表达雪花莲外源凝集素基因的油菜转基因植株的获得

利用农杆菌素LBA4404（pCAMBIA3300RG)转化优良甘蓝型油菜恢复系W723的下胚轴节段.pCAM-BIA3300RG含有Rssl启动子引导的雪花莲外源凝集素基因（gna）和CaMV-35S启动子引导的除草剂抗性基因（bar）。经过两轮除草剂（2.5mg/L bialaphos）筛选（两周/轮）,除草剂抗性再生芽被传入生根培养基中生根,对根系旺盛生长的植株中所含gna基因进行PCR分析,PCR分析证实了这些植株确为转基因植株,利用Western印迹法对随机选择的5株含gna基因的转基因植株的分析发现,其中4株表达了gna基因。目前正对这些表达gna基因的转基因植株进行后代遗传分离分析。     

农杆菌介导IPT和etr1-1双基因转化番茄

建立了番茄子叶高频再生体系，优化了农杆菌介导的外源基因转化番茄的条件，将IPt和etrl-1双基因导入番茄中，以除草剂PPT作为筛选标记，得到3株转化再生植株．通过PCR，RT-PCR检测证明etrl-1和IPT基因已整合到转基因番茄植株的基因组中，并在转录水平有一定表达．     

油菜耐热基因（BnTR1）转化多年生黑麦草丛生芽的研究

油菜耐热基因(BnTR1)克隆自油菜,编码一种E3连接酶.为验证它的抗逆功能,本研究用携带BnTR1和新霉素磷酸转移酶(neomycin phosphotransferase,nptⅡ)基因的植物表达载体pCAMBIA2301-TR1的根癌农杆菌EHA105,对多年生黑麦草丛生芽进行遗传转化.经共培养和巴龙霉素筛选培养,获得了175株抗性植株.通过PCR和RT-PCR检测,获得62株转基因植株.结果表明,外源BnTR1基因已整合到转基因植株基因组中并在转录水平上表达.初步抗旱检测表明,BnTR1基因能明显提高转基因植株的抗逆能力.     

基于Gene-Deletor系统的安全复合性状转基因烟草研究

本研究利用农杆菌介导法将带有水稻花粉特异启动子籼稻花粉过敏原基因(OSIPA)启动子驱动的Gene-Deletor外源基因清除系统,水稻Os GA3ox2(D18)启动子驱动水稻Os GA2ox1基因,玉米Ubiquitin启动子驱动BAR::GUS融合基因为筛选标记基因以及水稻Actin1启动子驱动驱动抗虫Cry1Ab基因复合性状植物表达载体p GM626-D18-Os GA2ox1遗传转化三星烟草。通过GUS组织化学染色及PCR分子鉴定,获得了43株转基因烟草植株。结果表明,水稻OsGA2ox1基因在烟草中表达能降低转基因烟草植株高度,但不影响植株生殖生长。转基因烟草对烟草斜纹夜蛾幼虫具有抗性,可抑制烟草斜纹夜蛾幼虫的取食与发育。以50 mg/L的除草剂Basta处理野生型和转基因烟草离体叶片,发现BAR基因提高了烟草抗除草剂的能力。对12株转基因烟草T0代花粉外源基因清除效率进行研究,发现部分烟草株系外源基因清除可达到100%,其他未完全清除外源基因株系的清除效率介于42.84%~99.97%之间。     

转基因玉米遗传转化的研究进展

转基因玉米是目前植物基因工程的研究热点之一,已有转基因抗虫玉米、抗除草剂玉米进入商品化生产。通过对转基因玉米遗传转化方法的研究,概括了目前在转基因玉米的遗传转化中所用的各种方法、及其优缺点,以及已取得的成果和遗传转化的检测分析方法的研究进展。     

Fibrillin转基因油菜的再生及其光合特性的检测

用根癌农杆菌共培养法，以带有1～2 mm子叶柄的完整子叶为转化受体，将连有35S启动子和NPTⅡ标记基因的Fibrillin cDNA导入甘蓝型油菜主要栽培品种“2005南系”中，获得抗卡那霉素的转基因植株.对部分经卡那霉素筛选得到的再生植株进行PCR检测，有来自3个株系的植株表现为阳性，初步证实外源基因已整合到植物细胞基因组中.将转基因植株炼苗后移入大田，在3月初到4月末油菜开花结果期间，对叶片和角果进行光合指标和叶绿素荧光指标的检测以及产量性状分析，发现转基因植株和非转基因植株之间存在明显差异.     

农杆菌介导牧草蔗42遗传转化体系的建立

将拟南芥叶片衰老特异启动子sag12与异戊烯基转移酶基因ipt构成的嵌合基因经农杆菌介导转入牧草蔗42中，建立了农杆菌转化牧草蔗42的遗传转化系统，已经获得转基因再生植株，PCR及Southern杂交检测证实ipt基因确实转移到牧草蔗42中，报告基因GUS在再生植株中也得到表达。     

苜蓿转基因研究进展

植物遗传转化是指利用分子生物学的手段将外源基因导入受体植物,使其获得新的性状.传统的育种方法进展缓慢,周期长,而且短期内性状表现不明显.转基因技术比传统的育种方法能快速有效地定向培育植物新品种,获得的新性状比较明显.因此,它已成为植物育种研究领域热点.目前常用的植物遗传转化法包括农杆菌介导,基因枪直接转化等方法,但农杆菌介导法由于外源基因整合的拷贝数少,重排程度低,转化效率高,是最常用的苜蓿遗传转化法.现在通过农杆菌介导法已获得了抗逆,抗除草剂和品质改良等新性状转基因苜蓿,为苜蓿遗传育种开辟了一条新的途径.     

苜蓿花叶病毒RNA2 3''''端cDNA转基因烟草及其抗病性研究

将本室克隆的编码复制酶基因3′端约1／2序列及其3′端非编码区的苜蓿花叶病毒中国分离株(AIMV-Ch)RNA：3′端cDNA，重组到植物表达载体pROKⅡ中，通过致瘤农杆菌(Agrobacterium tumefaciens)介导，以叶圆片为转化材料，转化普通烟草，并获得了转基因植株．经卡那霉素抗性选择、PCR检测目的基因证明．AIMV RNA：3′端基因已整合到转基因烟草的基因组DNA中．转基因植物的攻毒试验表明转基因植株对苜蓿花叶病毒产生高水平的抗性．     

外源基因1Dx5在转基因小麦后代中的表达

采用PCR和SDS-PAGE技术,分析研究了转基因小麦后代外源品质基因1Dx5的遗传分离及表达规律,结果显示:转基因小麦后代的遗传分离完全符合孟得尔遗传分离规律,转基因植株都达到了纯合状态,1Dx5基因在转基因植株中已经稳定整合并稳定遗传,并发现了超表达的差异表达现象,在转基因植株中没有发现基因沉默现象.     

Expression of two insect-resistant genes crylA ( b&c )/GNA in transgenic tobacco plants results in added protection against both cotton bollworm and aphids

The synthesized Bacillus thuringiensis insecticidal protein gene crylA(b&c) and the synthesized gene GNA, (the mannose specific lectin from snowdrop ( Galanthus nivalis)), tumefaciens have been inserted into plant expression vector pGW4BAI. Leave stripes of Nico-tiana tabacum var. K326 have been transformed with Agrobacterium tumefaciens strain LBA4404 harboring the plant expression vector. 28 kanamycin resistant tobacco plants have been obtained. PCR and Southern blot analyses show that the foreign crylA and GNA genes have been inserted into the genome of transformed tobacco plants. Haemagglutination assays show that GNA has a functional activity. Leaf disc bioassays against cotton bollworm ( H. armigera) show that the transgenic tobacco plants have a high insecticidal activity. The inhibition of aphid population in leaf disc bioassays against Myzus persicae shows that the fecundity of aphid on transgenic plants is lower than that on untransformed plants; the aphid population on the transgenic tobacco plants is 25%-70% that on untransformed tobacco plants. ELISA analysis of CrylA protein in tobcco leaves provides similar data to bioassay results. Through the two bioassays against H. armigera and M. persicae, several transgenic tobacco plants showing high insect-resistant activities to both pests have been obtained.     

From pollen actin to crop male sterility

Actin plays an important role in the life activity of animal and plant cells. Pollen cells have plenty of actin whose structure and characteristics are very similar to the animal actin. The nucleotide sequence and amino acid sequence of plant actin gene are very similar to those of the animal gene. The content of pollen actin from male sterile plants is much more lower than that from its maintainer plants. The expression of actin gene is organ-specific during the plant development. The expression quantity of actin gene in pollen is much more higher than those from root, stem and leaf. The expression plasmid of the anti-sense actin gene was constructed, transferred to the protoplasts of wheat and tomato to inhibit the expression of actin gene in pollen and thus the male sterile plants of wheat and tomato were obtained. The actin in pollens from the transgenic plants was reduced significantly, whereas the pistil was not affected. This study might pave a new way to breeding male sterile lines for the application of hybrid vigor of wheat and tomato.     

Expression of two insect-resistant genescryIA (b&c)/GNA in transgenic tobacco plants results in added protection against both cotton bollworm and aphids

The synthesizedBacillus thuringiensis insecticidal protein gene cryIA(b&c) and the synthesized geneGNA, (the mannose specific lectin from snowdrop (Galanthus nivalis)), tumefaciens have been inserted into plant expression vector pGW4BAI. Leave stripes ofNicotiana tabacum var. K326 have been transformed withAgrobacterium tumefaciens strain LBA4404 harboring the plant expression vector. 28 kanamycin resistant tobacco plants have been obtained. PCR and Southern blot analyses show that the foreigncryIA andGNA genes have been inserted into the genome of transformed tobacco plants. Haemagglutination assays show thatGNA has a functional activity. Leaf disc bioassays against cotton bollworm (H. armigera) show that the transgenic tobacco plants have a high insecticidal activity. The inhibition of aphid population in leaf disc bioassays againstMyzus persicae shows that the fecundity of aphid on transgenic plants is lower than that on untransformed plants; the aphid population on the transgenic tobacco plants is 25%–70% that on untransformed tobacco plants. ELISA analysis of ClyIA protein in tobcco leaves provides similar data to bioassay results. Through the two bioassays againstH. armigera andM. persicae, several transgenic tobacco plants showing high insect-resistant activities to both pests have been obtained.     

Expression of lysine-rich protein gene and analysis of lysine content in transgenic wheat

Expression vector pBPC102, which carries winged bean lysine-rich protein (wblrp) gene and dihydropicolinate synthase (DHDPS) gene, was transferred into hexaploid winter wheat cv. Jinghua No.l, Jing411, You899 and Yangnongl5 explants of immature inflorescence and immature embryos by particle bombardment. More than 100 transgenic plants were obtained under the selection of s-(2-aminoethyl)-L-cysteine (AEC). Confirmed transgenic plants of To and TI generation by PCR and PCR-Southern blotting analyses showed successful integration of wblrp gene into wheat genome. Analysis of transgenic plant lines of T2 by Northern dot-blotting showed good expression of wblrp gene in offspring seed. The content of free lysine in leaves, contents of bound lysine and total proteins in seeds of T2 transgenie wheat lines were determined and analyzed. Among 34 tested transgenic lines, levels of free lysine content in leaves of 9 transgenic lines are 2~3times higher than un-trans-formed wild-type cultivars. Among 17 analyzed transgenic lines, bound lysine content of 4 transgenic lines is more than 10% higher than that of wild-type cultivars. Our research suggests that introducing wblrp gene into wheat is an effective way to improve its nutrition quality.     

Introduction of rice chitinase gene into wheat via low energy Ar+ beam implantation

A chitinase gene (RCH8) in plasmid vector pCAMBIA1308 was delivered into 3 wheat cultivars (Yangmai 158, Wan 9210, Wanmai 32) by low energy Ar⁺ beam-mediated method. Preliminary calli from treated mature embryos were first selected on hygromycin (Hm, 20 or 30 mg/L) containing medium. After the resistant calli formed, they were transferred to the regeneration medium with 10 or 20 mg/L Hm. All the three wheat varieties obtained transgenic plants. PCR and PCR-Southern assays showed that most plants regenerated from the resistant calli were positive transgenic plants. Southern blot of the positive green plants confirmed stable integration of alien DNA into wheat genome. The plant transformation frequencies varied with the variety and ion dose implanted. Wanmai 32 possessed the highest transformation frequency, reaching 3.8% at a suitable implantation dose. The transformation frequency of Yangmai 158 and Wan 9210 varied from 0.5% to 2.5% and from 0.5% to 1.4%, respectively. Progeny test for resistance to wheat scab showed that the leaf extract of R₁ generation inhibited the growth of wheat scab strain R₀ and F₁₅.     

线粒体定位序列介导的绿色荧光蛋白基因在烟草中表达的分析

由于绿色荧光蛋白可在活组织或细胞中直接检出 ,因而近年已在转基因植物的研究中用作报告基因 ,这样可在植物生长的任何阶段进行活体筛选和鉴定。本研究利用线粒体定位序列对改良 gfp基因在转基因烟草中的表达进行了观察 ,结果表明 :将GFP直接在细胞质中大量表达会对植物细胞产生毒性 ,从而影响植物细胞的分化 ,而将其定位在线粒体中 ,则从转化细胞产生植株的频率明显增高。     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize (Zea mays L.)

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutin1 gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors (pUUOAH). The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of Cry1Ah protein in the construct containing the ubi1 intron (pUUOAH) was 20% higher than that of the intronless construct (pUOAH). Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubi1 intron was higher than that of the intronless construct. These results indicated that the maize ubi1 intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently     

泛素启动子在转基因植物中的应用

启动子是基因表达调控的重要元件,在转基因植物中,选择高效率的启动子是高效率表达外源基因的关键.泛素启动子以其启动效率高、甲基化程度相对较低、遗传性状稳定等特点而成为目前研究较多的启动子之一.综述了泛素启动子研究现状,包括泛素基因的结构特点、泛素启动子的高效机制及在转基因植物中的应用和存在的问题.     

小麦遗传转化方法研究进展

目前小麦遗传转化尽管有多种方法,但转化效率仍然很低,一个重要原因是遗传转化方法尚不成熟,因此建立合适的转化方法是小麦遗传转化成功的关键.本文综述了小麦基因枪转化、农杆菌介导的遗传转化和花粉管通道法等几种重要遗传转化方法研究的最新进展,分析了各种方法的基本原理、优缺点及其影响因素.最后对小麦遗传转化研究中存在的主要问题进行了总结,并展望今后的研究重点与发展趋势.     

Transformation of GbSGT1 gene into banana by an Agrobacterium-mediated approach

SGT1 is a homologue of the yeast ubiquitin ligase-associated protein. It controls some protein degradation and activates defense pathway in plants. Cotton GbSGT1 gene (Gossypium barbadense) has been isolated and characterized in previous work. In this study, the plant expression vector pBSGT1 with bar gene as a selection agent was constructed and transgenic banana was obtained via Agrobacterium-mediated transformation with the assistance of particle bombardment and screened with PCR and Basta spreading on banana plant leaves. Estimating of transgenic banana plants for resistance to Panama wilt is in progress.