农杆菌介导玉米胚性愈伤的遗传转化研究

利用3种不同类型的农杆菌菌株C58、LBA4404和EHA105携带外源GUS基因分别侵染玉米自交系齐319和18(红)胚性愈伤.结果显示,不同的菌株和自交系间的搭配,其遗传转化效率差异很大,GUS瞬时表达率呈极显著差异(F=24.92**),抗性愈伤率也呈极显著差异(F=19.43**).其中,EHAl05-齐319组合遗传转化效率最高,其GUS瞬时表达率平均为55.5%,最高可迭71.1%;其抗性愈伤率平均为14.4%,最高可达20%;对22株转基因To代抗性植株进行PCR检测,其中PCR呈阳性植株有11株,阳性率为50%.进一步对此22株To代抗性植株进行叶片组织化学染色分析,结果显示,PCR呈阳性的植株中均有GUS基因表达.从而证明,外源GUS基因在转基因玉米To代植株中得到稳定表达,而且验证了PCR检测结果和GUS表达分析结果的一致性.     

普通小麦品种Alondra＇s遗传转化体系的建立

小麦（Triticum aestivum）幼胚愈伤组织的诱导和分化再生有高度依赖基因型特征。为了建立和优化Alondra’s的高效再生及遗传转化体系,为小麦遗传转化提供更多的受体基因型,以Alondra’s的幼胚为外植体,研究了培养基种类、不同激素配比等对其幼胚愈伤组织诱导及再生的影响。结果表明,在使用N6培养基时,添加3mg·L^-1的2,4-D并附加1000mg·L^-1的CH对愈伤组织的诱导效果较好;添加4mg·L^-1的ZT、不附加IAA对愈伤组织的分化效果最好。通过构建植物表达载体pCAMBIA1301-220.6,利用基因枪法将HYG基因导入Alondra’s幼胚愈伤组织中,以建立Alondra’s的高效遗传转化体系。结果在含100mg·L^-1潮霉素的选择培养基上进行筛选、分化,获得了30棵抗性植株。经PCR检测,其中5株为阳性转基因植株,转化率为0.5%。Alondra＇s遗传转化体系的建立丰富了小麦遗传转化的基因型,为小麦品种的转基因改良和在不同背景下研究基因的功能奠定了良好的基础。     

优良大麦品种花30幼胚遗传转化体系的优化

以大麦花培基因型花30的幼胚为外植体,设置不同的培养基类型、不同激素配比及碳源,研究其对幼胚愈伤组织诱导及绿苗分化的影响,以此建立和优化一个适于优良大麦品种遗传转化的高效组织培养体系。结果表明:在N6、MS和B5的组合改良培养基下,以蔗糖为碳源,附加2mg/L 2,4-D、1mg/L ABA时,有最高的愈伤组织诱导率,且愈伤质量最好。Cu2+的添加具有抑制幼胚直接发芽成苗和改善愈伤组织质量的双重功效。添加2mg/L 6-BA对愈伤组织的分化效果比较理想。为了提高农杆菌介导转化大麦外源基因的瞬时表达率和优化遗传转化体系,利用花30幼胚产生的愈伤组织为受体材料,通过检测GUS基因的瞬时表达情况,研究了农杆菌介导的大麦遗传转化中菌液的浓度、侵染时间以及共培养天数对遗传转化的影响,结果表明:当菌液浓度OD600=0.5的条件下,侵染15min,共培养2d表现出最佳的GUS瞬时表达率。     

普通小麦品种Alondra's遗传转化体系的建立

小麦(Triticum aestivum)幼胚愈伤组织的诱导和分化再生有高度依赖基因型特征。为了建立和优化Alondra’s的高效再生及遗传转化体系,为小麦遗传转化提供更多的受体基因型,以Alondra’s的幼胚为外植体,研究了培养基种类、不同激素配比等对其幼胚愈伤组织诱导及再生的影响。结果表明,在使用N6培养基时,添加3mg·L-1的2,4-D并附加1000mg·L-1的CH对愈伤组织的诱导效果较好;添加4mg·L-1的ZT、不附加IAA对愈伤组织的分化效果最好。通过构建植物表达载体pCAMBIA1301-220.6,利用基因枪法将HYG基因导入Alondra’s幼胚愈伤组织中,以建立Alondra’s的高效遗传转化体系。结果在含100mg·L-1潮霉素的选择培养基上进行筛选、分化,获得了30棵抗性植株。经PCR检测,其中5株为阳性转基因植株,转化率为0.5%。Alondra's遗传转化体系的建立丰富了小麦遗传转化的基因型,为小麦品种的转基因改良和在不同背景下研究基因的功能奠定了良好的基础。     

高羊茅和黑麦草农杆菌介导转化体系的研究

利用C58C1农杆菌菌系（携带的表达载体上含GUS基因和nptII基因）感染4个草坪草品种追寻者、爱神特、腾跃和守门员成熟胚来源的愈伤组织,共培养后部分愈伤组织进行X-Gluc组织化学染色检测,其余愈伤组织在含G418 10-25 mg/L的MS改良培养上先后筛选抗性愈伤组织和分化抗性再生植株,对移栽成活的144棵抗性再生植株分别进行了ELISA检测、PCR检测和组织化学染色检测。愈伤组织阶段X-Gluc染色检测结果表明,4个草坪草品种GUS基因瞬间表达率8.6%～46.9%,爱神特愈伤组织对农杆菌侵染最为敏感,其次是腾跃和守门员,追寻者最不敏感;ELISA检测结果表明,45株呈现阳性,证明nptII基因已转入草坪草并已表达;PCR检测结果与ELISA检测结果一致,表明nptII基因确实已经整合到了草坪草基因组中,且没有发生沉默现象;转基因植株X-Gluc染色检测结果表明,GUS基因在43株中得到了稳定表达,在2株中发生了沉默现象。4个草坪草品种抗性再生植株分化率0～43.5%,转化率0～21.5 %。结果还表明,GUS基因瞬间表达率与稳定转化率在草坪草上很不一致,不能作为衡量基因型转化效果的指标。     

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。     

小麦抗白粉病相关基因的转化

利用玉米花青素苷合成调节基因C1-Lc作为报告基因, 通过瞬间表达后愈伤组织表面红色斑点的统计分析, 优化了小麦幼胚愈伤组织的基因枪转化参数。小麦Beclin1类似基因TaTBL和硫代硫酸硫转移酶基因TaTST是2个在白粉菌诱导条件下具有增强表达特性的抗病相关基因。本实验进一步利用基因枪将ubi强启动子控制下的2个基因导入到小麦品种扬麦158的幼胚愈伤组织细胞中, 使用除草剂经两轮选择培养基上的筛选和再生获得抗性植株, 进一步通过抗性植株的PCR分析获得转TaTBL基因植株5株, 转TaTST基因植株6株。转基因植株离体叶片的人工接种实验表明, 外源基因的导入不同程度上增强了植株的白粉病抗性, 表现为延缓了白粉菌的发育。利用玉米花青素苷合成调节基因C1-Lc作为报告基因,通过瞬间表达后愈伤组织表面红色斑点的统计分析,优化了小麦幼胚愈伤组织的基因枪转化参数。小麦Beclin1类似基因TaTBL和硫代硫酸硫转移酶基因TaTST是两个在白粉菌诱导条件下具有增强表达特性的抗病相关基因。本实验进一步利用基因枪将ubi强启动子控制下的两个基因导入到小麦品种扬麦158的幼胚愈伤组织细胞中,使用除草剂经两轮选择培养基上的筛选和再生获得抗性植株,进一步通过抗性植株的PCR分析获得转TaTBL基因植株5株,转TaTST基因植株6株。转基因植株离体叶片的人工接种实验表明,外源基因的导入不同程度上增强了植株的白粉病抗性,表现为延缓了白粉菌的发育。     

CHI-PAT双价基因遗传转化贵州禾来拢

以贵州禾来拢幼胚为转化受体,用农杆菌介导法将几丁质酶和抗除草剂抗性双价基因(CHI-PAT)导入来拢幼胚,筛选出抗性愈伤组织并获得抗性植株.抗性植株经GUS组织化学及PCR检测呈阳性,转基因植株对50 mg/L的Basta溶液有抗性.初步证明CHI和PAT基因已整合进了水稻基因组中.     

Transgeni根癌农杆菌介导的小麦转基因植株再生(英文)

根癌农杆菌菌株Ａｇｌ Ⅰ的Ｔｉ 质粒ｐＵＮＮ－２ 带有Ｕｂｉ１ 启动子驱动的ｎｐｔ Ⅱ基因。７ 种基因型小麦幼胚或胚性愈伤组织用于农杆菌介导的转化实验。经过不同浓度巴龙霉素的筛选,３ 种基因型小麦产生抗性愈伤组织并再生植株。再生植株经ＰＣＲ 和Ｓｏｕｔｈｅｒｎ 杂交鉴定为转基因植株,转化频率（ 再生转基因植株的小麦愈伤组织数／ 用于转化实验的愈伤组织数） 为３．７％ ～５ ．９％ 。小麦基因型及转化材料的起始生理状态是影响ＴＤＮＡ转移的重要因素。     

根癌土壤杆菌介导的咖啡转化

用五种不同的根癌土壤杆菌转化咖啡栽培品种小粒种的成熟胚培养物,筛选出对咖啡侵染力较强的菌株Gv3111(pTiB6s3)和A281(pTiB0542)。在无激素的Ms培养基(MsD)上得到淡黄色、松脆、生长迅速的瘤性愈伤组织,继代培养4—6周后,产生大量的畸胎芽。转化体经Opine检测,分别含有特异的章鱼碱或农杆碱。GV3111转化体的DNA分子杂交结果表明,在咖啡的基因组内有T-DNA的插入。用带有GUS基因和NPT I基因的双元载体系统pBI121／Gv3111和pBl121／A281转化咖啡子叶,转化后第三天可以检测到Gus基因的瞬时表达。在含卡那霉素(Kanamycin,Km)的Ms选择培养基上,得到抗性愈伤组织,这些愈伤组织仍能检测到GUS基因的表达·     

Transformation of Brazilian eliteIndica-type rice (Oryza sativa L.) by electroporation of shoot apex explants

We have generated transgenic plants of a Brazilian elite Indica-type rice by electroporation of shoot apices. This approach avoids a callus phase and produces 0.4–13.8% resistant plants. Transgenic plantlets were transferred to soil a few weeks after explant electroporation. Root segments from plantlets obtained from transformation experiments with pAHC25 plasmid were GUS positive. Integration of the introduced gene into the genome was demonstrated by PPT and antibiotic screening as well as by PCR and Southern blot hybridization of genomic DNA isolated from R₂ plantlets.     

Production of herbicide-resistant transgenic sweet potato plants through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> method

Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] were produced through Agrobacterium-mediated transformation system. Embryogenic calli derived from shoot apical meristems were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3301 vector containing the bar gene encoding phosphinothricin N-acetyltransferase (PAT) and the gusA gene encoding β-glucuronidase (GUS). The PPT-resistant calli and plants were selected with 5 and 2.5 mg l⁻¹ PPT, respectively. Soil-grown plants were obtained 28–36 weeks after Agrobacterium-mediated transformation. Genetic transformation of the regenerated plants growing under selection was demonstrated by PCR, and Southern blot analysis revealed that one to three copies of the transgene were integrated into the plant genome of each transgenic plant. Expression of the bar gene in transgenic plants was confirmed by RT-PCR and application of herbicide. Transgenic plants sprayed with Basta containing 900 mg l⁻¹ of glufosinate ammonium remained green and healthy. The transformation frequency was 2.8% determined by herbicide application which was high when compared to our previous biolistic method. In addition, possible problems with multiple copies of transgene were also discussed. We therefore report here a successful and reliable Agrobacterium-mediated transformation of the bar gene conferring herbicide-resistance and this method may be useful for routine transformation and has the potential to develop new varieties of sweet potato with several important genes for value-added traits such as enhanced tolerance to the herbicide Basta.     

基因枪法介导GNA基因遗传转化甘蔗的研究

目的:将含有雪花莲外源凝集素(GNA)基因的植物表达载体用基因枪法分别导入一个果蔗和一个糖蔗品种中,以期获得转基因植株。方法:将GNA基因插入到植物表达载体上,构建出不同选择标记、不同启动子的表达载体,并用基因枪法将之导入甘蔗胚性愈伤组织,分别在G418、PPT和Hyg的选择压力下,筛选抗性植株,并进行分子杂交鉴定。结果:通过斑点杂交和PCR-Southern杂交证明GNA基因已整合到甘蔗基因组中。结论:用基因枪法成功获得了含有GNA基因的甘蔗转化株,为培育抗甘蔗绵蚜(Ceratovacuna lanigeraZehnther)的新品种提供了基础。     

Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts

We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2–4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was varified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T_o plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T₁ and T₂ progenies. Enzyme analyses on T₁ progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T_o plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.     

Efficient regeneration and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated stable transformation of perennial ryegrass

We utilized gene transfer technology for genetic perennial ryegrass improvement, efficient regeneration, and Agrobacterium-mediated transformation of phosphinothricin acetyltransferase gene (bar). Four growth regulator combinations were compared and intact seeds of six turf-type cultivars as mature embryo sources were tested to optimize the regeneration conditions. Callus formation and regeneration were observed in all seeds. The highest callus formation frequency was observed in the seeds cultured on MS medium supplemented with 9 mg/l 2,4-D, without benzyladenine. Cv. TopGun revealed the highest callus induction and regeneration frequencies of 96 and 48.9%, respectively. By using an optimized regeneration system, embryogenic calli were transformed by an Agrobacterium strain LBA4404 containing the plasmid pCAMBIA3301. After the selection of the potentially transgenic calli with phosphinothricin, a herbicide, 22 transgenic resistant plants were regenerated. With PCR, Southern-blot hybridizations, and GUS expression techniques, we confirmed that some regenerants were transgenic. Two of the tested transgenic plants showed herbicide resistance. Our results indicated that embryogenic calli from mature seeds can be directly used for perennial ryegrass efficient regeneration and transformation and this protocol is applicable for genetic engineering of herbicide-resistant plants. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 590–596. The text was submitted by the authors in English.     

AtNHX1基因对草木樨状黄芪的转化和耐盐性表达研究

应用RT-PCR技术从100mmol/LNaCl胁迫处理的拟南芥幼中克隆得到编码液泡膜Na /H 逆向转运蛋白的AtNHX1基因cDNA 编码ORF.并在该ORF上游分别插入CaMV 35启动子和TMV RNA5'UTR的Ω片段,而在下游插入NOS polyA构建真核表达盒,进而将该表达盒插入双元植物表达栽体pNT质粒的T-DNA区构建了携带AtNHX1 基因的植物表达载体质粒pNT-AtNHX1.将pNT-AtNHX1 导入农杆菌LBA4404,用农杆菌介导法将AtNHX1 基因导入豆科牧草草木樨状黄芪中,共获得103株Kan抗性再生植株.通过对农杆菌茵液浓度、侵染时间和乙酰丁香酮浓度等影响转化效率的因素进行优化,初步建立了稳定的草木樨状黄芪农杆菌转化体系.经过PCR检测、Southern杂交和RT-PCR检测表明,AtNHX1 基因已被成功整合到草木樨状黄芪基因组中,并且能够正常转录.野生型和转基因株系诱发的愈伤组织进行耐盐生长实验,结果显示相同盐胁迫条件下,转基因愈伤组织的相对生长率显著高于野生型愈伤组织.施加梯度NaCl胁迫后,植株叶片K ,Na 含量和叶片相对电导率测定结果显示,转基因植物叶片比野生型积累更多的Na 和K ,维持较高的K /Na ;转基因株系叶片相对电导率显著低于野生型.上述结果表明,AtNHX1 基因的导入和表达在提高草木樨状黄芪耐盐性的同时减轻了盐胁迫对植物细胞膜的伤害.关键词: AtNHX1 草木樨状黄芪农杆菌遗传转化耐盐性.     

Stably transformed callus of wheat by electroporation-induced direct gene transfer

Fertile plants of wheat have been regenerated from protoplasts in several laboratories. The objective of this study was to develop a transformation system using protoplasts as target cells. Protoplasts were isolated from cell suspensions initiated from an anther-derived callus. The protoplasts were transformed by electroporation using pBARGUS or pBAS, both carrying the Basta resistance (BAR) gene. A total of 2,761 calli were produced from electroporation transformed protoplasts in 3 independent experiments. Six calli survived selective culture on 10 mg/l phosphinothricin (PPT), a concentration that completely inhibited the growth of non-transformed wheat callus. Five PPT resistant calli showed phosphinothricin acetyltransferase (PAT) activity, whereas the sixth probably was a mutant. The transformed wheat calli could tolerate PPT concentrations up to 2,560 mg/l. Southern blot analyses confirmed the integration of the BAR gene in wheat genomes. The integrated DNA sequence may have partially methylated and tandemly repeated at least once. These results demonstrate the production of stably transformed wheat calli by electroporation-mediated direct gene transfer into protoplasts.     

农杆菌介导的雪花莲凝集素基因转入玉米骨干自交系

以农杆菌AGL0介导,将雪花莲凝集素基因转入玉米骨干自交系齐319和掖515胚性愈伤组织细胞,从筛选后的抗性愈伤组织获得再生植株。农杆菌浓度和共培养时间均能显著影响侵染后玉米愈伤组织的抗性频率。在农杆菌浓度OD600 0．2～0．3,共培养时间3d时,侵染后玉米愈伤组织的抗性频率最高,平均约4％。对再生植株及其子代基因组DNA的PCR及Southern杂交分析表明雪花莲凝集素基因已经整合到玉米基因组中,并遗传给后代。在蚜虫人工接种试验中,转基因植株上蚜虫的繁殖力为非转基因对照植株上的50％,这表明转基因植株抗蚜性显著增强。     

Development of a modified transformation platform for apomixis candidate genes research in Paspalum notatum (bahiagrass)

The aim of this work was to improve existing transformation protocols and to transform specific genotypes of Paspalum notatum (bahiagrass) for functional analyses of candidate genes involved in reproduction. Three different explants were assayed for in vitro plant regeneration: mature seeds, mature embryos, and shoot meristems. Plant regeneration was achieved with all explant types, but mature seeds produced the optimal rate (78.0%) and were easiest to manipulate. A method based on serial re-induction of calli from meristems of the regenerated lines was also developed, which could be useful in plant breeding strategies pursuing somaclonal variation. Transient transformation experiments were performed on calli obtained from mature seeds using a compressed helium gene gun. Transient transformation constructs included anthocyanin-synthesis genes cloned under the CAMV 35S promoter and an enhanced green fluorescent protein gene (egfp) driven by the rice actin1 (act1) promoter. Selection curves for ammonium glufosinate were developed in order to determine the optimal selective pressure for stable transformation (1.0 mg/L). Stable co-transformation experiments were carried out with two different constructs containing: (1) the reporter egfp gene cloned under the rice act1 promoter and (2) the selector bar gene driven by the ubiquitin promoter. A total of 27 (64.2%) transgenic plants out of 42 resistant plants analyzed were obtained. The presence of the transgenes in regenerated plants was confirmed by polymerase chain reaction and DNA gel blot analysis. Gene expression was demonstrated by eGFP fluorescence detection and in vivo assays for ammonium glufosinate tolerance. This platform is being used to generate transgenic plants of P. notatum to analyze the function of apomixis-associated candidate genes.     

MDMV CP基因的克隆及其转基因玉米的研究

用RT-PCR方法分离了玉米矮花叶病毒外壳蛋白基因(MDMV CP),并且利用基因枪法将该基因导入玉米优良自交系18-599红、18-599白幼胚诱导的愈伤组织中。转化的愈伤组织在Bialaphos浓度(PPT)为8mg／L、10mg/L、5mg／L的筛选压下经过3次抗性筛选后,分别再生出可育植株12株和6株。PCR和Southem检测结果说明CP基因已整合到玉米自交系基因组中。对T1代转基因植株进行病毒人工接种试验,结果表明对照植株全部表现为感染玉米矮花叶病的典型症状,而转基因植株后代呈现不同程度的抗性。