棉铃虫HaHR3基因的克隆及其植物RNA干扰载体的构建

采用RT-PCR方法克隆了棉铃虫蜕皮调节转录因子HaHR3基因,该基因含有1671 bp的完整开放阅读框。序列分析表明,HaHR3与多种昆虫蜕皮调节转录因子高度同源。利用生物信息学方法对HaHR3的结构特征进行分析发现,HaHR3具有蜕皮调节转录因子超家族的典型特征,包括两个锌指结构和一个DNA结合结构域,不存在信号肽序列和N糖基化位点。选择HaHR3基因的部分片段构建了RNAi中间载体pRNAi1017-HaHR3sa,再将HaHR3正反向序列亚克隆至植物表达载体pCAMBIA2300-35S-OCS,成功构建了由35s启动子调控的HaHR3基因的正反向RNA干扰载体pCAM-RNAi-HaHR3。这一载体的成功构建为下一步通过植物介导的RNA干扰技术防治棉铃虫打下了坚实的基础。     

利用DAD1反义片段转化创建菜薹可调控雄性不育材料

 菜薹因为没有好的雄性不育材料或自交不亲和系,至今尚无一代杂种用于生产,根据拟南芥及白菜型油菜的花药不开裂基因DAD1的保守序列设计引物,扩增菜薹的DAD1基因片段（DAD1F）,构建反义DAD1F植物表达载体,用农杆菌介导法转化菜薹,对转基因植株进行分子检测,鉴定其雄性不育性并进行育性恢复试验.克隆得到的菜薹的DAD1基因片段大小为678 bp,命名为BrcpDAD1F,其序列与拟南芥和白菜型油菜的DAD1高度同源,同源率分别为88％和99％;共得到了12株转基因植株,有6株在转录水平上得到表达,表现为雄性不育,花器官畸形,花粉活力低,萌发率不到10％,且开花后不能结角果或结空角果,或者得到极少种子但种子不萌发;用对照的花粉给转基因植株授粉可使其正常结实.以500 μmol·L^-1茉莉酸甲酯处理可使其雄性不育得到恢复,花粉可以在柱头和培养基上萌发,具有受精能力。T₁代可育株与不育株的比例都呈1:3分离, T₂代不同株系的育性分离比例不同,有些株系继续呈1:3的分离,有些株系全是可育株或全是不育株,说明反义抑制呈单基因稳定遗传。     

锦鲤保护性组织RNA提取及引物扩增研究

从养殖锦鲤各种组织中提取完整的RNA, 是分子生物学研究锦鲤应激表达、体色变异、生长发育等功能基因表达的关键实验。在不伤害养殖锦鲤个体健康的情况下, 取用鱼体的鳍条、鳞片、鳃丝、血液4种保护性组织,可以最大程度地减少对珍贵锦鲤个体的损伤, 并达到研究相关基因表达的目的。本研究利用TR izo l法提取RNA, 获得的各样品RNA条带完整、纯度高。结果表明, 4 种保护性组织提取的RNA 经过逆转录反应, 得到了高质量的CDNA。内标基因( B- actin)、热激蛋白基因( hsp70)、金属硫蛋白基因(MT)扩增得到清晰的条带, 通过NCB I数据上的BLAST比对证明, 各序列的同源性在95%以上。利用本研究的方法, 可以充分保护养殖锦鲤个体的完好性, 用于进一步的分子生物学研究。     

Sinomenine reduces MPP+-induced damage in SK-N-SH cells via ANRIL/miR-626 signaling pathway

AIM To investigate the effect of sinomenine (SN) on the damage of human neuroblastoma SK-N-SH cells induced by 1-methyl-4-4 phenylpyridine (MPP⁺) and its mechanism for exploring the pathogenesis of Parkinson disease. METHODS SN was used to treat MPP⁺-induced SK-N-SH cells. The levels of malondialdehyde (MDA) and glutathione (GSH) in cell culture supernatants were measured by ELISA. The apoptosis was analyzed by flow cytometry. The protein expression levels of Bcl-2 and Bax were determined by Western blot. The expression levels of long noncoding RNA ANRIL and microRNA-626 (miR-626) were detected by RT-qPCR. Dual-luciferase reporter assay was used to evaluate the relationship between ANRIL and miR-626. After ANRIL small interfering RNA was transfected into SK-N-SH cells, the effects of ANRIL expression knock-down on MPP⁺-induced SK-N-SH cell apoptosis, the protein expression levels of Bcl-2 and Bax, and the levels of MDA and GSH in cell culture supernatants were examined. RESULTS After treatment with MPP⁺, the apoptotic rate, Bax protein level and ANRIL expression in SK-N-SH cells were increased (P<0.05), and the Bcl-2 protein level and miR-626 expression were decreased (P<0.05). The level of MDA in cell culture supernatants was increased (P<0.05), and the level of GSH was decreased (P<0.05). After SN treatment or ANRIL expression knock-down, decreased apoptotic rate, Bax protein level and ANRIL expression (P<0.05), and increased Bcl-2 protein level and miR-626 expression in MPP⁺-induced SK-N-SH cells were observed (P<0.05). The level of MDA in the cell culture supernatants was decreased (P<0.05), and the level of GSH was increased (P<0.05). CONCLUSION SN attenuates MPP⁺-induced damage in SK-N-SH cells by regulating ANRIL/miR-626 signaling pathway.     

Progress in roles of MNX1 antisense RNA 1 in malignant tumor

MNX1 antisense RNA 1 （MNX1-AS1） is a newly discovered long non-coding RNA （lncRNA）, which is highly dysregulated in various carcinomas and its expression level is closely related to the overall survival and prognosis of patients. MNX1-AS1 regulates the occurrence and development of carcinomas by endogenous competitive adsorption of miRNA, regulating cell cycle, inducing epithelial mesenchymal transformation and activating multiple signaling pathways. The in-depth study of the carcinogenesis of MNX1-AS1 is useful for the early diagnosis, targeted therapy and prognostic assessment of relevant carcinomas. This article reviews the roles of MNX1-AS1 in malignant tumor.     

Effect of lncRNA FEZF1-AS1 on viability and apoptosis of LPS-induced vascular endothelial cells by regulating miR-363-3p expression

AIM To investigate the mechanism of long noncoding RNA （lncRNA） FEZF1-AS1 regulating microRNA-363-3p （miR-363-3p） on the viability and apoptosis of lipopolysaocharide （LPS）-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells （HUVECs） were cultured in vitro. pcDNA-NC, pcDNA-FEZF1-AS1, anti-miR-NC, anti-miR-363-3p, miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1, Ki67 and cleaved caspase-3. RESULTS Compared with control group, the expression level of FEZF1-AS1 in LPS group was significantly reduced （P<0.05）, and the expression level of miR-363-3p was significantly increased （P<0.05）. Compared with pcDNA-NC+LPS group, the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Compared with anti-miR-NC+LPS group, the cell viability in anti-miR-363-3p+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group, the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced （P<0.05）, the apoptotic rate was significantly increased （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly reduced （P<0.05）, and the protein level of cleaved caspase-3 was significantly increased （P<0.05）. CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.     

水稻齿叶矮缩病毒Pns10蛋白在水稻原生质体内的表达

【目的】水稻齿叶矮缩病毒(Rice ragged stunt virus,RRSV)Pns10蛋白在介体昆虫细胞内可形成类似病毒原质(viroplasm)的内含体,是RRSV侵染介体所必需。然而Pns10蛋白在水稻寄主中是否具有类似功能及其表达情况如何未见报道。【方法】利用大肠杆菌系统表达Pns10蛋白,免疫家兔制备多克隆抗体;通过水稻原生质体病毒侵染体系,利用免疫荧光技术分析Pns10蛋白在水稻原生质体内的分布情况,利用实时定量PCR技术和Western blot技术分别检测Pns10 RNA和Pns10蛋白在水稻原生质体内的积累情况。【结果】将Pns10基因克隆到Gateway系统原核表达载体p DEST17中,IPTG诱导表达成功后,制备融合蛋白抗血清。Western blot检测显示,该抗血清可检测感病水稻叶片中的Pns10蛋白。病毒侵染水稻原生质体后,Pns10蛋白可形成类似病毒原质的内含体;Pns10 RNA在病毒接种8 h后开始积累,24 h后达到最大值,随后开始下降;Pns10蛋白在24 h后开始表达,之后维持较高水平,60 h后略有下降。【结论】成功获得了Pns10抗血清;Pns10在水稻原生质体内成功表达,可形成类似病毒原质的内含体,并且Pns10 RNA的表达先于其蛋白的表达。     

寄主诱导的基因沉默提高植物真菌病害抗性研究进展

寄主诱导的基因沉默(host-induced gene silencing,HIGS)以病原菌生长发育、产孢繁殖、侵染或致病过程中的关键基因作为靶点,在寄主植物中表达针对靶基因的RNAi构建体,在病原菌侵染植物的过程中,摄入相应的ds RNA或si RNA分子,通过识别、结合特异核苷酸序列,干扰病菌靶基因表达,从而抑制病菌侵染和扩展,使植物表现抗病。这项技术为培育基于病原菌特异序列的植物抗病性奠定了基础,显示了巨大的应用潜力。本文综述了利用HIGS技术提高植物真菌病抗性的最新研究进展,总结了国内外利用这项技术改良植物真菌病害抗性的主要策略、技术路线,展望了发展应用前景。