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1.
通过PCR从苋属植物千穗谷(Amaranthus hypochondriacus)的总DNA中扩增出苋菜凝集素(AHA)的核基因片段。序列分析结果表明该基因为2453bp,含有一1538bp的内含子和两个分别为212bp和703bp的外显子。采取反向PCR的方法获得仅含该基因的编码区克隆。以此为基础与二元表达载体pBin438构建含内含子与不含内含子AHA基因的植物表达载体pBAHAg和pBAHAc并通过土壤农杆菌介导转化了烟草。转化再生植株的PCR和Southern blot分析表明,AHA基因已整合到烟草的染色体中,有单拷贝和多拷贝的整合。用与AHA蛋白高度同源的ACA蛋白的抗血清进行了免疫斑点(Immunodot blot)检测,结果初步表明转基因烟草有AHA蛋白的表达。虫试结果表明转pBAHAg和pBAHAc烟草对蚜虫的平均抑制率分别达57.2%和48.8%,有的高达90%以上。含内含子和不含内含子的AHA基因在转基因植株中的抗蚜性不同。  相似文献   

2.
二次转化获得整合phbA、phbB、phbC基因的转基因烟草   总被引:1,自引:0,他引:1  
将携有导肽序列的phbB(编码乙酰乙酰CoA原酶)和phbC(编码PHB合酶)连入pBIB-HYG得到组成型表达载体pZCB。用冻融法转入根癌土壤杆菌(Agrobacterium tumefaciens(Smith et Townsend)Conn)并由其介导转化已整合且表达phbA(编码3-酮硫裂解酶)基因并具有卡那霉素抗性的转基因烟草(Nicotiana tabacum L.)。通过二次转化可避开传统杂交育种,在5个月内获得整合PHB合成所需3个基因的转基因烟草。所获转基因植株表型正常,经PCR、PCR-Southern、RT-PCR-DNA交交检测确定有50株烟草稳定整合phbB、phbC基因,其中6.67%的植株可在转录水平表达双基因。  相似文献   

3.
转基因烟草表达霍乱毒素B亚单位的研究   总被引:10,自引:0,他引:10  
将霍乱毒素B亚单位(CTB)基因克隆到质粒pBin438中,分别构建植物表达载体pBI-CTB、pBI-SPCTB和pBI-CTBER。采用叶盘法分别转化烟草K326,各表达载体得到了一批较基因植株。转基因烟草的PCR和Southern blot分析表明CTB基因整合到了烟草基因组中。转基因植株的ELISA和Western blot分析表明pBI-SPCTB和pBI-CTBER的转基因植株能有效表  相似文献   

4.
在克隆了马铃薯X病毒(PVX)、马铃薯Y 病毒(PVY)和马铃薯卷叶病毒(PLRV)的外壳蛋白基因的基础上,构建同时包含PVX和PVY 与PVY 和PLRV 两个外壳蛋白基因植物表达框架的表达载体,通过农杆菌(Agrobacterium tumefaciens)介导转化烟草(Nicotianatabacum )和生产上常用的几个马铃薯(Solanum tuberosum )优良品种:“Favorita”、“虎头”、“克4”。经PCR检测证明外源基因已整合到植物的染色体上,得到批量转基因植株。在转PVX+PVY 外壳蛋白基因的烟草上接种PVX (5 μg/m L)、PVY(20 μg/m L)病毒,得到有一定抗性的植株  相似文献   

5.
用合成的crylAc基因与绿色荧光蛋白基因(GFP)构成融合蛋白基因,然后和改造的GNA基因构建双价抗虫基因植物表达载体pBGbfg,经根癌农杆菌介导转化了烟草。在紫外灯照射下,观察到转基因植株叶片中有较强的绿色荧光;经抗虫试验、PCR、Southern blot和Western blot等检测,表明该重组植物表达载体能够在转基因植物中有效表达外源基因,转基因植株绿色荧光的表型与其抗虫性密切相关。从而成功地建立了以绿色荧光蛋白基因与抗虫基因组成的融合基因转化系统,简化了抗虫转基因植物筛选程序,有助于快速获得双价抗虫转基因植株。  相似文献   

6.
在大肠杆菌中表达了马铃薯Y病毒中国分离物(PVY-C)复制酶NIb基因,并制备了其抗血清。利用PCR定点突变方法使NIb基因移码-1位,构建了移码-1位NIb基因(UN)的植物表达载体。通过土壤农杆菌(Agrobacterium tumefaciens LBA4404)介导转化烟草NC89,获得51株再生植株。对再生植株的分子检测结果表明,转基因烟草中检测到UN基因相应的RNA转录产物,3推测该基  相似文献   

7.
用合成的cry1Ac基因与绿色荧光蛋白基因(GFP)构成融合蛋白基因,然后和改造的GNA基因构建双价抗虫基因植物表达载体pBGbfg,经根癌农杆菌介导转化了烟草.在紫外灯照射下,观察到转基因植株叶片中有较强的绿色荧光;经抗虫试验、PCR、Southern blot和Western blot等检测,表明该重组植物表达载体能够在转基因植物中有效表达外源基因,转基因植株绿色荧光的表型与其抗虫性密切相关.从而成功地建立了以绿色荧光蛋白基因与抗虫基因组成的融合基因转化系统,简化了抗虫转基因植物筛选程序,有助于快速获得双价抗虫转基因植株.  相似文献   

8.
黄瓜花叶病毒(CMV)运动蛋白基因介导的抗病性   总被引:4,自引:0,他引:4  
利用Fny_CMV株系RNA3cDNA克隆,构建了含有全长和编码区缺失501个核苷酸的运动蛋白(MP)基因植物表达载体pBMPR和pBMPK。在土壤农杆菌(Agrobacteriumtumefaciens(SmithetTownsend)Conn)LBA4404介导下转化烟草(NicotianatabacumL.)品种“NC89”,分别经Southernbloting、RT_PCR或Westernbloting分析,外源基因已整合到再生植株中并得到表达。抗病性分析表明,含有缺失型MP基因的R0代转基因植株抗性较好,接种50d后,10株转化植株中仍有5株不表现症状。在自然发病条件下,这5个含有缺失型MP基因转基因株系在R1代都表现了一定的抗病性。抗性主要表现为症状出现推迟,严重度减轻。利用PCR筛选、种子卡那霉素抗性试验和温室抗病性测定等方法,初步认为R2代转基因烟草K_6_5株系为转基因抗病纯合系。而含有全长MP基因的R0代转化植株,前期没有表现明显的抗病性,但在接种40d后部分发病植株有恢复健康的趋势。  相似文献   

9.
金属硫蛋白有α、β两个结构域(dom ain),其中α结构域优先结合Cd2+ 和Hg2+ .小鼠αα突变体在大肠杆菌中已经构建并得到表达,其转基因植株已得到,可在Cd300(300 μm ol/L)中生长.为了进一步提高外源基因在烟草中的表达量,首先用PCR 的方法设计引物,在基因翻译起始密码子ATG 附近加入植物偏爱的碱基组合AACAATG.另外,将该突变体基因插入具有双35 S(CaMV35S)强启动子的植物双元表达载体pGPTVd35S-BAR中,获得了带有αα突变体的植物双元表达载体.通过农杆菌介导的叶盘转化法转化烟草NC89,获得了抗除草剂的转基因植株.经PCR-Southern 和蛋白Dot-blotting 检测,证明了αα突变体在烟草中的嵌合与表达.抗重金属实验证明转基因烟草可以在Cd400(400 μm ol/L)中生长.  相似文献   

10.
转雪花莲外源凝集素基因烟草对桃蚜的抑制作用   总被引:31,自引:0,他引:31  
将编码雪花莲外源凝集素成熟蛋白的cDNA GNA12和其前体蛋白cDNA GNA34插入到二元载体pBin438的双倍增强子CaMV 35S启动子或二元载体pBcop1的CoYMV启动子下游,分别构建成植物表达载体pBGna12、pBGna34\,pBCGna12和pBCGna34。土壤农杆菌介导的转化再生植株的PCR和Southern blot分析表明,GNA基因已整合到烟草DNA中。Western blot分析发现pBGna34和pBCGna34的转基因植株能有效地表达外源GNA,表达量约占可溶性总蛋白的0.08%~0.15%,并且前体蛋白基因编码的蛋白在植物体内进行了正确的加工;而pBGna12和pBCGna12的植株几乎检测不到外源GNA的表达。有效表达外源GNA的pBGna34和pBCGna34的转基因植株具有较强的抗蚜活性,平均能够抑制桃蚜(Myzus persicae)45%~60%蚜口密度,有的高达90%以上。在转基因烟草中含双倍增强子的CaMV 35S启动子与韧皮部特异表达的CoYMV启动子介导GNA基因表达具有相似的强度,但它们的抗蚜活性存在差异。  相似文献   

11.
转双抗虫基因烟草的研究   总被引:22,自引:3,他引:19  
用改造的雪花莲凝集素基因GNAmm与合成的苏云金芽孢杆菌(Bt)毒蛋白cry1Ac基因构建了带有双价基因的植物表达载体,在该表达载体中这两个基因的转录分别受笋瓜PP2启动子(SPP2P)和CaMV 35S启动子的调控。通过根癌土壤杆菌介导转化法,获得了一批抗卡那霉素的转化再生烟草植株。PCR检测及基因组DNA Southern blot\,Slot blot杂交分析的结果表明Gna基因和Bt基因已整合到烟草总DNA中。用Bt毒蛋白抗血清进行Western blot分析,转基因植株均有Bt杀虫蛋白的不同程度的表达。对转化再生烟草的虫试结果表明,在所受试的19株烟草中60%的植株上的棉铃虫在5天内死亡率达到100%,而且存活幼虫的生长发育受到明显抑制;蚜虫抑制生长试验表明,多数转化再生植株具有较强的抗蚜活性,平均能够抑制桃蚜50%~60%的蚜口密度,有的高达80%以上。以上结果表明利用这两个改造过的抗虫基因可以获得既抗虫又耐蚜的转双抗虫转基因植物。  相似文献   

12.
两种凝集素基因在转基因烟草中表达的研究   总被引:10,自引:0,他引:10  
构建了含尾穗苋凝集素基因(ACA)的cDNA序列和改造后的雪花莲凝集素基因(GNA)的植物表达载体pBACG。在此表达载体中,ACA和GNA基因的表达分别由35S启动子和CoYMV启动子控制。通过农杆菌介导,将ACA和GNA基因转化到烟草中,经卡那霉素筛选获得60株转化再生植株。对PCR检测呈阳性的50株植株进行接蚜虫实验,结果表明,其平均抑虫率达83.9%。Southern blotting分析表明,ACA和GNA基因都已整合到烟草基因组中。Western blotting结果显示这两个基因在不同植株中都可表达其相应的蛋白质,但表达水平不同。部分Western blotting分析呈阳性植株的抗蚜性与T0代相近,达85.3%,说明这两个基因的抗蚜功能可以稳定遗传。  相似文献   

13.
雪花莲凝集素基因(gna)的改造及其抗蚜性   总被引:21,自引:1,他引:20  
用定点突变方法对编码雪花莲凝集素(Galanthus nivalis agglutinin,GNA)前体蛋白的DNA序列进行了改造和转基因烟草9Nicotana tabacum L.)抗蚜性的研究。结果表明,将GNA编码序列中含有的稀有密码子改造后,GNA的表达水平从占总可溶性蛋白的0.17%增加到0.25%,转基因烟草的抗蚜性也随之增强,从平均抑制桃蚜(Myzus per-sicae(Sulzer))虫口密度63.7%显地提高到71.0%。  相似文献   

14.
Yao J  Pang Y  Qi H  Wan B  Zhao X  Kong W  Sun X  Tang K 《Transgenic research》2003,12(6):715-722
Tobacco leaf discs were transformed with a plasmid, pBIPTA, containing the selectable marker neomycin phosphotransferase gene (nptII) and Pinellia ternata agglutinin gene (pta) via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic tobacco plants were regenerated. PCR and Southern blot analyses confirmed that the pta gene had integrated into the plant genome and northern blot analysis revealed transgene expression at various levels in transgenic plants. Genetic analysis confirmed Mendelian segregation of the transgene in T1 progeny. Insect bioassays showed that transgenic plants expressing PTA inhibited significantly the growth of peach potato aphid (Myzus persicae Sulzer). This is the first report that transgenic plants expressing pta confer enhanced resistance to aphids. Our study indicates that the pta gene can be used as a supplement to the snowdrop (Galanthus nivalis) lectin gene (gna) in the control of aphids, a sap-sucking insect pest causing significant yield losses of crops.  相似文献   

15.
To investigate the possible function of the agglutinin from Amaranthus caudatus L. (ACA) in plant defending against insect pests, ACA cDNA was cloned by RT-PCR and the 5‘ and 3‘ sequences were confirmed by rapid amplification of cDNA ends (RACE). The phloem-specific expression vector of ACA gene, pBCACAc, was constructed based on the plant binary vector pBC438 and transfered into tobacco plants via Agrobacterium-mediated transformation method. Results from PCR and Southern blotting analysis showed that AOA gene was integrated into the genomes of transformed plants and the transgene integration varied from one to four estimated copies per genome. Western blotting analysis indicated that ACA gene was transcribed and translated in the transgenic plants. The bioassay of Myzus persicae Sulzer on detached leaves demonstrated that the 78% transgenic tobacco plants displayed an average aphid-resistant rate of more than 75%. Some apterous progeny of M. persicae were found dead on the resistant plants. These results indicate that ACA gene should be an effective aphid-resistant gene and could be valuable for application in crop breeding for aphid resistance.  相似文献   

16.
The first intron (EPI) of rice 5-enolpyruvylshikimate 3-phosphate synthase gene was isolated by PCR from one clone with genomic EPSP synthase gene. Sequence analysis showed that the first intron is 704 bp in length with 36.2% G+C content. To investigate its effect on expression of foreign gene, we inserted the first intron between CaMV35S promoter and β-glucuronidase (GUS) gene. The transient expression results showed that GUS could be expressed effectively with EPI. The GUS activity in transgenic tobacco shows that the EPI can greatly enhance the expression level of β-glucuronidase (P < 0.01) compared with transgenic tobacco without the first intron, and 3-to 6-fold increase in GUS activity in some transgenic tobaccos. Northern blot indicated the first intron was spliced from GUS pre-mRNA, and the steady-state mRNA levels of GUS with EPI in transgenic tobaccos were higher than that in transgenic tobacco without EPI, which suggested that the first intron of EPSP was a non-translated intron.  相似文献   

17.
豇豆胰蛋白酶抑制剂基因转化芥菜及抗虫鉴定   总被引:3,自引:0,他引:3       下载免费PDF全文
用农杆菌介导将豇豆胰蛋白酶抑制剂 (CpTI)基因导入芥菜 ,获得了Kan抗性植株 .经PCR扩增、PCR Southern印迹和Northern印迹分析 ,转化再生植株大部分呈阳性 ,而非转化的再生植株均为阴性 ,证明CpTI基因已存在于芥菜基因组中 .在室内进行了喂虫试验 ,结果表明转基因芥菜抗虫性明显高于对照 ,转基因植株之间存在抗虫性差异  相似文献   

18.
The first intron of rice EPSP synthase enhances expression of foreign gene   总被引:5,自引:0,他引:5  
Translatable exon sequences in pre-mRNA often are separated by non-coding introns in eu-karyotic genomes. The removal of non-coding introns from pre-mRNA and the splicing together of translatable exons sequence is an essential requirement of gene expression. DNA size of introns in a gene is 5—10 times larger than that of exon, which can store more information and is helpful for a gene during evolution[1]. In many experiments on gene expression, it is indispensable for a gene to be expresse…  相似文献   

19.

Background

RNA silencing is an important mechanism for regulation of endogenous gene expression and defense against genomic intruders in plants. This natural defense system was adopted to generate virus-resistant plants even before the mechanism of RNA silencing was unveiled. With the clarification of that mechanism, transgenic antiviral plants were developed that expressed artificial virus-specific hairpin RNAs (hpRNAs) or microRNAs (amiRNAs) in host plants. Previous works also showed that plant-mediated RNA silencing technology could be a practical method for constructing insect-resistant plants by expressing hpRNAs targeting essential genes of insects.

Methodology/Principal findings

In this study, we chose aphid Myzus persicae of order Hemiptera as a target insect. To screen for aphid genes vulnerable to attack by plant-mediated RNA silencing to establish plant aphid resistance, we selected nine genes of M. persicae as silencing targets, and constructed their hpRNA-expressing vectors. For the acetylcholinesterase 2 coding gene (MpAChE2), two amiRNA-expressing vectors were also constructed. The vectors were transformed into tobacco plants (Nicotiana tabacum cv. Xanti). Insect challenge assays showed that most of the transgenic plants gained aphid resistance, among which those expressing hpRNAs targeting V-type proton ATPase subunit E-like (V-ATPaseE) or tubulin folding cofactor D (TBCD) genes displayed stronger aphicidal activity. The transgenic plants expressing amiRNAs targeting two different sites in the MpAChE2 gene exhibited better aphid resistance than the plants expressing MpAChE2-specific hpRNA.

Conclusions/Significance

Our results indicated that plant-mediated insect-RNA silencing might be an effective way to develop plants resistant to insects with piercing-sucking mouthparts, and both the selection of vulnerable target genes and the biogenetic type of the small RNAs were crucial for the effectiveness of aphid control. The expression of insect-specific amiRNA is a promising and preferable approach to engineer plants resistant to aphids and, possibly, to other plant-infesting insects.  相似文献   

20.
菠菜甜菜碱醛脱氢酶基因在烟草中的表达   总被引:74,自引:0,他引:74  
质粒pLS9含有1.5kb的编码菠菜甜菜碱醛脱氢酶(BADH)基因。经限制酶切后克隆到植物表达载体的35S启动子和PolyA终止子之间。经农杆菌介导转化烟草,获得90多株抗卡那霉素再生植株。经PCR检测证明60%以上再生植株含有BADH基因。转基因植株经Western blot,BADH酶活性测定,BADH酶活性特异性染色法检查和耐盐性分析,证明菠菜BADH基因在烟草正常表达。在叶绿体和胞液中均有BADH酶存在。转基因植株能耐较高浓度盐。  相似文献   

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