Transformation of wheat with the HMW-GS <Emphasis Type="Italic">1Bx14</Emphasis> gene without markers

High molecular weight (HMW) glutenin polypeptides are critical contributors to the visco/elastic properties responsible for the processing characteristics and utilizations of wheat flour. In order to improve bread making quality of flour and produce transgenic plants free of selectable markers, a linear DNA construct consisting of a minimal expression cassette with the HMW-GS 1Bx14 gene was transformed into wheat cultivar Mianyang19 by microprojectile bombardment. The transform ants were selected by PCR instead of herbicidal markers. Seven transgenic plants were identified from a total of 1219 transformants, yielding a transformation frequency of 0.28%. An SDS-PAGE analysis confirmed that the 1Bx14 gene was expressed in three T1 seeds of the transgenic plants. Our results demonstrated that it is feasible to obtain marker-free trans-formants using the linear-expression-cassette-transformation approach coupled with PCR selection.     

水稻谷蛋白GluA-2基因在小麦胚乳中的表达(英文)

水稻(Oryza sativa L.)谷蛋白(Glutelin)约占水稻储藏蛋白总量的80％,谷蛋白赖氨酸含量较高并易于被人体消化吸收。为了提高小麦(Triticum aestivum L. )的营养品质,将水稻谷蛋白GluA-2基因的cDNA序列导入小麦栽培品种Bobwhite(T. aestivum cv. Bobwhite)。共轰击了600个小麦幼胚,经PCR和Southern杂交鉴定,共获得4棵转GluA-2基因小麦;SDS-PAGE分析表明,GluA-2基因在3棵转基因植株及其后代中表达,在1棵转基因植株中未表达,但其内源的高分子量麦谷蛋白亚基Bx7和By9含量显著降低,并且可遗传至T_代。     

高分子量麦谷蛋白1Bx14亚基AS-PCR标记

根据已发表的1Bx14亚基的基因序列在不同位点设计了10对特异引物,从中筛选出1对引物,对HMW-GS在Glu-1Bx位点已知的10个小麦品种进行了PCR扩增.结果表明,具有1Bx14亚基的4个品种都能扩增出1条1 256 bp左右的特异带.用这一特异标记对山东省种植面积较大的40个品种进行PCR扩增(即等位专一PCR,AS-PCR),发现仅有5个品种携带1Bx14亚基.该AS-PCR标记可用于检测小麦品种在该位点的亚基组成,与SDS-PAGE相比,可显著提高检测的准确性和效率,可为种质鉴定和育种工作提供参考.     

小麦HMW-GS 1Bx14基因特异标记体系的建立

比较1Bx14及其它已知HMW-GS基因的启动子和编码区,根据其不同点设计出1Bx14基因特异扩增引物。以8种已知HMW-GS组成的小麦DNA为模板进行PCR扩增。结果表明：具有1Bx14亚基的品种扩增出1条400bp左朽特异条带。结合该特异标记和已报道的1Dx5特异标记对2个F2杂交群体进行检测,从184个F2单株中筛选出111个同时含有1Bx14和1Dx5基因的单株。该研究结果可为种质鉴定和亚基整合育种提供参考。     

基因枪法获得无选择标记的1Dx5基因表达的转基因小麦

利用基因枪将无选择标记的优质高分子量麦谷蛋白亚基基因1Dx5导入新疆耐盐小麦品种新冬26,为利用优质基因进行小麦品质改良奠定基础。构建无选择标记的线性1Dx5表达框。利用基因枪将其转入不含该亚基的小麦品种新冬26幼胚盾片中,经PCR二分法筛选,从转化的1 000块幼胚盾片中共获得3株转基因阳性植株,转化效率0.3%。利用SDS-PAGE分析目的基因在转基因后代籽粒中的表达。转基因植株后代种子分析表明,1Dx5在转基因后代部分种子中表达。本研究成功地将无选择标记的线性1Dx5片段导入普通小麦新冬26中,并在后代部分种子中得到了表达。为利用优质亚基基因改良小麦加工品质奠定基础。     

水稻谷蛋白GluA-2基因在小麦胚乳中的表达

水稻(Oryza sativa L.)谷蛋白(Glutelin)约占水稻储藏蛋白总量的80%,谷蛋白赖氨酸含量较高并易于被人体消化吸收.为了提高小麦(Triticum aestivum L.)的营养品质,将水稻谷蛋白GluA-2基因的cDNA序列导人小麦栽培品种Bobwhite(T. aestivum cv.Bobwhite).共轰击了600个小麦幼胚,经PCR和Southern杂交鉴定,共获得4棵转GluA-2基因小麦;SDS-PAGE分析表明,GluA-2基因在3棵转基因植株及其后代中表达,在1棵转基因植株中未表达,但其内源的高分子量麦谷蛋白亚基Bx7和By9含量显著降低,并且可遗传至T1代.     

农杆菌介导的半夏凝集素基因(Pinellia Ternate Agglutinin Gene,pta)对小麦的遗传转化及鉴定

以甘肃主要推广春小麦品种陇春22幼胚为转基因受体材料,建立了农杆菌介导的小麦遗传转化体系。以预培养4天的幼胚愈伤组织为受体,C58c1农杆菌菌株为供体,将含有半夏凝集素基因的重组质粒pBIpta转入了小麦,经G418 25 mg/L抗性筛选、PCR检测和荧光定量PCR检测共获得转基因植株3株,外源基因的插入拷贝数分别为2、1、3。同时对转基因小麦的T₁代植株进行了PCR检测和抗虫性分析,表明半夏凝集素基因在转基因植株的后代中得到了遗传并有一定的抗蚜虫作用。     

转TaDREB基因提高芦荟抗低温特性的研究

以库拉索芦荟(Aloe vera L.)幼茎为外植体,利用农杆菌介导法将携带小麦功能基因TaDREB的表达载体pBIR1转化库拉索芦荟,在添加3.0mg/L BA、250mg/L Carbenicillin和15~25mg/L G418的MS培养基上诱导、筛选丛生芽,连续筛选几代后,将2.0~3.0cm高的抗性再生芽转移到1/2MS生根培养基上壮苗,共获得58株生长良好的抗性植株。根据标记基因npt Ⅱ及目的基因TaDREB的序列设计上游引物,对所有抗性植株进行PCR检测,共获得3株PCR阳性植株,结果表明目的基因的转化效率为0.5%。将转基因阳性植株置于4℃低温处理2周,20℃冷冻处理30min,发现对照植株发生严重冻害,而转基因植株生长良好,抗低温特性明显提高。对低温胁迫条件下培养14天的转基因植株SOD、POD酶活性进行分析,结果表明低温胁迫下转基因植株SOD、POD活性均呈降－升－升－升趋势变化,与非转基因植株的变化趋势降－升－降－降有所不同。进一步利用电导率法检测,结果转基因植株电导率的平均值为0.456,明显低于对照的0.685;说明转基因芦荟细胞膜的稳定性得到很好的改善,是其抗低温特性提高的内在生理基础。     

Expression of Cholera Toxin B Subunit in Transgenic Rice Endosperm

The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1% of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced only in the rice endosperm demonstrated strong affinity for G_M1-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine.     

脱水应答转录因子CBF1的克隆与转基因小麦的分子检测

根据已发表的小麦(T.aestivum)转录因子CBF1基因序列(GenBank Accession No.AF376136),设计引物从小麦品种‘京花1号’叶片中克隆出该基因,用拟南芥RD29B基因为启动子构建含CBF1基因的逆境诱导表达载体pBAC127F(6 967 bp),以‘99-92’、‘5-98’、‘104’和‘轮选987’等冬小麦品种(系)的幼穗和幼胚为材料,基因枪转化该表达载体。经筛选与植株再生,共获得14株转基因植株及其后代株系。这14个株系经PCR分析和点杂交检测,最终确认了5-98-40、5-98-41这2个株系为转基因株系,结果表明拟南芥RD29B启动子调控下的转录因子CBF1基因已稳定整合到转基因植株中。     

羽衣甘蓝胁迫应答基因BoRS1转化烟草的初步研究

将克隆于羽衣甘蓝的胁迫应答基因BoRS1连入中间载体p35S-2300：：gus：：noster相应位点,成功地构建了含BoRS1基因的植物双元表达载体p35S-2300：：BoRS1：：noster,并通过农杆菌介导法对烟草进行了遗传转化。PCR检测结果表明目的基因BoRS1已成功地导入并整合到烟草基因组中。RT-PCR分析显示,在不同的转基因烟草植株中BoRS1表达量存在差异。转BoRS1烟草的耐干性和甘露醇胁迫研究表明,BoRS1基因的表达对提高植物抗干旱胁迫能力有一定的作用。     

An approach for isolating high-molecular-weight glutenin subunit genes using monoclonal antibodies.

High-molecular-weight glutenin subunits (HMW-GSs) play an important role in the breadmaking quality of wheat flour. In China, cultivars such as Triticum aestivum 'Xiaoyan No. 6' carrying the 1Bx14 and 1By15 glutenin subunits usually have attributes that result in high-quality bread and noodles. HMW-GS 1Bx14 and 1By15 were isolated by preparative sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and used as an antigen to immunize BALB/c mice. A resulting monoclonal antibody belonging to the IgG1 subclass was shown to bind to all HMW-GSs of Triticum aestivum cultivars, but did not bind to other storage proteins of wheat seeds in a Western blot analysis. After screening a complementary DNA expression library from immature seeds of 'Xiaoyan No. 6' using the monoclonal antibody, the HMW-GS 1By15 gene was isolated and fully sequenced. The deduced amino acid sequence showed an extra stretch of 15 amino acid repeats consisting of a hexapeptide and a nonapeptide in the repetitive domain of this y-type HMW subunit. Bacterial expression of a modified 1By15 gene, in which the coding sequence for the signal peptide was removed and a BamHI site eliminated, gave rise to a protein with mobility identical to that of HMW-GSs extracted from seeds of 'Xiaoyan No. 6' via SDS-PAGE. This approach for isolating genes using specific monoclonal antibody against HMW-GS genes is a good alternative to the extensively used polymerase chain reaction (PCR) technology based on sequence homology of HMW-GSs in wheat and its relatives.     

Molecular characterization of high molecular weight glutenin subunit allele 1Bx23 in common wheat introduced from hexaploid triticale

High molecular weight glutenin subunit (HMW-GS) 1Bx23, an x-type subset encoded by Glu-B1p, which is only distributed in Triticum turgidum, was successfully transferred from hexaploid triticale to common wheat line SY95-71. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that subunit 1Bx23 has a faster mobility than subunit 1Bx7 and 1Bx20, but slower than 1Bx17. Primers designed from the conserved regions in wheat HMW-GS gene promoter and coding sequences were used to amplify the genomic DNA of SY95-71. Total nucleotide sequences of 3426 bp including an open reading frame of 2385 bp and upstream sequence of 1038 bp were obtained. Compared with the reported gene sequences of Glu-B1-1 alleles, including 1Bx7, 1Bx14, 1Bx20 and 1Bx17, the promoter region of the 1Bx23 was displayed close to 1Bx7 and 1Bx17. The deduced amino acid sequence of coding region of 1Bx23 exhibited 34, 30, 20 and 22 amino acid substitutions from that of 1Bx14, 1Bx20, 1Bx7 and 1Bx17, respectively. A phylogenetic tree based on the nucleotide sequence alignment of the Glu-1Bx alleles shows that the 1Bx23 are apparently clustered with 1Bx7 and 1Bx17, and more ancient than 1Bx14 and 1Bx20, suggesting that the evolution speeds are different among Glu-1Bx genes. Additionally, the potential use of wheat line SY95-71 to further screen the quality contribution of unique subunit 1Bx23 is also discussed.     

利用反义RNA技术分析春化相关基因VER17在冬小麦花发育过程中的功能

为了研究小麦春化相关基因VER17的功能,应用反义RNA技术,将VER17基因的反义片段构建到载体pBI121上,通过花粉管通道法获取转基因小麦.对T0代转基因植株GUS染色以及PCR等分子鉴定,得到14株含反义VERJ7基因片段的阳性转基因植株.对T0代和T1代的表型观察结果显示,VER17反义转基因植株开花时间延迟,并且穗的顶部和基部小花出现明显的退化.表明春化相关基因VER17在小麦发育过程中可能起到促进植物开花以及穗顶端和基部花发育的作用,减少小花退化,同时对雄蕊的发育也有影响.     

Introduction of a rice blight resistance gene, Xa21, into five Chinese rice varieties through an Agrobacterium-mediated system

A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium-mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3∶1 ratio in the selfed T1 generation when one copy of the gene was integrated in the transformants. Inoculation tests displayed that transgenic T0 plants and Xa21 PCR-positive T1 plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.     

一种基于PCR技术鉴定单拷贝转基因烟草的方法

为了鉴定携带单拷贝外源基因的转基因烟草植株,以烟草核基因组上已知的单拷贝内源基因（RNR2）为内参,转基因烟草植株基因组DNA为模板,在同一PCR反应体系中扩增内源基因（RNR2）和外源目的基因（NPTⅡ）。反应产物在琼脂糖凝胶上电泳,获得了预期大小的两条特异性扩增条带。经ImageJ软件捕捉分析两条目的条带的灰度比,当T1代转基因烟草植株中外源基因与内源基因的扩增条带灰度比为1时,所检测植株即为单拷贝外源基因的转基因烟草植株。孟德尔经典遗传学方法证实了上述检测结果高度可信。     

Potato flour viscosity improvement is associated with the expression of a wheat LMW-glutenin gene

It has been previously shown that expression of a high-molecular-weight glutenin (HMW-GS) in transgenic wheat seeds resulted in the improvement of flour functional properties. In this study, potato flour viscosity was improved through a specific expression of a low-molecular-weight glutenin (LMW-GS-MB1) gene in tuber. The resulting construct was introduced into potato leaf explants (Solanum tuberosum cv Kennebec) through Agrobacterium tumefaciens-mediated gene transfer. Southern and Northern analysis of transgenic potato confirmed that the integration of LMW-GS-MB1 in genomic DNA was stable and its mRNA was abundant in transgenic line 16 tubers. Western blot analysis of line 16 extract shows a LMW-GS subunit accumulation in tuber. To demonstrate the capacity of transgenic lines to produce tubers with improved flour functional properties, transgenic lines 9 and 16 exhibiting, respectively, moderate and high expression of LMW-GS-MB1 mRNA and nontransgenic plants were transferred to field plots. The mean viscosity value of flour obtained from the field-grown tubers of transgenic line 16 exhibited a 3-fold increase in viscosity at 23 degrees C when compared to flour from nontransgenic tubers.