量。对T4转基因株系进行拷贝数鉴定、目的基因表达量检测、农艺性状及纤维品质考察。【结果】成功构建GhFAD2-1的RNA干涉载体并转入棉花,转基因株系目的基因GhFAD2-1的表达量显著低于对照,具有高油酸、低亚油酸的表型并且能稳定遗传给后代;转基因株系可将棉花种子油酸提高224.1%,将亚油酸降低237.5%。与对照材料相比,转基因株系的农艺性状、纤维品质没有明显差异。【结论】验证了棉花GhFAD2-1基因的功能并且为培育高油酸棉花品种提供了帮助。

Exploration and Validation of the Potential Downstream Genes Underlying ipa1-2D Locus for Rice Panicle Branching

In recent years,some super hybrid rice varieties were bred with strong culms and large panicles,which are mainly contributed by the ipa1-2D locus.A gain-of-function allele of OsSPL14 is the ipa1-2D and it can greatly increase the panicle primary branch number.However,the key downstream genes mediating this trait variation are not fully explored.In this study,we developed high-quality near-isogenic lines(NILs)with a difference of only 30 kb chromosomal segment covering the ipa1-2D locus.Using the NILs,we explored the impact of ipa1-2D on five sequential stages of early inflorescence development,and found that the locus can greatly enhance the initiation of primary branch meristems.A transcriptomic analysis was performed to unveil the downstream molecular network of ipa1-2D,and 87 genes were found differentially expressed,many of which are involved in metabolism and catalysis processes.In addition,transgenic lines of overexpression and RNA interference were generated to shape different levels of OsSPL14.They were also used to validate the expression variation explored by transcriptome.Based on the gene annotation,twelve potential downstream targets of ipa1-2D were selected,and their expression variation was confirmed by qRT-PCR analysis both in NILs and transgenic lines.This research expands the molecular network underlying ipa1-2D and provides novel gene information which might be involved in the control of panicle branching.We discussed the potential function of identified genes and highlighted their values for future function exploration and breeding application.

Expressions of MMP-2,-9,TIMP-1,-2,-3 mRNA in Rat Uterus during Estrous Cycle

Zymography and in situ hybridization were used to investigate matrixmetalloproteinase -2, -9 (MMP -2, MMP-9) activities and expressions of MMP -2, -9 and TIMP1, -2, -3 (tissue inhibitors of matrix metallo-proteinases) mRNA in the rat uterus during estrouscycle. The relative activity was semiquanted by using densitometric analysis. The MMP-2(67 kDa) activity in every stage during estrpus cycle was detected by zymography. MMP-2activity was highest at proestrus; higher at estrus and metaestrus; lowest at diestrus. Throughin situ hybridization, MMP -2, -9, TIMP -1~ -3 mRNA mainly in hasal stroma cells of uterineendometrium were detected. The positive signals of MMP -2 and -9 mRNAs in hasal stromacells were shown stronger at proestrus, estrus and metaestrus while they showed the weakest atdiestrus. The expression of MMP -2 mRNA coincided with MMP -2 activity change. MMP-2and -9 mRNAs were also highly expressed in uterine circular muscle at estrus. Weak signals ofMMP -9 mRNA were detected in uterine luminal and glandular epithelial cells at estrus.TIMP -1 mRNA in hasal stroma cells was shown as the strongest expression at estrus andmetaestrus; stronger at proestrus and the weakest at diestrus. TIMP-2 mRNA in basal stromacells was stronger at estrus and diestrus; weaker at proestrus and metaestrus. TIMP -1 and -2mRNAs were also highly expressed in uterine luminal and glandular epithelial cells at estrus.TIMP -3 mRNA in hasal stroma cells revealed the strongest expression at estrus; stronger atdiestrus and metaestrus and showed the weakest at proestrus. The mRNA was also highlyexpressed in uterine circular muscle at estrus. In short, our present results provide evidencethat MMP -2, -9 and TIMP -1~ -3 were involved in rat uterine endometrium reconstructionduring estrous cycle.

Induction of anti-hepatoma immunity by recombinant retrovirus expressing B7-1 /B7-2 costimulatory molecules

Objective: To construct recombinant R7-l/B7-2 retrovirus vectors and observe the effects of B7-l/R7-2 gene expression on in ho and in for immune response against against murine hepatoma. Methods: The recombinant retrovirus vectors expressing B7-1/B7-2 were constructed by gene cloning technology to produce retrovirus-infected PE501 and PA317 cell lines and murine hepatoma Hepal-6. The expression of R7-l/B7-2 was detected by fluorescence activated cell soning analysis (FACS). B7-l/B7-2 positive Hepal-6 Cell lines were used in inducing anti-hepatoma immunity in ho and in the. Results: In contrast to the excessive growth of parental Hemal-6 tumor, the growth of B7-l/B7-2-positive Hepal-6 inoculated into syngenic mice regressed. B7-1/R7-2-positive or cytokine-treated Hepal-6 alone could only induce mild cytototicity; in contrast, B7-1/B7-2-positive Hemal-6 treated with cytokine-stimulated spleen cells and activated the cytotoxicity effectively. Immunity in mice with R7-1/B7-2-positive tumor cells or cytokine-beated Hepal-6 only provided partial protection against parental Hepa1-6 tumor, whereas pretreatment of the transfected tumor cells with IFN-r and TNF-a induced complete immunity protection in vivo. Mice receiving inoculation of cytokine-treated B7-l/R7-2-positive Hemal-6 cells presented regression of the establoshed pental tUmor and survived for more than l00 d, while those untreated mice died within 40 d. Conclu sions: B7-l/R7-2 expression is necessary but not sufficient in inducing anti-hepatoma immune response, whereas it is efficient when combined with the beatment of IFN-γ and TNF-a.

The 5’-Untranslated Region of the C9orf72 mRNA Exhibits a Phylogenetic Alignment to the Cis-Aconitase Iron-Responsive Element;Novel Therapies for Amytrophic Lateral Sclerosis

The hexanucleotide repeat mutation in the intron-1 of the chromosome 9 open reading frame (C9orf72) is a frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Altered RNA folding plays a role in ALS pathogenesis in two ways: non-ATG translation of the repeat can lead to aggregates of the known C9orf72 specific dipeptide polymer, whereas the repeat also can form neurotoxic RNA inclusions that dose-responsively kill motor neurons. We report the presence of a homology in the 5’untranslated region (UTR) of the messenger RNA encoding C9orf72 with the iron responsive elements (IRE) that control expression of iron-associated transcripts and predict that this RNA structure may iron-dependently regulate C9orf72 translation. We previously report altered serum ferritin levels track with severity of ALS in patients. Here, we conduct bioinformatics analyses to determine the secondary structure of the 5’UTR in C9orf72 mRNA and find it aligned with IREs in the human mitochondrial cis-aconitase and L and H-ferritin transcripts. Comparison of the role of RNA repeats in Friedriech’s ataxia and fragile X mental retardation suggests the utility of RNA based therapies for treatment of ALS. Antisense oligonucleotides (ASO) have been reported to therapeutically target these GGGGCC repeats. At the same time, because the function of C9orf72 is unknown, knockdown strategies carry some risk of inducing or compounding haploinsufficiency. We propose, for consideration, an approach that may enhance its therapeutic dynamic range by increasing the 5’UTR driven translation of C9orf72 protein to compensate for any potential ALS-specific or ASO-induced haploinsufficieny.

结肠癌组织SATB2、SIRT1、HOTAIR表达及与预后的相关性

目的研究结肠癌组织特定富含AT碱基DNA序列结合蛋白2(SATB2)、沉默信息调节因子1(SIRT1)和同源盒基因转录反义RNA(HOTAIR)表达水平及与预后的关系。方法选取2014年1月—2016年6月我院102例结肠癌,根据患者预后分为死亡组和存活组,检测2组肿瘤组织SATB2、SIRT1及HOTAIR表达水平,并分析其与患者预后的关系。结果本组中位随访时间为72个月,102例随访期间死亡67例(65.69%),中位总体生存期(OS)为63个月。死亡组肿瘤最大径和AJCC分期Ⅲ+Ⅳ期患者占比均大于存活组,肿瘤分化程度低于存活组,差异有统计学意义(P<0.05,P<0.01)。死亡组SATB2阳性率低于存活组,SIRT1阳性率和HOTAIR mRNA相对表达量高于存活组,差异有统计学意义(P<0.01)。多因素Cox回归分析显示,肿瘤低分化、AJCC分期Ⅲ+Ⅳ期、SIRT1阳性和HOTAIR mRNA为结肠癌患者死亡的危险因素,SATB2阳性为结肠癌患者死亡的保护因素(P<0.05,P<0.01)。SATB2阳性患者中位OS(69个月)长于SATB2阴性患者(54个月),SIRT1阳性患者中位OS(57个月)短于SIRT1阴性患者(78个月),HOTAIR mRNA≥47.85患者中位OS(57个月)短于HOTAIR mRNA<47.85患者(78个月),差异有统计学意义(P<0.05,P<0.01)。结论结肠癌组织SATB2、SIRT1和HOTAIR表达均为影响患者预后的重要因素。

大豆fad3c基因沉默载体的构建

构建大豆fad3c基因的沉默载体,旨在为培育生物安全的高油酸低亚麻酸转基因大豆奠定基础。从高蛋白大豆黑农35中克隆大豆种子特异表达启动子GY1;从高油大豆黑农37中克隆fad2-1b基因内含子1;从5个转基因受体大豆中克隆fad3c基因片段,作为正向臂和反向臂。构建筛选标记基因为bar,启动子为GY1,内含子为fad2-1b基因内含子1的双T-DNA载体。将双T-DNA载体转化大肠杆菌DH5α,酶切和序列分析鉴定表明扩增得到的重组质粒正确,命名为pDT-GFAD3I。

Study of the SH3-domain GRB2-1ike 2 gene expression in laryngeal carcinoma

Background Laryngeal carcinoma is a common malignant tumor of the upper respiratory tract, and in 95% of cases the tumor is laryngeal squamous cell carcinoma （LSCC）. The abnormity of SH3-domain GRB2-1ike 2 （SH3GL2） gene was found in LSCC. In order to clarify the relationship between SH3GL2 gene and LSCC, we evaluated the expression of the SH3GL2 gene in LSCC. Method Real-time PCR, immunohistochemistry and Western blotting were used to detect the mRNA and protein expression and find the various rules of SH3GL2 gene in LSCC. Results The result of real-time PCR showed that the expression level of SH3GL2 mRNA in LSCC tissue was apparently down-regulated; immunohistochemical analysis showed that SH3GL2 protein was mainly located in cytoplasm, the rate of positive cells and SH3GL2 protein expression level were fluctuated with the pathological classification of LSCC; the result of Western blotting showed that SH3GL2 protein was down-regulated significantly in LSCC samples, especially in metastatic lymph nodes. Conclusions These results suggest that SH3GL2 is a LSCC related gene and its expression level is fluctuated with the pathological classification which indicate that SH3GL2 participates in the development and progression of LSCC. And it may be considered as a novel tumor marker to find both a new anti-oncogene and relative factors of invasion and metastasis of laryngeal carcinoma.

Free fatty acids, glucose, and insulin in type 2 diabetes mellitus

Xu et al used the HOMA2 model to estimate theβ-cell function and insulin resistance levels in an individual from simultaneously measured fasting plasma glucose and fasting plasma insulin levels.This method is based on the assumption that the glucose-insulin axis is central for the metabolic activities,which led to type 2 diabetes.However,significant downregulation of both the NKX2-1 gene and the TPD52L3 gene force an increase in the release of free fatty acids(FFAs)into the blood circulation,which leads to a marked reduction in membrane flexibility.These data favor a FFA-glucose-insulin axis.The authors are invited to extend their study with the introduction of the saturation index(number of carbon-carbon double bonds per 100 fatty-acyl chains),as observed in erythrocytes.

On four closely related hypotrichous ciliates(Protozoa,Ciliophora,Spirotrichea):molecular characters,interspecific relationships and phylogeny defined with multigene sequence information

In order to clarify the phylogeny and relationships of the most confused hypotrichous ciliates,Holosticha-complex,four closely related holostichids（five populations）,Holosticha bradburyae,H.diademata,Anteholosticha sp.,and A.manca,were compared and analyzed using ITS2 secondary structures,ITS1-5.8S-ITS2 region and SSrRNA gene sequences.The ITS1-5.8S-ITS2 region sequences of these four species were first sequenced,and they shared sequence identities ranging from 68.0% to 90.1%,while two populations of Anteholosticha sp.differed in three nucleotides（sequence identity 99.8%）.There were several minor differences among ITS2 secondary structures of these species,while two populations of Anteholosticha sp.had the identical secondary structure.Phylogenetic trees inferred from the ITS1-5.8S-ITS2 region sequences of stichotrichs using multiple algorithms（Neighbor-Joining,Maximum Parsimony and Bayesian） revealed similar topologies.The results show that：（1） Holosticha bradburyae and H.diademata firmly clustered together with strong bootstrap supports,forming a sister clade with Anteholosticha sp.,（2） Anteholosticha appeared to be a paraphyletic assemblage,in which the morphotype A.manca was more closely related to Diaxonella trimarginata than to its congener Anteholosticha sp.Phylogenetic analyses based on the SSrRNA gene and the combined sequences of SSrRNA gene and ITS1-5.8S-ITS2 region revealed the similar relationships between Holosticha and Anteholosticha,nevertheless their positions within the subclass Stichotrichia differed from each other inferred from different genes.

Long non-coding RNA SAP30-2:1 is downregulated in congenital heart disease and regulates cell proliferation by targeting HAND2

Congenital heart disease(CHD)is the most common birth defect worldwide.Long non-coding RNAs(lncRNAs)have been implicated in many diseases.However,their involvement in CHD is not well understood.This study aimed to investigate the role of dysregulated lncRNAs in CHD.We used Gene Expression Omnibus data mining,bioinformatics analysis,and analysis of clinical tissue samples and observed that the novel lncRNA SAP30-2:1 with unknown function was significantly downregulated in damaged cardiac tissues from patients with CHD.Knockdown of lncRNA SAP30-2:1 inhibited the proliferation of human embryonic kidney and AC16 cells and decreased the expression of heart and neural crest derivatives expressed 2(HAND2).Moreover,lncRNA SAP30-2:1 was associated with HAND2 by RNA immunoprecipitation.Overall,these results suggest that lncRNA SAP30-2:1 may be involved in heart development through affecting cell proliferation via targeting HAND2 and may thus represent a novel therapeutic target for CHD.

Endothelin-1, an important mitogen of smooth muscle cells of spontaneously hypertensive rats

OBJECTIVE: To study the features of vascular smooth muscle cell (VSMC) proliferation induced by endothelin-1 (ET-1). METHODS: VSMCs of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats were cultured and treated with ET-1. Basic fibroblast growth factor (bFGF) gene expression was measured using both Northern blot and an enzyme-linked immunoassay. RESULTS: ET-1 resulted in an increase in bFGF transcripts at 8 - 24 h; bFGF levels were significantly higher in VSMCs treated with ET-1 than in those not treated. However, VSMCs growth responses in SHR and WKY were different. Smooth muscle cells of SHR were hyper-responsive to ET-1. Maximal bFGF mRNA levels were elevated 3.5-fold at 4 h of stimulation in WKY and 8-fold at 8h in SHR4. Moreover, the proliferation of VSMCs induced by ET-1 was inhibited by antisense phosphorothioate oligodeoxynucleotides (10 micromol/L AS-bFGF) but not sense bFGF oligomers at the same concentrations, being reduced by 80% in SHR and 40% in WKY vs control, respectively. Furthermore, the effect of AS-bFGF oligomers on SHR SMC proliferation is significantly greater than on WKY SMC proliferation. CONCLUSION: ET-1 may be required for exaggerated vascular growth responses in SHR and bFGF may be involved.