油菜种子低亚油酸和亚麻酸含量异交稳定技术的建立

为降低油菜种子中的亚油酸和亚麻酸含量并使之在异交条件下保持基本稳定,以高油品种CY2作为受体亲本,采用RNA干涉技术沉默油菜内源Bn FAD2基因的表达,抑制油酸脱饱和反应。育成的3个独立转基因株系的亚油酸和亚麻酸含量下降到6%以下,其中亚油酸含量较受体亲本降低78.2%~86.5%,亚麻酸含量降低53.4%~65.8%。将3个转基因株系与2个亚油酸和亚麻酸含量较高的常规品种正反交,F1种子中这2种脂肪酸的含量依然保持在与转基因亲本相仿的低水平。由此可知,通过RNA干涉技术沉默Bn FAD2基因的表达可赋予油菜低亚油酸和亚麻酸含量的异交稳定特性。这一特性的获得对于今后培育异交稳定的高油酸油菜新品种具有重要意义。     

蓝莓不同品种花芽形成过程中内源激素变化

采用酶联免疫吸附测定法测定5个蓝莓品种在花芽形成期间花芽中4种内源激素含量，研究内源激素对蓝莓花芽分化的影响，为人工调控蓝莓花芽分化奠定理论基础。研究结果表明，5个蓝莓品种在花芽形成过程中，花芽中GAs和ZR含量呈先升高后下降，再升高的变化趋势，在花芽分化初期达到最大值，之后一直保持在较低水平；IAA含量呈先下降后升高的趋势，在花芽分化中期达到最小值，之后含量迅速上升；ABA含量呈先下降后升高，再下降的趋势，在花芽分化末期达到最大值。在花芽形成过程中，5个蓝莓品种花芽中ABA/GAs比值均表现为先下降后升高；ZR/GAs和ZR/IAA的变化趋势相同，均呈先升高后下降的‘M’形变化；ABA/IAA的变化趋势与ZR/GAs相反。     

Effects of Smo gene silencing on cell activity and apoptosis of human cervical carcinoma HeLa cells

AIM: To investigate the effect of Hedgehog (Hh) signaling pathway on the viability and apoptosis of cervical carcinoma cells by shRNA technique to knock down Smoothened (Smo) gene. METHODS: Smo shRNA was used to transfect the cervical carcinoma HeLa cells. The expression of Smo and Gli1 at mRNA and protein levels in the HeLa cells was determined by RT-PCR and Western blot, respectively. The effect of Smo gene silencing on the growth of the cells was measured by MTT assay. The apoptosis and cell cycle were determined by flow cytometry. RESULTS: Compared with control group, the mRNA and protein expression of Smo and Gli1 were evenly reduced obviously after transfected with Smo shRNA for 72 h (P<0.05). The viability of HeLa cells transfected with Smo shRNA was significantly inhibited. The percentages of the cells in G₀/G₁ phase and early apoptosis rate were obviously higher in Smo shRNA transfection group than those in control group. CONCLUSION: Smo gene silencing effectively inhibits the cell growth and induces the apoptosis of human cervical carcinoma cells.     

Screening of lentiviral vectors carrying effective siRNA targeting S1P2 gene

AIM:To screen the lentiviral vector carrying siRNA with higher efficiency of suppressing the sphingosine-1-phosphate receptor 2(S1P2) gene expression in the primarily cultured corpus cavernosum smooth muscle cells of spontaneously hypertensive rats (SHR).METHODS:SHR and SD rats (n=5 each) were used for primarily culturing corpus cavernosum smooth muscle cells.The cells were randomly divided into 6 groups:SHR siRNA-1,SHR siRNA-2,SHR siRNA-3,SHR GFP,SHR control (SHR non-transfection group),and SD control (SD rat control group).Each group had 5 samples with 1.0×10⁵ cells of each sample.At 72 h after transfection (MOI=60) with lentiviral vectors carrying S1P2 siRNA into the SHR corpus cavernosum smooth muscle cells,the expression of GFP was observed under fluorescence microscope.The protein expression of S1P2,ROCK1,ROCK2 and eNOS in the corpus cavernosum smooth muscle cells,and the mRNA expression of S1P2,ROCK1 and ROCK2 were determined by by Western blot and RT-PCR.RESULTS:The transfection efficiency of the corpus cavernosum smooth muscle cells in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SHR GFP groups were>80%.Compared with SHR control group,the mRNA levels and the protein expression of S1P2,ROCK1 and ROCK2 in SHR GFP group showed no remarkable changes,while those in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SD control groups were significantly lower than those in SHR control group (P<0.05).The protein expression of eNOS in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SHR GFP groups were not significantly changed as compared with SHR control group,but that in SD control group was significantly higher than that in SHR control group.CONCLUSION:Three groups of siRNA lentiviral vectors targeting S1P2 inhibit the expression of S1P2 in the corpus cavernosum smooth muscle cells of SHR,and by silencing the S1P2 expression,the expression of ROCK1 and ROCK2 is inhibited.Among them,siRNA-1 has the highest inhibitory efficiency.     

Effects of CENP-W down-regulation on human glioma U87 cells

AIM: To study the effect of centromere protein W (CENP-W) down-regulation on human glioma U87 cells.METHODS: Small interfering RNA (siRNA) was used to inhibit the expression of CENP-W in the U87 cells. The interference effect of siRNA was evaluated by RT-qPCR and Western blot. The proliferation of the cells was analyzed by MTT assay, BrdU staining and colony formation experiment. Transwell chamber assay was used to detect the invasion ability of the cells. The cell migration ability was measured by a scratch test. The changes of the cell cycle distribution and apoptosis were analyzed by flow cytometry.RESULTS: The results of MTT assay, colony formation experiment and BrdU staining showed that the cell proliferation and colony formation abilities in experimental group were significantly lower than those in control group and negative control group. The results of Transwell and scratch experiments showed that the migration and invasion abilities in experimental group were weaker than those in blank control group and negative control group. The results of flow cytometry analysis showed that the cell cycle distribution in experimental group was arrested in G₀/G₁ phase. The percentage of apoptotic cells in experimental group was higher than that in control group (P<0.05).CONCLUSION: Down-regulation of CENP-W expression inhibits the proliferation, migration and invasion of human glioma cells and promotes the apoptosis of the cells, suggesting that CENP-W may be a potential target of gene therapy for human glioma.     

吉林延龄草种子内源抑制物质初步研究

为探讨吉林延龄草种子内源抑制物质生理活性及种子中抑制物质的去除方法,采用乙酸乙酯、甲醇、水提取吉林延龄草种子并测定其提取物对白菜、水稻幼苗生长的抑制活性,用温水(25,35,45℃)浸种并测定各批次浸提液抑制活性,结果表明,吉林延龄草种子浸提液(乙酸乙酯提取液除外)对白菜幼根及胚轴,水稻幼根均有显著抑制作用,且抑制物质的活性随着提取物浓度的增加而增强,吉林延龄草种子水提取液(C)对白菜和水稻幼苗生长的抑制活性高于甲醇提取液(B)的生物活性,吉林延龄草种子乙酸乙酯提取液对白菜和水稻幼苗无显著抑制作用(p＞0.05),吉林延龄草种子经乙酸乙酯、甲醇溶液提取后,再经蒸馏水提取获得的水提液Ⅱ(E)对白菜和水稻幼苗生长的抑制活性减弱;温水浸泡可将其内源抑制物质浸提出来,且随着浸种时间的增加,浸提液对白菜幼根及胚轴的抑制作用呈现先增加而后逐渐降低的趋势.25℃蒸馏水浸泡种子第5天浸提液抑制作用达到最大,对白菜幼根及胚轴的抑制作用分别达到65.89％和25.69％,35℃蒸馏水浸泡种子第3天浸提液抑制作用达到最大,对白菜幼根及胚轴的抑制作用分别达到82.14％和59.93％,45℃蒸馏水浸泡种子第2天浸提液抑制作用达到最大,对白菜幼根及胚轴的抑制作用分别达到90.62％和83.86％.综合分析表明,吉林延龄草种子中存在活性较高的内源抑制物,用45℃温水浸泡可有效除去种子内源抑制物质.     

基于18S rRNA的鸡组织滴虫病PCR诊断方法的建立

河南某鸡场约4周龄鸡疑似发生鸡组织滴虫病.根据鸡组织滴虫18 S rRNA序列设计引物,提取肝脏、盲肠内客物寄生虫DNA,采用PCR方法检测.结果表明,PCR扩增到与预期大小一致的产物,经测序比对,证实为鸡组织滴虫感染.所建立的PCR方法具有灵敏、特异、快速等优点,不仅能用于鸡组织滴虫病临床诊断,还能用于开展流行病学研...     

葡萄果实cDNA文库的构建及鉴定

构建葡萄果实cDNA文库是研究果实发育相关基因表达调控的基础工作.本实验从巨峰葡萄果实中提取了高质量的RNA,利用MMLV反转录酶把总RNA中的mRNA反转录成cDNA第一链,用特异引物进行LD-PCR扩增合成双链cDNA.将分级纯化的带有粘性末端双链cDNA与λTrip Ⅰ Ex2载体连接,重组载体体外包埋成为cDNA原始文库.初步鉴定原始文库的滴度为1.32×106 pfu/mL,文库扩增后滴度达1.45×1010 pfu/mL.从扩增文库中随机挑取250个克隆进行PCR鉴定,结果表明重组率为98.7%,插入片段大小在0.5～2.0 kb之间,因此,本研究成功构建了葡萄果实cDNA文库.     

化控种衣剂对大豆幼苗侧根原基的发生 和内源激素含量的影响

以垦农4号为试验材料,利用垂直板发芽试验研究了化控种衣剂 (HK)和常规种衣剂(ND)对大豆幼苗侧根原基发生的影响,并对侧根原基发生过程中植物内源激素含量的动态变化进行了相应的分析。结果表明：(1)种衣剂包衣后,侧根原基发生区长度明显比对照(不包衣的种子为对照,用CK表示)增加,从大到小依次为：HK＞ND＞CK;(2)种衣剂处理加快了侧根原基的发生进程,总的趋势是HK＞ND＞CK,经F检验表明,处理和对照差异极显著;(3)侧根原基的发生状况与侧根原基发生区内源激素的含量有一定的关系,侧根的发生是几种植物内源激素相互协调、综合作用的结果,其中生长素类物质在诱导侧根原基的发生过程中起主导作用;(4)应用种衣剂可以提高侧根原基发生的数量,缩短侧根原基的发生时间,其中以化控种衣剂效果最好。     

百合总RNA提取方法的比较和分析

以卷丹、麝香百合品种富田和离体鲜切花的花蕾为材料，比较Trizol法、异硫氰酸胍法、CTAB改进法和SDS改进法提取总RNA的效果，结果表明，SDS改进法能有效去除多糖，提取的RNA中28S rRNA亮度约为18S rRNA的两倍，OD260/OD280值介于1．7-2．2之间，卷丹RNA得率为132．52μg／g，富田RNA得率为186．88μg／g。进一步用SDS改进法分别提取富田外轮花瓣、内轮花瓣、雄蕊、雌蕊、叶和茎的RNA，同样可以获得完整的纯度高的RNA，其中28S rRNA亮度约为18S rRNA的两倍，OD260／0D280值介于1．7～2．2之间，说明此方法适用于百合各个组织RNA的提取。经RT—PCR获得了花发育基因的特异性条带，说明用SDS改进法从百合中提取的RNA质量好、产率高、完整性强，完全适合于百合进一步的分子生物学研究。