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.     

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 ubiqutinl 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 CrylAh protein in the construct containing the ubil 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 ubil intron was higher than that of the intronless construct. These results indicated that the maize ubil intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently     

Transgenic tobacco plants expressing synthetic Cry1Ac and Cry1Ie genes are more toxic to cotton bollworm than those containing one gene

Transgenic tobacco plants carrying CrylAc, Crylle or both genes were obtained. In the leaves of transgenic plants carrying both genes, the contents of CrylAc and Crylle proteins were 0.173% and 0.131% of the total proteins, respectively. CrylAc protein content was 0.182 % and Cry1 le protein content was 0.124% of the total proteins in the leaves of transgenic plants containing only one Bt gene. Fresh leaves of transgenic tobacco and wild-type plants were used for the insect bioassay against wild-type and Cry1Ac-resistant cotton bollworm （Helicoverpa armigera）. The bioassay results showed that transgenic plants carrying both genes were significantly more toxic to wild-type and CrylAc-resistant cotton bollworm than those carrying CrylAc or Crylle alone. This study indicates that the higher toxicity of transgenic tobacco plants carrying both genes is caused by the cooperative function of both Bt proteins, thus providing a potential way to delay the development of insect resistance to transgenic crops.     

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     

Transgenic tobacco plants expressing synthetic Cry1Ac and Cry1Ie genes are more toxic to cotton bollworm than those containing one gene

Transgenic tobacco plants carrying Cry1Ac, Cry1Ie or both genes were obtained. In the leaves of transgenic plants carrying both genes, the contents of Cry1Ac and Cry1Ie proteins were 0.173% and 0.131% of the total proteins, respectively. Cry1Ac protein content was 0.182% and Cry1Ie protein con- tent was 0.124% of the total proteins in the leaves of transgenic plants containing only one Bt gene. Fresh leaves of transgenic tobacco and wild-type plants were used for the insect bioassay against wild-type and Cry1Ac-resistant cotton bollworm (Helicoverpa armigera). The bioassay results showed that transgenic plants carrying both genes were significantly more toxic to wild-type and Cry1Ac-resistant cotton bollworm than those carrying Cry1Ac or Cry1Ie alone. This study indicates that the higher toxicity of transgenic tobacco plants carrying both genes is caused by the cooperative function of both Bt proteins, thus providing a potential way to delay the development of insect resis- tance to transgenic crops.     

Salt tolerance conferred by over-expression of OsNHX1 gene in Poplar 84K

OsNHX1 gene (Na^＋/H^＋ antiporter gene of Oryza sativa L.) was introduced into Poplar 84K with Agrobacterium tumefaciens-mediated transformation. PCR, Southern and Northern blot analysis showed that OsNHX1 gene was incorporated successfully into the genome of Poplar 84K and expressed in these transgenic plants. Salt tolerance test showed that three lines of transgenic plants grew normally in the presence of 2OO mmol/L NaCI, while the Na^＋ content in the leaves of the transgenic plants grown at 2OO mmol/L NaCl was significantly higher than that in plants grown at 0mmol/L NaCI. The osmotic potential in the transgenic plants with high salinity treatment was lower than that of control plants. Our results demonstrate the potential use of these transgenic plants for agricultural use in saline soils.     

Overexpression of phospholipase <Emphasis Type="Italic">D</Emphasis>α gene enhances drought and salt tolerance of <Emphasis Type="Italic">Populus tomentosa</Emphasis>

The cDNA of AtPLDa （Arabidopsis thaliana Phospholipase Da） gene was introduced into P. tomentosa （Populus tomentosa） under the control of the Cauliflower mosaic virus 35S promoter. Southern and Northern blot analyses suggested that the AtPLDa gene has been transferred into the P. tomentosa genome. No obvious morphological or developmental difference was observed between the transgenic and wild-type （WT） plants. Drought and salt tolerance and gene expression of seedlings of several transgenic lines and WT plants （control） were studied. The results showed that the rhizogenesis rate and the average root-length of transgenic lines were significantly higher than WT plants after mannitol and NaCI treatment under the same growth conditions. Northern blot analysis indicated that the higher the PLDa expression in the transgenic plants, the more tolerant the transgenic plants are to drought and salt treatment. Meanwhile, another group of these transgenic lines and WT plants （control） were treated with PEG6000 and NaCI separately. The contents of chlorophylls and the activities of some anti- oxidant enzymes （superoxide dismutase, guaiacol peroxidase and catalase） as well as malondialdehyde and relative electrical conductivity were analyzed. Altogether, our results demonstrated that overexpression of the PLDa gene can enhance the drought and salt tolerance in transgenic P. tomentosa plants.     

Overexpression of phospholipase Dα gene enhances drought and salt tolerance of Populus tomentosa

The cDNA of AtPLDα (Arabidopsis thaliana Phospholipase Dα) gene was introduced into P. tomentosa (Populus tomentosa) under the control of the Cauliflower mosaic virus 35S promoter. Southern and Northern blot analyses suggested that the AtPLDα gene has been transferred into the P. tomentosa genome. No obvious morphological or developmental difference was observed between the transgenic and wild-type (WT) plants. Drought and salt tolerance and gene expression of seedlings of several transgenic lines and WT plants (control) were studied. The results showed that the rhizogenesis rate and the average root-length of transgenic lines were significantly higher than WT plants after mannitol and NaCl treatment under the same growth conditions. Northern blot analysis indicated that the higher the PLDα expression in the transgenic plants, the more tolerant the transgenic plants are to drought and salt treatment. Meanwhile, another group of these transgenic lines and WT plants (control) were treated with PEG6000 and NaCl separately. The contents of chlorophylls and the activities of some anti-oxidant enzymes (superoxide dismutase, guaiacol peroxidase and catalase) as well as malondialde-hyde and relative electrical conductivity were analyzed. Altogether, our results demonstrated that overexpression of the PLDα gene can enhance the drought and salt tolerance in transgenic P. tomen-tosa plants.     

小麦与高冰草体细胞杂种F5代麦谷蛋白及SDS沉降值分析

通过对普通小麦与高冰草体细胞杂种F5代 175个株系的麦谷蛋白及SDS沉降值分析 ,发现杂种F5代有 10种不同的麦谷蛋白组成 ,并出现了 1Ax1,1Ax2 ,1Bx13,1By16 ,1By8,1Dx5等 6种不存在于亲本小麦中的高分子量麦谷蛋白亚基及 1Bx13 1By16 ,1Bx7 1By8,1Dx5 1Dy12等 3种新亚基组合 ,部分杂种中出现了高冰草的麦谷蛋白特征谱带 ;杂种株系的SDS沉降值表现出较大的差异 ,其中一些杂种的SDS沉降值明显高于亲本普通小麦 .结果表明 ,通过体细胞杂交有可能将野生禾草的优良性状引入普通小麦 ,创造出多种不同组成及组合的新种质 ,为小麦品质育种开辟新途径     

转基因小麦T1代中外源基因的检测和分析

该研究采用Multiplex PCR和SDS-PAGE技术对转1 Dx5基因(ORF)T1代小麦进行了检测和分析.结果显示,Multiplex PCR能够扩增出转基因T1代材料中1 Dx2基因和1 Dx5基因的特征片段,表明外源基因已整合到受体基因组中;SDS-PAGE检测到一新的蛋白质亚基X,表明外源基因的插入引起了转基因T1代籽粒中HMW-GS组成的变化.该研究不仅验证了线性基因片段遗传转化策略的可行性,而且印证了普通小麦Glu-1D等位基因的多重PCR分子标记体系的有效性,为培育安全型的转基因作物新种质打下坚实的基础,加快小麦分子育种进程.     

高粱总DNA导入春小麦新品系高分子量麦谷蛋白亚基的变化

通过花粉管通道法将高粱总DNA导入春麦甘麦8号、陇春13号和陇春10号,经过多代选择获得了5个稳定遗传新品系.在高分子量麦谷蛋白亚基分析中,甘麦8号后代89144的高分子量麦谷蛋白亚基发生突变,较其受体多了5 10亚基,而少了2 12亚基;其他几个转基因后代与其受体比较,高分子量麦谷蛋白亚基组成未发生变化;但是各亚基的相对质量分数有较大变化.高分子量麦谷蛋白亚基的组成和各亚基质量分数的变化直接影响小麦品质.本研究对外源总DNA花粉管通道法导入小麦在改良小麦品质方面的作用进行了讨论.     

农杆菌介导的双价抗虫基因对番茄遗传转化的研究

采用农杆菌介导法将含有苏云金芽孢杆菌毒蛋白基因(CryIAc)与半夏凝集素抗虫基因(Pta)的高效植物表达载体pCAMBIA3300转入番茄品系Micro Tom的子叶外植体中。经过共培养、除草剂筛选和分化再生,获得了24个具有除草剂抗性的株系。再将转化后的番茄植株经过PCR检测和Southern Blot检测,确定检测后呈阳性反应的株系为8个。通过小菜蛾幼虫初步抗性试验证明,转基因株系表现出较强的抗虫性。实验结果为进一步研究番茄抗虫性和培育抗虫番茄新品种奠定了重要基础。     

Inheritance and expression of multiple disease and insect resistance genes in transgenic rice

2—3 anti-fungal disease genes are coinserted with hygromycin phosphotransferase in the same vector. Two insecticidal genes and PPT acetyl transferase genes are placed in another one. The vectors are co-delivered to rice embryonic cellus tissue at a molar ratio of 1︰1 using the particle gun method. 55 independent regenerated lines have been obtained through screening for hygromycin resistance. Of these, 70% transgenic plants harbor 6—7 foreign genes. The genes on the same vectors are always co-delivered to rice plant. Northern blot analysis has indicated that the multiple foreign genes give stable expression. In the 6 transgenic plants carrying 6—7 foreign genes, multiple foreign genes tend to integrate in 1 or 2 genetic loci. Progeny segregation is consistent with Mendel’s 3︰1 segregation law. 8 homozygous R₁ transgenic plants harboring 2—3 anti-fungal and 2 insecticidal genes are selected from large number of transgenic progeny screening for hygromycin and Basta resistance.     

Generation of transgenic wheat resistant to wheat yellow mosaic virus and identification of gene silence induced by virus infection

The plasmid containing the promoter Act1, the coat protein (cp) gene of wheat yellow mosaic virus (WYMV) and the selectable bar gene, was delivered via particle bombardment, directly into immature embryos of a wheat cultivars. PCR and PCR-RFLP were employed to screen the existence of the cp gene in T0 and T1 generations. Seeds from the positive T1 plants were sowed in fields heavily contaminated with WYMV to detect their resistance. In field trial of virus infection, one of the transgenic wheat lines, P8-T2, exhibited highly disease-resistance. Western blot and RT-PCR analysis showed that the expression level of cp gene in the resistant transgenic line was reduced greatly compared to those susceptible to WYMV infection. This provided evidence to presume that the resistance obtained by the transgenic wheat line was stimulated by the mechanism of the virus induced gene silencing.     

Identification of a novel HMW glutenin subunit and comparison of its amino acid sequence with those of homologous subunits

Aegilops tauschii is the donor of the D genome of common wheat (Triticum aestivum). Genetic variation of HMW glutenin subunits encoded by the Glu-1Dt locus of Ae. tauschii has been found to be higher than that specified by the Glu-1D locus in common wheat. In the present note, we report the identification of a novel HMW glutenin subunit, Dy13t, from Ae. tauschii. The newly identified subunit possessed an electrophoretic mobility that was faster than that of the Dy12 subunit of common wheat. The complete ORF of encoding the Dy13t subunit contained 624 codons (excluding the stop codons). The amino acid sequence deduced from the Dy13t gene ORF was the shortest among those of the previously reported subunits derived by the D genome. A further comparison of Dy13t amino acid sequence with those of the subunits characterized from the A, B, D, R genomes of Triticeae showed that the smaller size of the Dy13t subunit was associated with a reduction in the size of its repetitive domain.     

野生二粒小麦高分子量谷蛋白亚基等位变异分析

本研究采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）方法,对从国内外引进的175份野生二粒小麦（Triticum dicoccoides）种质资源高分子量谷蛋白亚基（HMW-GS）Glu1-等位基因组成进行了鉴定和分析.结果表明,175份野生二粒小麦中,Glu-1B位点上的HMW-GS有32种变异类型,比普通面包小麦Glu-1B位点编码的HMW-GS的变异类型丰富得多,其中7＋8和14.1＋15.1亚基分布频率最高（占20.00%）;Glu-1A位点编码的HMW-GS有4种变异类型：1、1^＊、2^＊和Null,一共鉴定了10个新的高分子量亚基.这些新的亚基和等位基因有可能成为面包小麦品质改良的候选基因资源.     

小麦谷蛋白亚基的快速提取分离及SDS-PAGE分析

用70％乙醇和50％异丙醇除去醇溶蛋白，以二硫苏糖醇(DTT)为还原剂，在50％异丙醇，0．08mol／L Tris-HCl(pH为8．0)缓冲液中提取总麦谷蛋白亚基，加入丙酮至浓度为40％时，高分子量麦谷蛋白亚基(HMW-GS)沉淀析出；继续加入丙酮至80％时，析出低分子量麦谷蛋白亚基(LMW-GS)．在3．3％浓缩胶和10％分离胶的不连续体系中进行SDSPAGE电泳，结果表明，该方法可以有效地除去醇溶蛋白对麦谷蛋白亚基电泳分析的背景干扰，HMW-GS和IMW-GS的提取、分离一步完成，操作简便，时间短，对大量小麦样品的HMW-GS和LMW-GS可以快速分离、提纯和鉴定．