苏云金芽孢杆菌cry1基因的克隆、植物表达载体的构建及转化大豆

此研究利用醋酸钠-抗生素加热法从土壤中分离获得1株产晶体的苏云金芽孢杆菌菌株（Bacillus thuringiensis）。通过已知cry1基因设计引物,以分离得到的菌株DNA为模板进行PCR扩增,将克隆到的目的片段进行测序。测序结果表明：该序列与cry1基因同源性达到90%~99%。将该基因连接到植物双元表达载体pBI121中,构建了该基因的植物表达载体,并将阳性重组质粒转化根癌农杆菌EHA105,利用农杆菌侵染子叶节的方法进行大豆的转化。通过初步的PCR验证,成功获得了转基因植株,建立了大豆的转化体系。     

苏云金芽胞杆菌cry2Ac4基因在 大肠杆菌中的表达

在大肠杆菌中表达苏云金芽胞杆菌（Bacillus thuringiensis,简称Bt）cry2Ac4基因;笔者以含有Bt WB9菌株cry2Ac4基因的质粒pMD2Ac为模板,利用cry2Ac4基因的特异引物对(ET-F/ET-R)扩增获得该基因,进而将cry2Ac4基因与pET-29a原核表达载体连接;成功构建了重组表达载体并转化大肠杆菌JM109,从阳性转化子中提取重组表达质粒pET-29a-cry2Ac4,再转化大肠杆菌BL21（DE3）pLysS,经IPTG诱导后,对诱导表达产物进行了SDS-PAGE检测;cry2Ac4基因编码的约70kDa蛋白在大肠杆菌中得到了高效表达。     

农杆菌介导的hIL-12基因转化马铃薯的研究

构建CaMv35S启动子驱动人白细胞介素12基因(hIL-12)的植物表达载体pBI121-hIL-12,通过三亲杂交法将重组载体导入根癌农杆菌菌株EHA105,农杆菌介导法转化马铃薯茎段,经卡那霉素抗性筛选,获得28株抗性植株。通过PCR检测,22株为阳性,从PCR阳性植株中随机选取5株,ELISA法检测茎和叶中hIL-12的表达量,其中2株与对照组相比有显著性差异(p<0.001),hIL-12的表达量分别为(3 714.0±48.8)pg/g和(3 465.0±185.0)pg/g,初步表明外源基因hIL-12已经整合进马铃薯基因组中并得到了表达,为进一步研究重组hIL-12的分离纯化和生物学活性奠定了试验基础。     

转Bt cry1Ah/2mG2-epsps双价基因抗虫/耐除草剂玉米研究

构建了含有Bt杀虫基因cry1Ah和耐草甘膦基因2mG2-epsps的双价植物表达载体pMAGUHM,通过基因枪轰击Q31×Z31杂交组合的胚性愈伤组织,以2mG2-epsps为筛选标记,通过1mmol/L草甘膦异丙胺盐筛选,获得T0代转化植株80株,其中PCR检测有36株为阳性植株,阳性率达45%。对得到的阳性植株的T1、T2代进行了跟踪检测,证明外源基因已整合到玉米基因组中并正确表达。草甘膦及玉米螟抗性检测结果表明,有5个转基因品系表现出具有耐草甘膦和抗虫的双重特性。     

转cry1C~*基因抗虫超级粳稻田间目的基因表达及抗螟虫性研究

为了研究转基因抗虫超级粳稻田间目的基因表达及抗螟虫性,将10个来自于不同独立抗性愈伤组织的转cry1C*基因抗虫超级粳稻品系种植于田间,利用实时荧光定量PCR方法检测孕穗期叶片、茎鞘和幼穗等器官目的基因mRNA,利用酶联免疫吸附(ELISA)法检测孕穗期叶片、茎鞘和幼穗及收获后糙米的Cry1C蛋白,利用田间目测调查二化螟危害的白穗率。结果显示,转基因超级粳稻不同品系及不同器官cry1C*基因田间表达量明显不同,cry1C*基因mRNA表达量:叶片茎鞘幼穗,蛋白质表达量:叶片茎鞘幼穗糙米。转基因超级粳稻田间目的基因表达,在mRNA水平和蛋白质水平,不同器官间存在正相关关系,各器官Cry1C蛋白质含量和糙米Bt蛋白质含量呈正相关。在本研究范围内,不论转基因粳稻植株Cry1C蛋白质含量高或低的品系,田间均表现为高抗二化螟。培育转基因抗虫粳稻品种时,应注意对目的基因适量表达的抗虫基因型的选择。     

维管组织特异表达启动子驱动RID1调控水稻抽穗期

水稻基因RID1突变后表现为"不开花"的表型,且RID1特异在幼叶中表达。本研究利用启动子RPP16,构建表达载体RPP16::RID1,转化rid1突变体,得到了19株转基因阳性植株,其中有9株表现为恢复抽穗,表明在维管组织中特异表达RID1可以恢复rid1突变体表型。RT-PCR检测结果显示,转基因单株中抽穗期基因Ehd1、Hd3a和RFT1的表达量与RID1表达量呈正相关,且RID1表达量高的转基因单株相应抽穗也比较早。     

改造Cry1Ac蛋白C端对转基因水稻抗二化螟虫的影响

二化螟虫的泛滥严重影响水稻的产量,cry1Ac基因是目前世界上应用最广泛和最高效的抗虫基因之一,但二化螟虫通过进化会逐渐对其产生抗性。通过改变蛋白结构来构建新的Cry蛋白是解决这一问题的有效途径之一。为分析改造Cry1Ac蛋白C端能否对转基因作物抗虫性产生影响,本研究采用Cry1Ja蛋白的C端替换Cry1Ac蛋白的C端,重组获得CryFLAc蛋白。将cry1Ac基因和编码CryFLAc蛋白的cryFLAc基因分别构建到pTF101.1-ubi植物表达载体上,利用农杆菌介导方法转化到吉林省水稻品种吉粳88中;采用PCR、RT-PCR、免疫检测试纸条检测的方法确定cry1Ac、cryFLAc基因整合和表达情况;通过室内和田间抗虫性测试评价CryFLAc和Cry1Ac蛋白对T1代转基因水稻抗虫性的影响。研究发现:cry1Ac、cryFLAc基因均成功整合到水稻基因组中,并稳定表达;转cryFLAc基因水稻抗二化螟水平达到抗性级别,转cry1Ac基因水稻达到高抗级别;转cry1Ac基因水稻二化螟抗性高于转cryFLAc基因水稻。结果表明:Cry1Ja蛋白的C端替换Cry1Ac蛋白的C端不会提高转基因水稻的抗虫性。上述研究为人工设计合理化改造Cry蛋白提供了新的理论依据。     

水稻OsAAA1基因超表达载体构建及遗传转化

为了构建带Flag标签的OsAAA₁超表达载体,获得阳性转基因植株并分析其OsAAA₁基因表达情况。以日本晴的cDNA为模板,将OsAAA₁克隆至pU1301-Flag载体;重组质粒通过PCR、酶切及测序鉴定正确后,以农杆菌为介导进行遗传转化至日本晴;转基因植株通过分子鉴定正确后,采用Real-time PCR检测OsAAA₁基因的mRNA表达水平变化。成功构建了OsAAA₁-pU1301-Flag重组载体;遗传转化后获得33株转基因植株;通过分子鉴定筛选出26株阳性转基因植株;荧光定量PCR结果分析发现23株阳性转基因植株OsAAA₁基因的表达出现不同程度上调。与日本晴相比,有8株表达量达到30倍以上,其中有5株表达量超过40倍。成功构建了带Flag标签的OsAAA₁超表达载体;遗传转化结果表明OsAAA₁-Flag能整合到日本晴的基因组DNA中,从而使OsAAA₁基因的表达水平增加。本研究为后续通过Flag标签,在水稻植物体内直接挖掘与OsAAA₁互作的功能蛋白奠定了基础。     

用核基质结合区(SAR)序列提高小麦最小表达框转基因表达的稳定性

采用最小表达框技术转化植物可以规避由骨架序列引起的安全风险。核基质结合区序列SAR (scaffold attachment region)可作为边界元件与核基质结合阻挡转基因片段邻近染色质区的作用与影响, 提高外源基因稳定性。本研究在最小表达框序列两端添加SAR序列, 提高小麦最小表达框转基因表达的稳定性, 提高转化基因的表达效率。首先, 以GUS为目的基因构建带有SAR序列的最小表达框, 以科农199为受体进行基因枪转化, 同时以不加SAR序列的最小表达框片段为对照。带有SAR序列的最小表达框片段共轰击857个幼胚, T₀代获得40株再生植株, PCR检测到16株阳性植株, 转化效率为1.87%; 对这16个阳性单株进行GUS染色, 15株显色; 从来自4个T₀阳性植株的18个T₁代植株中随机选取18株进行PCR和GUS染色检测, 有15株表现为阳性。不带SAR序列的对照片段轰击1012个幼胚, 获得31株再生植株, 其中5株PCR阳性, 转化效率0.49%, 这5个阳性植株中仅2株为GUS染色阳性; 来自于5个T₀代PCR阳性株系的10个T₁代单株中没有发现PCR和GUS染色阳性株。表明SAR序列可以提高基因枪转基因效率和目的基因表达稳定性。为了创制抗旱转基因小麦, 以来自大豆的抗旱相关转录因子基因GmDREB3为目的基因, Bar基因为筛选标记基因, 转化受体小麦济麦22, 共轰击6045个幼胚, 获得再生植株130株, PCR检测阳性植株30株, 转化效率为0.50%; 随机选取6株PCR阳性植株进行RT-PCR分析, 其中5株可检测到外源基因的转录。进一步对这5株RT-PCR阳性植株插入片段完整性进行分析, 其中4株插入片段基本完整。通过real-time PCR分析, 发现T₀代6个RT-PCR阳性植株的外源GmDREB3的拷贝数为1~3个。以上结果证明, 在最小表达框两端加上SAR序列后可以提高小麦最小表达框转基因表达的稳定性。     

大肠杆菌中的表达

 在大肠杆菌中表达苏云金芽胞杆菌（Bacillus thuringiensis,简称Bt）cry2Ac4基因;笔者以含有Bt WB9菌株cry2Ac4基因的质粒pMD2Ac为模板,利用cry2Ac4基因的特异引物对(ET-F/ET-R)扩增获得该基因,进而将cry2Ac4基因与pET-29a原核表达载体连接;成功构建了重组表达载体并转化大肠杆菌JM109,从阳性转化子中提取重组表达质粒pET-29a-cry2Ac4,再转化大肠杆菌BL21（DE3）pLysS,经IPTG诱导后,对诱导表达产物进行了SDS-PAGE检测;cry2Ac4基因编码的约70kDa蛋白在大肠杆菌中得到了高效表达。     

phaC1、phaC2基因双价植物表达载体的构建及其转基因烟草的获得

利用聚合酶链式反应(PCR)技术，从类产碱假单胞菌YSl(Pseudomonas psuedoalcaligenese YSl)染色体DNA中扩增并克隆了调控短链与中链PHA生物合成的两个关键酶基因：phaCl、phaC2基因。同时利用套叠PCR技术对phaCl基因进行了改造，经过基因拼接构建了植物表达载体pC3C1(嵌合phaCl)、pC3C2(嵌合phaC2)和pC3C1C2(嵌合phaCl和phaC2双基因)。将构建好的嵌合phaC1和phaC2双基因的植物高效表达载体pC3C1C2，用冻融法转入根癌土壤杆菌(Agrobacterium tumefaciens EHAl05)并且通过农杆菌介导的叶盘法转化烟草(Nicotiana tobacum Honghuadajinyuan)。2个月后获得了一批卡那霉素抗性烟草植株，抗性植株大田生长表型正常，生长速度相对缓慢。抗性植株经过PCR、PCR-Southern、Southern检测初步确定有32％烟草稳定整合了phaCl和phaC2。用氯仿一次氯酸钠直接从部分Southern检测阳性转化植株中抽提纯化得到PHA，在产物合成水平确证有25．6％的转基因植株。     

根癌农杆菌介导获得稗草Ecppc转基因小麦的研究

采用携带pUbi-Ecppc质粒的3个根癌农杆菌菌株（LBA4404、EHA105和C58c1）,对经过预培养10~12 d的春小麦品种扬麦158、Bobwhite和扬麦12的幼胚愈伤组织进行了遗传转化。对筛选中的抗生素浓度、菌液浓度、共培养温度和时间、受体基因型、菌株-质粒组合等影响转化的重要因素进行了研究。首次将单子叶野生C₄植物稗草的磷酸烯醇式丙酮酸羧化酶基因（Ecppc）导入小麦受体基因型,并得到具有潮霉素（Hyg）抗性的转化植株。从816块共培养愈伤组织中转化得到34株抗性植株,其中14株PCR检测为阳性。扬麦158的转化效率达3.03%。Southern和RT-PCR分析表明外源基因已整合到小麦基因组并得到正确的转录。生理学检测显示,转基因小麦植株的光合速率和PEPC活性都有所提高。说明Ubiqintin基因启动子控制的稗草PEPC cDNA基因在小麦中可以正确表达和起到一定的生理作用。这些工作为进一步探讨PEPC对小麦光合作用及其他生理过程的影响奠定了基础。     

苏云金芽孢杆菌cry1Ea基因的克隆及 生物信息学分析

不同来源的苏云金芽孢杆菌能产生多种多样的晶体(Cry)蛋白。基于这个特性,人们可以通过基因工程的手段向工程菌中转入编码多种Cry毒素的基因来控制虫害。通过DNA重组技术,从Bt HZM2菌株中克隆出了cry1Ea基因,对其进行了生物信息学分析,同源比对结果表明,cry1Ea8基因的核苷酸序列与已知cry1Ea的同源性为99.77%~99.91%,对应的氨基酸序列同源性为99.49%~99.74%。对cry1Ea8基因的分析还揭示出了cry1Ea8及其编码蛋白的一些生物和理化性质。结构域预测表明,Cry1Ea8由3个结构域组成,其中N-末端螺旋状结构域与膜插入与孔隙形成有关,而第二和第三个结构域与受体的结合有关。该研究为转基因抗虫植物和微生物杀虫工程菌的构建提供了新的基因来源。     

转cry1C基因抗虫超级粳稻田间目的基因表达及抗螟虫性

为了研究转基因抗虫超级粳稻田间目的基因表达及抗螟虫性,将10个来自于不同独立抗性愈伤组织的转cry1C*基因抗虫超级粳稻品系种植于田间,利用实时荧光定量PCR方法检测孕穗期叶片、茎鞘和幼穗等器官目的基因mRNA,利用酶联免疫吸附(ELISA)法检测孕穗期叶片、茎鞘和幼穗及收获后糙米的Cry1C蛋白,利用田间目测调查二化螟危害的白穗率。结果及分析显示,转基因超级粳稻不同品系及不同器官cry1C*基因田间表达量明显不同,cry1C*基因mRNA表达量叶片＞茎鞘＞幼穗,蛋白质表达量叶片＞茎鞘＞幼穗＞糙米。转基因超级粳稻田间目的基因表达,在mRNA水平和蛋白质水平,不同器官间存在正相关关系,各器官Cry1C蛋白质含量和糙米Bt蛋白质含量呈正相关。在本研究范围内,不论转基因粳稻植株Cry1C蛋白质含量高或低的品系,田间均表现为高抗二化螟。培育转基因抗虫粳稻品种时,注意对目的基因适量表达的抗虫基因型的选择。     

Transformation of a large number of potato varieties: genotype-dependent variation in efficiency and somaclonal variability

Transformation of potato is a genotype dependent process as was shown by experiments conducted with 16 varieties. Not all genotypes could be transformed with a single procedure hence two different procedures were attempted for all 16 varieties in a pilot experiment. Large differences in regeneration capacity of putative transformants were observed with the two protocols. Regeneration capacity and transformation efficiency were not correlated. All varieties were transformed with the same construct, composed of a kanamycin resistance gene and an antisense gene coding for granule-bound starch synthase. This led to different percentages of plants with the desired maximum effect (i.e. amylose-free starch) ranging from less than 1 percent to 23.3 percent. It was shown that variety-dependent phenotypic variation occurred ranging from 1 to 21%. Field experiments, conducted over a number of years, using plants with different degrees of antisense effect (from no detectable effect to maximum effect) showed that most transformants would have a decreased yield and starch content as determined by specific gravity measurements. However, these negative effects can be overcome by selecting the proper transgenic plants. Molecular characterisation of transformants using PCR, showed that 90% of the analysed transgenic plants, belonging to all effect classes, contained vector DNA sequences since they contained either the NPTIII gene or the trfA gene. The other 10% of the transgenic plants had no insertion of vector DNA. This revised version was published online in August 2006 with corrections to the Cover Date.     

Enhanced resistance to a lepidopteran pest in transgenic sugar beet plants expressing synthetic <Emphasis Type="Italic">cry1Ab</Emphasis> gene

Two diploid sugar beet genotypes of agronomical importance were transformed using Agrobactrium tumefaciens harboring pBI35Scry containing a synthetic cry1Ab gene. Leaf blade with attached shoot bases, a highly regenerative tissue, were used as explant substratum for transformation. PCR screening with cry1Ab-specific primers showed the presence of transgene in more than 50% of the regenerated kanamycin-resistant plants after treatment with the antibiotic. A transformation rate of 8.8–12.2% (depending on genotype) was achieved as revealed by genomic DNA dot blotting. The intact integration of transgene cassette into the genome was furthermore confirmed by Southern blot analysis. The expression of the cry1Ab gene encoding a truncated endotoxin (67 kDa) at about 0.1% of total soluble protein was achieved in the leaves of transgenic plants as shown by Western blot analysis. Bioassays under in vitro conditions with Spodoptera littoralis, one of the most important pests in sugar beet fields, demonstrated enhanced resistance against this pest. The inheritance of the inserted transgene was confirmed in F₁ plants obtained through crossing of T₀ plants with a cytoplasmic male sterile line. Transgenic plants are currently grown in a greenhouse and will be subjected to further bioassay analyses against other lepidopteran pests of sugar beet.     

农杆菌介导加幼苗直接形成将Bt-cry1A(b)基因转入印度棉花

利用农杆菌介导法用Bt-cry1A(b)基因对印度栽培棉种进行非基因依赖型遗传转化和植株再生.将印度栽培棉品种Anjali(LRK-516)和LRA-5166与携带Bt-cry1A(b)+nptⅡ基因的根癌农杆菌LBA4404共培养,在含100tμg@ml-1卡那霉素的筛选培养基上获得了可能的转基因直接再生苗.细菌浓度、共培养时间、感染组织的阶段和大小、标记筛选浓度、培养基成分和激素等都影响转化效果和效率.对程序进行了优化.经PCR、Southern杂交、ELISA等方法分别检测,证实了Bt-cry基因的插入和表达.Southern分析表明转化植株存在3～5个基因拷贝,但CRY蛋白表达量非常低(为叶蛋白的0.003%～0.004%)且生化抗虫性很弱或基本不影响鳞翅目昆虫.尽管如此,该方案仍适用于其它CRY基因或重要经济型基因进行非基因依赖型遗传转化和再生,产生转基因棉花.     

Agronomic and morphological characterization of Agrobacterium-transformed Bt rice plants

The agronomic and morphological characteristics of Agrobacterium-transformed rice plants carrying the synthetic cry1Ab or cry1Ac gene were investigated. Tremendous variations in plant height, seed fertility, grain size and other traits were seen in 80 T₁ lines, derived from 80 T₀ plants of 9 rice varieties. On average, about 33% T₁ lines had either morphological or agronomic variant plants. Most of the variations in T₁ plants had no significant correlation with transgene insertion and were proved heritable to their progenies. Genetic analysis in T₃ or T₄ generations showed some simple mutations such as chlorophyll deficiency and stunted plants were independent of transgene insertion and seemed to be controlled by a pair of single genes. However, in two independent transgenic progenies of Xiushui 11, all plants homozygous for transgenes showed dwarfism while all hemizygous and null segregants had normal plant heights. Two advanced homozygous Bt lines, KMD1 and KMD2, were developed from these two progenies. Comparison of the agronomic traits of KMD1 and KMD2 with their parent displayed marked differences among them in terms of seedling growth, tillering ability, yield components and yield potential. The genetic variation observed was generally not linked to the transgene locus and was ascribed to somaclonal variation, but other causes might also exist in particular cases. The results are discussed in the context of choosing appropriate transformation methodology for rice breeding programs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Amenability of castor to an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation strategy using a <Emphasis Type="Italic">cry1AcF</Emphasis> gene for insect tolerance

Agrobacterium tumefaciens mediated in planta transformation protocol was developed for castor, Ricinus communis. Two-day-old seedlings were infected with Agrobacterium strain EHA105/pBinBt8 harboring cry1AcF and established in the greenhouse. Screening the T₁ generation seedlings on 300 mg L⁻¹ kanamycin identified the putative transformants. Molecular and expression analysis confirmed the transgenic nature and identified high-expressing plants. Western blot analysis confirmed the co-integration of the nptII gene in the selected transgenic plants. Bioassay against Spodoptera litura corroborated with high expression and identified five promising effective lines. Analysis of the T₂ generation plants proved the stability of the transgene indicating the feasibility of the method.