Construction of B7x Gene Overexpression Lentiviral-based Vector

To construct overexpression lentiviral-based vector carrying rat B7x gene,B7x gene precursor sequences amplified by polymerse chain reaction（PCR） were ligated with pLVTHM to generate pLVTHM-B7x gene expression lentiviral-based vector.The positive clones were selected to be submitted to DNA sequencing.HEK293T cells were co-transfected with pLVTHM-B7x and two packaging plasmids psPAX2 and pMD2G to produce lentivirus which express B7x gene.The mRNA expression levels of B7x gene and virus titer were detected by real-time PCR in HEK293T cells.The B7x protein levels were detected by Western blot analysis in HEK293T cells.The identification of restriction enzyme digestion and DNA sequencing confirmed that pLVTHM-B7x lentiviral-based vector was successfully constructed.Green fluorescence was observed in HEK293T packaging cells by means of an inverted fluorescence microscope and the virus titer measured was 2×108 TU/mL.It will establish the foundation for studing deeply the biological function of B7x to construct successfully B7x expression lentiviral-based vector.

Effects of Heterologously Overexpressing PIP5K-Family Genes in Arabidopsis on Inflorescence Development

Castor is one of the top 10 oil crops in the world and has extremely valuable uses.Castor inflorescences directly affect yield,so the study of inflorescence development is very important in increasing castor yield.Our previous studies have shown that the PIP5K gene family(PIP5Ks)is associated with inflorescence development.In this study,to determine the function of each PIP5K gene in castor,a female Lm-type castor line,aLmAB2,was used to determine the relative expression levels of the PIP5Ks in castor inflorescences.Six PIP5K genes were heterologously overexpressed in Arabidopsis thaliana,the relative expression of each gene and the effect on plants was determined in A.thaliana,and the relationships among the PIP5Ks in castor were inferred.The expression levels of the PIP5Ks in the female Lm-type castor line aLmAB2 were analyzed.The relative expression levels of the PIP5K9 and PIP5K11 genes were high(p<0.05)in isofemale inflorescences,and those of PIP5K1,PIP5K2,PIP5K6,and PIP5K8 were high(p<0.05)in female inflorescences but low(p<0.05)in bisexual inflorescences.The PIP5Ks were heterologously overexpressed in A.thaliana,and T3-generation plants with stable genetic resistance,i.e.,AT-PIP5K^(+)plants(AT-PIP5K1^(+),AT-PIP5K2^(+),AT-PIP5K6^(+),AT-PIP5K8^(+),AT-PIP5K9^(+),and ATPIP5K11^(+) plants),were obtained.Biological tests of the AT-PIP5K+plants showed that the growth of the main stem was significantly delayed in AT-PIP5K+plants compared with Columbia wild-type(WT)A.thaliana plants;the PIP5K1 and PIP5K2 genes promoted lateral stem growth and flower and silique development;and the PIP5K6,PIP5K8,PIP5K9 and PIP5K11 genes inhibited lateral stem growth and flower and silique development.The correlations among PIP5Ks in castor suggest that there may be a synergistic relationship among PIP5K1,PIP5K2,and PIP5K6 in castor inflorescences,and PIP5K8,PIP5K9,and PIP5K11 are complementary to the other three genes.

Overexpression of a Foreign Bt Gene in Cotton Affects the Low-Molecular-Weight Components in Root Exudates

Most research in the past using genetically modified crops (GM crops) has focused on the ecological safety of foreign gene (i.e., the gene flow), gene products (for example, Bt (Bacillus thuringiensis) protein), and the safety of transgenic food for humans. In this study, changes in both the species and amounts of low-molecular-weight components in cotton (Gossypium hirsutum L.) root exudates after foreign Bt gene overexpression were investigated under different nutritional conditions. Transgenic cotton containing Bt (Bt-cotton), supplemented with all the mineral nutrients, secreted more organic acids than the wild-type cotton (WT). When nitrogen was removed from the full-nutrient solution, the amount of organic acids secretion of Bt-cotton was lesser than that of WT. The roots of the transgenic cotton secreted lesser amounts of amino acids and soluble sugars than the WT roots in the full-nutrient solution. Deficiencies of P and K caused a large increase in the total amino acid and soluble sugar secretions of both Bt-cotton and WT, with larger increases observed in Bt-cotton. Because transferring the foreign Bt gene into cotton can result in alterations in the components of the root exudates, with the effect varying depending on the nutritional status, the cultivation of genetically modified crops, such as Bt-cotton, in soil environments should be more carefully assessed, and the possible effects as a result of the alterations in the root exudate components should be considered.

Profiling influences of gene overexpression on heterologous resveratrol production in Saccharomyces cerevisiae

Metabolic engineering of heterologous resver- atrol production in Saccharomyces cerevisiae faces challenges as the precursor L-tyrosine is stringently regulated by a complex biosynthetic system. We over- expressed the main gene targets in the upstream pathways to investigate their influences on the downstream resver- atrol production. Single-gene overexpression and DNA assembly-directed multigene overexpression affect the production of resveratrol as well as its precursor p-coumaric acid. Finally, the collaboration of selected gene targets leads to an optimal resveratrol production of 66.144-3.74 mg.L-1, 2.27 times higher than the initial production in YPD medium （4% glucose）. The newly discovered gene targets TRP1 expressing phosphoribosy- lanthranilate isomerase, AR03 expressing 3-deoxy-D- arabino-heptulosonate-7-phosphate synthase, and 4CL expressing 4-coumaryl-CoA ligase show notable positive impacts on resveratrol production in S. cerevisiae.

Cloning and Overexpressing Apo-phycoerythrocyanin α-subunit Gene of Mastigocladus laminosus in E.coli

By PCR method, apo phycoerythrocyanin α subunit gene (pecA) of Mastigocladus laminosus (M. laminosus) was amplified from its genomic DNA, and then cloned in pBluescript. The pecA gene was subcloned into the expression vector pGEMD, and then transformed into E.coli BL21 (DE3). After induction, a new protein of molecular weight 19×10 3 existing in inclusion body was overexpressed. The expressed product was confirmed to be apo phycoerythrocyanin α subunit by Dot ELISA.

Overexpression of the <i>Rosa rugosa RrGT1</i>Gene Induces Anthocyanin Accumulation in Tobacco

Rosa rugosa has always been an important plant in landscape application, and the improvements and innovations about its flower color are particularly important. Glycosylation modification fulfills an important role in increasing the stability and solubility of anthocyanin in plants. In this study, based on the transcriptional database of R. rugosa, a gene with full length cDNA of 1161 bp, encoding 386 amino acids, designated as RrGT1, were isolated from flowers of R. rugosa “Zizhi” and then functionally characterized. Sequence alignments with the NCBI database show that the RrGT1 protein is a member of the GTB superfamily and has typical conserved amino acid residues called PSPG that are crucial for RrGT1 enzyme activity. RrGT1 transcripts were detected in five flowering stages and seven tissues of R. rugosa “Zizhi” and their expression patterns corresponded with the accumulation of anthocyanins. Additionally, the in vivo function of RrGT1 was investigated via its overexpression in tobacco. Transgenic tobacco plants expressing RrGT1 induced anthocyanin accumulation in flowers, indicating that RrGT1 could encode a functional glycosyltransferase (GT) protein for anthocyanin biosynthesis and could function in other species. Therefore, we speculated that glycosylation of RrGT1 played a crucial role in anthocyanin biosynthesis in R. rugosa.

In vivo protective effect of late embryogenesis abundant protein(ApSK3 dehydrin)on Agapanthus praecox to promote post-cryopreservation survival

Dehydrins(DHNs),as members of the late embryogenesis abundant protein family,play critical roles in the protection of seeds from dehydration and plant adaptation to multiple abiotic stresses.Vitrification is a basic method in plant cryopreservation and is characterized by forming a glassy state to prevent lethal ice crystals produced during cryogenic storage.In this study,ApSK3 type DHN was genetically transformed into embryogenic calluses(EC)of Agapanthus praecox by overexpression(OE)and RNA interference(RNAi)techniques to evaluate the in vivo protective effect of DHNs during cryopreservation.The cell viability showed a completely opposite trend in OE and RNAi cell lines,the cell relative death ratio was decreased by 20.0%in ApSK3-OE EC and significantly increased by 66.15%in ApSK3-RNAi cells after cryopreservation.Overexpression of ApSK3 increased the content of non-enzymatic antioxidants(AsA and GSH)and up-regulated the expression of CAT,SOD,POD,and GPX genes,while ApSK3-RNAi cells decreased antioxidant enzyme activities and FeSOD,POD,and APX genes expression during cryopreservation.These findings suggest that ApSK3 affects ROS metabolism through chelating metal ions(Cu^(2+)and Fe^(3+)),alleviates H_(2)O_(2)and OH·excessive generation,activates the antioxidant system,and improves cellular REDOX balance and membrane lipid peroxidation damage of plant cells during cryopreservation.DHNs can effectively improve cell stress tolerance and have great potential for in vivo or in vitro applications in plant cryopreservation.

THE EXPRESSION OF CDK4 GENE IN HUMAN BREAST CANCER

Purpose: To analyze the genetic changes of CDK4gene and expression of CDK4 mRNA in the breastcancer.Materials and methods: We obtained a fragment ofCDK4 gene, from many kinds of human cancer ornormal tissues by PCR .Taking it as a probe, using themethods of Southern and Dot blotting, We analyzed therole of CDK4 gene in the occurrence and developmentof human breast cancer.Results: CDK4 gene appeared amplification and itsmRNA overexpressed clearly in the breast cellline(MDA231). In human breast cancer tissues, CDK4mRNA expressed much more than normal breasttissues(p<0.05).Conclusion: in the tumorigenesis process, theregulative role of CDK4 is important. its abnormalamplification or expression will cause cell division cycleinto a disorder one, then the cell may enter an unlimitedproliferation cycle.

甘蔗赤霉素合成基因ScGA3ox的功能初步研究

近年来在拟南芥、水稻等模式植物中对赤霉素生物合成途径的研究日益完善,而重要糖料作物甘蔗中的赤霉素生物合成途径尚未明确,为了进一步验证ScGA3ox的功能,开展了甘蔗赤霉素合成基因ScGA3ox的功能初步研究。本文通过构建甘蔗GA3氧化酶基因ScGA3ox的植物过量表达载体,并将该基因通过农杆菌介导法转化到拟南芥中来快速验证其在植株生长中的功能。结果表明:本研究成功构建了ScGA3ox过量表达载体,并转化拟南芥获得过表达转基因T2株系。ScGA3ox过量表达对拟南芥的表型产生了影响:ScGA3ox转基因拟南芥株系与野生植株(WT)在株高上存在较为明显的差异,ScGA3ox-1和ScGA3ox-2株系分别比野生植株矮5.04和2.33cm。甘蔗GA3氧化酶基因ScGA3ox对植物生长有重要影响,但其具体功能研究有待深入开展。本研究结果将为进一步解析ScGA3ox基因调控甘蔗节间长度的分子机理提供了理论支撑。

棉花凝集素类受体蛋白激酶基因GhLecRK1响应棉蚜危害的功能分析

【目的】克隆棉花(Gossgpium hirsutum)凝集素类受体蛋白激酶(LecRK1)基因GhLecRK1,分析其在响应棉蚜危害中的功能,为培育棉花抗蚜新品种提供理论参考。【方法】在棉蚜诱导的棉花叶片全转录组测序数据分析基础上,克隆响应棉蚜危害的GhLecRK1基因,并对其序列进行生物信息学分析。采用实时荧光定量PCR检测GhLecRK1基因在棉蚜诱导下的表达模式,运用瞬时过表达技术研究其在棉花对棉蚜防御响应中的功能。【结果】GhLecRK1基因开放阅读框(ORF)长度1233 bp,编码410个氨基酸残基,理论等电点(pI)8.36,预测蛋白质分子量45948 Da,由具有1个α-甘露糖凝集素结合结构域、1个S-糖蛋白结构域(SLG)和1个纤溶酶原/苹果/线虫结构域(PAN)组成,属G型凝集素类受体蛋白激酶。同源序列比对分析结果表明,GhLecRK1与其他植物的G型LecRLKs序列具有高度相似性(94.39%)。系统发育分析结果表明,GhLecRK1与Gossypium raimondii的Gr012449778.1亲缘关系最近。在棉蚜取食诱导后24、48和72 h的棉花植株的子叶、根、茎中GhLecRK1基因的表达量均有不同程度上调,但是仅有子叶在棉蚜取食24 h时GhLecRK1基因相对表达量显著上调(P<0.05,下同)。选择性和非选择性试验结果显示,瞬时过表达GhLecRK1基因棉花上棉蚜的数量均少于WT植株和pMD植株,且在取食48 h时过表达GhLecRK1基因棉花上棉蚜蜜露的分泌量显著减少。【结论】GhLecRK1基因响应棉花对棉蚜的防御反应,GhLecRK1基因过量表达后可增强棉花对棉蚜的抗性。

大白菜BrCYP83B1基因的克隆及表达分析

【目的】细胞色素P450家族是十字花科植物硫苷合成重要的酶系,其中CYP83亚家族主要参与核心结构的合成,旨在探究大白菜(Brassica rapa ssp.pekinensis)CYP83B1基因的功能。【方法】利用RT-PCR技术克隆BrCYP83B1基因,通过生物信息学软件分析其编码蛋白理化性质、同源性及启动子顺式作用元件,利用RT-qPCR技术分析BrCYP83B1的表达模式,并构建其植物超表达载体。【结果】BrCYP83B1 cDNA序列全长为1500 bp,编码499个氨基酸,编码蛋白属于细胞色素P450超家族,主要定位于细胞质,二级结构主要由α-螺旋和无规则卷曲构成,与甘蓝型油菜、青花菜的CYP83B1蛋白具有较高的同源性。启动子分析表明,该基因启动子区域包含水杨酸、脱落酸及茉莉酸甲酯等激素响应的顺式作用元件,说明BrCYP83B1基因表达可能受激素调控。RT-qPCR分析结果表明,BrCYP83B1基因在大白菜的根、茎、叶、花和果中均有表达,且以叶中的表达量最高;茉莉酸甲酯够显著促进该基因的表达,而水杨酸处理对其表达具有一定的抑制作用,脱落酸处理下基因先上调后又下调。【结论】BrCYP83B1可能参与大白菜对激素的响应调控。

过表达BIRC5基因对氧糖剥夺/复氧诱导的脑微血管内皮细胞活性及VEGF表达的影响

目的探讨过表达BIRC5基因对氧糖剥夺/复氧(OGD/R)诱导的小鼠脑微血管内皮细胞损伤的保护作用,并分析其可能的机制。方法将小鼠源性脑微血管内皮细胞,根据不同干预方式分为正常对照组(细胞正常培养)、细胞损伤组(细胞正常培养24 h,随后OGD3h/R3h损伤细胞)、BIRC5干预组(细胞预先转染腺病毒-BIRC5质粒并培养24 h,随后OGD3h/R3h处理)和阴性对照组(细胞预先转染腺病毒空质粒并培养24 h,随后OGD3h/R3h处理)。采用激光共聚焦显微镜观察各组细胞形态学变化;用MTT法和流式细胞仪分别检测各组细胞存活率和凋亡率;用RT-PCR和免疫印迹法分别检测各组细胞BIRC5和VEGF mRNA及蛋白表达。结果正常对照组的细胞骨架微丝彼此连接,分布规则,丝网状有序排列;细胞损伤组和阴性对照组的细胞微丝断裂,收缩变短或移向周边,微丝网状排列紊乱,可见细胞外形皱缩,间隙加大,少部分微丝缺失出现空隙;BIRC5干预组的细胞微丝连接,形态成长梭形,排列较规则,可见细胞间隙缩小,显示过表达BIRC5基因能够减轻损伤细胞的骨架微丝紊乱。与正常对照组相比,细胞损伤组、BIRC5干预组及阴性对照组细胞存活率降低,而细胞凋亡率增高,差异均有统计学意义(P<0.05);与细胞损伤组相比,BIRC5干预组的细胞存活率增高,而细胞凋亡率降低,差异有统计学意义(P<0.05)。与正常对照组相比,细胞损伤组、BIRC5干预组和阴性对照组的BIRC5和VEGF的mRNA及蛋白表达降低,差异有统计学意义(P<0.05);与细胞损伤组相比,BIRC5干预组细胞BIRC5和VEGF的mRNA及蛋白表达增高,差异有统计学意义(P<0.05)。结论过表达BIRC5基因对OGD/R诱导的脑微血管内皮细胞损伤具有保护作用,机制可能与上调VEGF表达有关,提示BIRC5调控VEGF表达促进血管新生可能是脑侧支循环建立和形成的重要机制之一。

‘红满堂’苹果MbbZIP43基因的克隆与功能研究

【目的】为探究碱性亮氨酸拉链(bZIP)转录因子在‘红满堂’苹果花青素代谢中的调控作用。【方法】从‘红满堂’苹果克隆得到一个bZIP基因,分析了其基因及编码蛋白序列特性,利用洋葱表皮细胞瞬时转化确定其编码蛋白亚细胞定位,利用实时荧光定量PCR(RT-qPCR)研究了该基因在不同成熟时期‘红满堂’果实中的表达情况,基于烟草叶片、苹果叶片以及苹果果实瞬时转化研究了其对花青素积累和花青素合成相关结构基因表达的调控作用。【结果】该基因不含内含子,与MdbZIP43(XP_008393381.1)相似度最高,故命名为MbbZIP43。其编码序列长为522 bp,可编码由173 aa组成、含有bZIP_plant_GBF1结构域、定位于细胞核的亲水蛋白。RT-qPCR分析结果显示,随着果实成熟,MbbZIP43在‘红满堂’果实中的表达水平呈“先升后降”的变化趋势,在花后11周的果实中表达量最高。相关性分析显示MbbZIP43基因表达量与总黄酮和花青素含量正相关。瞬时过表达该基因的烟草叶片、苹果叶片和果皮中花青素含量分别提高了17.42%、25.66%和48.99%,过表达MbbZIP43的苹果叶片中花青素合成结构基因CHI、F3'H、DFR和UFGT分别提高了2.27倍、1.84倍、2.39倍和2.89倍;过表达MbbZIP43的苹果果皮中CHI、F3'H、DFR和UFGT分别提高了1.79倍、1.70倍、1.35倍和1.51倍,说明MbbZIP43可以通过上调这些结构基因的表达正调控苹果花青素合成。【结论】MbbZIP43可通过激活花青素合成相关结构基因CHI和UFGT等的表达进而促进花青素的积累。

核酸酶基因LoENDONUCLEASE1在东方百合花朵衰老过程中的功能分析

为明确百合(Lilium spp.)花朵衰老发生的分子机制,在东方百合‘西伯利亚’(Lilium oriental hybrid‘Siberia’)花被片中克隆了一个衰老相关基因LoENDONUCLEASE 1(SAG10),利用qRT-PCR进行基因表达分析,并通过农杆菌介导的拟南芥稳定表达系统和百合花被片瞬时表达体系验证LoENDONUCLEASE 1基因功能。结果显示,LoENDONUCLEASE 1基因开放阅读框(ORF)为921 bp,编码306个氨基酸;LoENDONU⁃CLEASE 1在百合花朵和叶片的衰老过程中特异表达,且受到脱落酸和水杨酸的诱导;拟南芥过表达LoENDO⁃NUCLEASE 1株系中叶片叶绿素含量显著低于对照株系,百合花被片中瞬时过表达LoENDONUCLEASE 1基因加速了百合花被片的衰老,且过表达花被片中丙二醛和离子渗透率含量显著高于对照。结果表明,LoEN⁃DONUCLEASE 1具有促进花朵和叶片衰老的功能。

TaEMF2调控小麦抽穗期的功能分析

适宜的抽穗期(开花时间)是作物获得高产和稳产的重要育种目标,EMF2是参与成花调控过程中的重要基因之一,但该基因在小麦中的功能仍不清楚。本研究从普通小麦中克隆了水稻OsEMF2b的直系同源基因TaEMF2,TaEMF2-2D是一个细胞核和细胞质定位蛋白。敲除TaEMF2会推迟小麦的抽穗期,过表达TaEMF2-2D会使小麦抽穗期提前。利用RT-qPCR检测小麦开花相关基因在TaEMF2转基因株系中的表达,结果表明,VRN1和VRN3在敲除转基因株系中的表达水平显著下调,在过表达株系中这2个基因的表达量则显著上调,表明TaEMF2可能通过调控VRN1和VRN3的表达来影响小麦的抽穗期。

龙胜凤鸡IGF2BP1基因的克隆、序列分析和过表达载体构建

为研究胰岛素样生长因子2 mRNA结合蛋白1(Insulin-like growth factor 2 mRNAbinding protein 1,IGF2BP1)基因的序列和在鸡肌肉发育中的功能,研究克隆IGF2BP1基因并进行生物信息学分析,构建IGF2BP1的过表达载体pEGFP-IGF2BP1,在成肌细胞验证载体的功能。结果显示:龙胜凤鸡IGF2BP1基因CDS区序列全长1 731 bp,编码576个氨基酸,与参考序列相比,存在6处同义突变;物种间同源性分析显示,龙胜凤鸡IGF2BP1基因与原鸡(Gallus gallus)、火鸡(Meleagris gallopavo)、绿头鸭(Anas platyrhynchos)、猪(Sus scrofa)、奶牛(Bos taurus)、小鼠(Mus musculus)的相似性分别为99.7%、94.7%、92.5%、82.4%、82.8%、82.7%;蛋白质二级、三级结构主要含有无规卷曲(45.83%)、α螺旋(27.26%)和延伸链(20.83%);经过酶切和测序鉴定显示,成功构建了pEGFP-IGF2BP1质粒,并且该质粒可在成肌细胞中表达绿色荧光,qRT-PCR结果显示IGF2BP1在试验组(转染pEGFP-IGF2BP1)的表达量极显著高于对照组(转染pEGFP-N1)(P<0.01)。结果表明,成功克隆IGF2BP1基因,构建的IGF2BP1基因过表达载体在成肌细胞中成功表达。

南美油藤PvWRI1转录因子的克隆及其在油脂合成中的功能分析

WRI1是一类具有APETALA2结构域的转录因子,参与油脂生物合成过程。本研究从新鲜南美油藤叶片中克隆PvWRI1基因组序列,并对其进行生物信息学分析。通过构建过表达载体,将PvWRI1基因转化到烟草中获得转基因阳性烟草植株,收获成熟种子,利用气相色谱法进行脂肪酸组分测定。并通过实时荧光定量PCR分析PvWRI1基因下游脂肪酸途径基因的表达水平。PvWRI1基因组DNA全长5072 bp,编码区长度为1312 bp,编码436个氨基酸,共存在7个外显子和6个内含子。疏水性分析显示该蛋白为亲水性蛋白,结构域分析该蛋白含有2个AP2/EREBP结构区和4个固有无序蛋白区,二级结构分析表明该蛋白相对保守。磷酸化位点预测N端含叶绿体转运肽特征。氨基酸同源性分析在不同物种中AP2结构域同源性较高,系统进化树显示PvWRI1与蓖麻同源性最高。脂肪酸组分测定结果表明,硬脂酸和亚麻酸含量显著增加,分别增加16.8%和7.5%。转基因种子的含油量由12.84%增加到24.06%,达1.87倍。同时,亚油酸/亚麻酸平衡比例下降7.8%。RT-qPCR结果显示过表达PvWRI1导致下游脂肪酸途径基因表达量均出现上调,其中WRI1基因极显著上调,FATA、KASI和FAD3基因显著上调。因此,PvWRI1作为调控脂肪酸生物合成的关键因子,对改良油料植物含油量和脂肪酸平衡具有指导作用。

IGF-1基因克隆及其产物的测序鉴定

基因过表达在基础实验和基因治疗应用中都是常用的手段,而质粒的扩增在基因过表达过程中是不可或缺的技术,如何规范、高效地对质粒进行扩增和提取是基因治疗应用的前提,也是困扰很多基础研究人员的难题。利用现有的实验技术,在降低实验成本基础上,建立过表达质粒扩增、提取的最优方案。首先采用传统方法制作LB培养基和感受态细胞,对转化后的质粒进行大量扩增培养,之后使用商业试剂盒对质粒进行提取,反复试验后对商业试剂盒质粒提取过程进行优化,提高效率,最终获得高纯度和浓度的质粒溶液。经超微量分光光度计、琼脂糖凝胶电泳检测和sanger测序等多种方式检测验证后与NCBI数据库中目标基因序列一致,提示扩增、提取成功。经优化后的质粒扩增、提取方案标准、高效,成本低廉,是质粒基因克隆的最佳选择。

杨树UGT198基因的生物信息学分析、基因克隆及功能初探

【目的】研究UGT198基因是否参与调控烟草抗虫性,为未来探索其参与调控杨树抗虫性奠定基础。【方法】前期通过分析公共转录组数据发现PtrUGT198基因在杨树响应虫害的转录组中高调表达,对其进行生物信息学及进化分析。以107杨为材料,克隆UGT198基因,构建过量表达载体,利用农杆菌介导法转化烟草获得过量表达转基因株系。通过棉铃虫饲虫试验初步验证UGT198基因是否参与调控抗虫性,分析UGT198基因在调控抗虫性中发挥的功能。【结果】PtrUGT198基因完整的ORF区为1440 bp,编码479个氨基酸,理论分子量为53.14 kDa,等电点为5.92,编码不稳定疏水性蛋白。PtrUGT198蛋白的二级结构α⁃螺旋占41.75%,β⁃折叠占14.61%,β⁃转角占7.10%,无规卷曲占6.53%。PtrUGT198蛋白不存在跨膜区,预测其不属于膜蛋白,为非分泌性蛋白,该蛋白的磷酸化修饰以丝氨酸为主。同源序列对比结果显示PtrUGT198蛋白的C末端存在高度保守的植物次生产物糖基转移酶(Plant secondary product glycosyltransferase,PSPG)基序。系统进化树分析发现,PtrUGT198蛋白与同属植物毛白杨、银白杨和胡杨亲缘关系最近,其次和模式植物烟草有较近的亲缘关系。成功克隆杨树UGT198基因,构建pNC⁃UGT198基因过量表达载体,并转化烟草获得过量表达转基因株系,进行饲虫实验,得到高表达量烟草相对抗虫。【结论】首次成功克隆了UGT198基因,并初步解析其调控抗虫性的功能,为深入了解UGT198基因调控杨树抗虫机理提供参考价值。

构建过表达TRPV1基因的Caco-2细胞株

采用慢病毒载体系统构建辣椒素受体基因TRPV1过表达的人结直肠腺癌细胞Caco-2稳定重组株.将双酶切后的慢病毒空载体pCDH和TRPV1全基因PCR产物通过T4 DNA Ligase连接,构建包含TRPV1基因的过表达载体pCDH-TRPV1.将过表达载体pCDH-TRPV1转化DH 5α感受态细菌,大量扩繁后提取过表达载体pCDH-TRPV1的质粒,与psPAX2和pMD两种含有慢病毒包装所必需元件的质粒混合,再与脂质体混合制备脂质体-载体混合液.将脂质体-载体混合液转染至单层的293T细胞中,培养48h进行病毒包装.收集富含慢病毒颗粒的293T细胞上清液,超离心纯化成浓缩病毒,然后再与polybrene一起感染单层Caco-2细胞,通过GFP绿荧光信号来筛选获得TRPV1基因过表达的稳定细胞株.通过Realtime PCR方法和Western-blot检测TRPV1的mRNA表达量及蛋白表达量,结果表明,Caco-2-TRPV1重组细胞株的TRPV1的mRNA表达量及蛋白表达量均显著高于Caco-2-GFP对照细胞(P<0.05).成功构建了TRPV1基因过表达的稳定细胞株,为后续辣椒素降脂机理的研究提供了正向调控细胞模型.