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1.
采用B6、MS和NB 3种培养基,以粳稻(Oryza sativa ssp.japonica)品种日本晴的成熟胚为受体材料进行组织培养,比较了3种培养基的效果.结果表明,在3种培养基中,NB培养基对愈伤组织的诱导效果最好,转化效率最高,最适合于日本晴成熟胚的组织培养.在此基础上,通过根癌农杆菌(Agrobacterium tumefaciens)导转化法将稻瘟菌蛋白激发子基因pcmG1导人日本晴基因组,获得了转基因水稻植株.PCR、Northern blot和Western blot分别证实了pcmG1基因的整合、转录和表达.遗传分析表明,外源基因在转基因日本晴后代中的分离符合3:1的理论比例.  相似文献   

2.
水稻卷窄叶突变相关基因OsCSLD4的RNAi研究及表达分析   总被引：1，自引：1，他引：0  
在图位克隆获得目的基因OsCSLD4的基础上,利用Invitrogen公司的Gateway系统将500bp的OsCSLD4基因cDNA编码序列以反向互补的方式插入pANDA35HK,构建了水稻卷窄叶突变相关基因OsCSLD4的干涉载体pANDAH05。利用农杆菌转化法将干涉载体pANDAH05及对照空载体pANDA35HK分别转化突变体原始亲本日本晴幼胚。转基因阳性植株表型观察结果显示:干涉载体pANDAH05转化日本晴幼胚后,T0代植株出现了类似于突变体1ah137的表型,叶片明显变窄,卷曲程度增加,株高降低;对照空载体pANDA35HK转化日本晴幼胚后,T0代植株仍保持野生型表型;Real-timePCR检测结果显示:日本晴转干涉载体pANDAH05后,目的基因表达量明显降低,干涉强度不同目的基因表达量降低程度不同,研究结果表明目的基因OsCSLD4在水稻叶片形态发育过程中起着非常重要的作用。  相似文献   

3.
水稻胚性愈伤诱导及其遗传转化的几个技术参数研究   总被引：3，自引：2，他引：1  
以日本晴成熟种子及幼嫩种子为材料诱导形成胚性愈伤组织,经农杆菌介导将水稻隐花色素基因的4个RNAi和2个反义RNA表达载体分别转化日本晴愈伤组织,再经抗性筛选和分化培养诱导形成植株,经分子检测筛选出阳性转基因苗。对胚性愈伤诱导率、抗性愈伤率、分化出苗率及转化率进行统计分析。结果表明,成熟种子、新鲜成熟种子及幼嫩种子愈伤诱导率均大于81%,平均为86.67%,其中以新鲜成熟种子愈伤诱导率最高,达96.89%;抗性愈伤率成熟胚为22.21%~40.71%,平均为28.49%,幼胚为36.79%~43.21%,平均为40%,幼胚的抗性愈伤率高于成熟胚;分化出苗率成熟胚为59.29%~80.43%,平均为71.59%,幼胚为63.16%~72.73%,平均67.95%,幼胚与成熟胚差异不明显;转化率(Gus检测阳性率)成熟胚为43.07%~81.08%,平均61.59%,幼胚为58.33%~76.67%,平均67.50%,幼胚转化率稍高于成熟胚。  相似文献   

4.
以甘薯品种栗子香的胚性悬浮细胞为受体材料, 用根癌农杆菌介导法,获得了表达bar基因的抗除草剂转基因甘薯植株。农杆菌菌株LBA4404携带含有bar基因的双元载体pCAMBIA3300。共计450个胚性细胞团用于遗传转化。在添加2 mg/L 2,4－D、100 mg/L Carb和0.5 mg/L PPT 的固体MS培养基上选择培养8周后,得到了19个PPT抗性愈伤组织。将这19个抗性愈伤组织转移到添加1 mg/L ABA、100 mg/L Carb和0.5 mg/L PPT 的固体MS 培养基上,其中的10个抗性愈伤组织共再生出103株拟转基因植株。PCR分析表明,其中的69株为转基因植株。Southern blot分析表明,bar基因稳定整合到转基因植株的基因组中。除草剂喷洒试验结果表明,转基因植株具有高度除草剂抗性。  相似文献   

5.
组成型表达转植酸酶基因(phyA2)玉米的获得   总被引：5，自引：0，他引：5  
本研究将黑曲霉（Aspergullus nige）来源的植酸酶基因（phyA2）,通过基因枪法转化玉米（Zea maysL.）幼胚,获得再生苗。RT-PCR和Western blot检测证明,植酸酶基因已经整合到玉米基因组中并在玉米中稳定表达和遗传。转基因植株根组织的酶活性测定及利用植酸作为唯一磷源的培养基生长实验证明,植酸酶基因在植株根组织表达并能够分泌到根围高效利用植酸磷。  相似文献   

6.
摘要：以生产上优良旱稻（Oryza sativa L. ）新品种旱稻297、旱稻10号等的幼胚愈伤组织为转化受体材料，用基因枪法把抗Basta除草剂的bar基因导入了这些品种的愈伤组织，经两轮Basta抗性筛选和分化获得了再生植株，对再生植株进行PCR扩增和Southern杂交检测，T0和T1代Basta抗性实验表明，bar基因已整合到旱稻的基因组DNA中，并在T1代继续表达。对各品种幼胚培养的诱导、分化培养基实验表明，MB和MS培养基可作为这5个品种的愈伤组织诱导培养基，改良的RMB2、RMS2培养基可显著地提高愈伤组织的分化频率。实验所获得的转基因植株和建立的遗传转化系统，为旱稻的抗除草剂分子育种和其它基因转化奠定了初步的基础。  相似文献   

7.
利用萌动的成熟胚作外植体进行基因转化,可有效提高高粱转基因材料选育的效率。为了建立并完善以高粱萌动种胚作为外植体的遗传转化体系,本研究以高粱材料Is-623为转化受体,质粒pM3301UbiSpCP4为外源基因供体,采用农杆菌介导法进行遗传转化,并对转化的关键因素进行分析。结果表明,麦草畏(dicamba)诱导高粱萌动的成熟胚产生愈伤组织适宜浓度约为5 mg·L-1;农杆菌侵染时间为2 h时遗传转化效率较高;经筛选共获得301块抗性愈伤组织,75个转化事件,经PCR、Southern blotting杂交分析、CP4-EPSPS蛋白检测试纸条检测及田间草甘膦耐性鉴定,最终获得草甘膦耐性等级1级株系6株,耐性等级2级株系3株的转基因高粱株系。本研究初步建立了以高粱萌动种胚作为外植体的遗传转化体系,为高粱遗传转化体系的研究和应用奠定了基础。  相似文献   

8.
基因枪转化MAR序列介导水稻bar基因的表达分析   总被引：7，自引：0，他引：7  
以水稻（Oryza sativa L.) 成熟胚愈伤组织作为水稻遗传转化受体，采用带有MAR（matrix attachment region)和不带MAR的两种植物表达质粒进行基因枪轰击转化bar基因，获得抗basta转基因水稻。分析了MAR序列介导在转基因水稻中对转基因表达的影响。研究结果表明，利用MAR序列介导bar基因表达，获得的转基因系的数量比对照提高66．7％，单块抗性愈伤组织再生植株数比对照提高27．3％，MAR序列介导bar基因表达的转基因系中，bar基因的表达水平比对照高，并且不同转基因系间bar基因表达差异比对照小。  相似文献   

9.
为了建立黑龙江粳稻组织培养高效再生体系,以东农427和东农428的成熟胚为材料,探讨预先浸胚、低温处理、培养基种类、凝固剂种类、ABA、谷氨酰胺、活性炭、硝酸银、2,4-D浓度以及6-BA浓度等因素对水稻再生体系建立的影响。结果表明,东农427经预先浸胚和低温处理可使培养力分别提高6%和15%,最适培养基为NB;ABA和谷氨酰胺分别适合在分化和诱导阶段添加,分化时添加活性炭和硝酸银分别使分化率提高38%和9%;最适2,4-D和6-BA分别为1mg·L~(-1)和4mg·L~(-1)。东农428经预先浸胚和低温处理可使培养力分别提高14%和9%,最适培养基为MS;ABA适合在诱导阶段添加,谷氨酰胺适合在整个培养阶段添加;分化时添加活性炭和硝酸银分别使分化率提高1%和9%;最适2,4-D和6-BA分别为1mg·L~(-1)和3mg·L~(-1)。2个品种的凝固剂在诱导阶段宜采用琼脂,分化阶段宜采用Phytagel。本研究为进一步优化水稻再生体系提供了依据,为后续水稻遗传转化奠定了基础。  相似文献   

10.
抗除草剂基因导入早稻（Oryza sativa）栽培品种   总被引：2，自引：0，他引：2  
以生产上优良旱稻(Oryza sativa L．)新品种旱稻297、旱稻10号等的幼胚愈伤组织为转化受体材料,用基因枪法把抗Basta除草剂的bar基因导入了这些品种的愈伤组织,经两轮Basta抗性筛选和分化获得了再生植株,对再生植株进行PCR扩增和Southern杂交检测,T0和T1代Basta抗性实验表明,bar基因已整合到旱稻的基因组DNA中,并在T1代继续表达。对各品种幼胚培养的诱导、分化培养基实验表明,MB和MS培养基可作为这5个品种的愈伤组织诱导培养基,改良的RMB2、RMS2培养基可显著地提高愈伤组织的分化频率。实验所获得的转基因植株和建立的遗传转化系统。为早稻的抗除草剂分子育种和其它基因转化奠定了初步的基础。  相似文献   

11.
We introduced the human cytochrome P450 gene CYP2B6 into rice plants (Oryza sativa L. cv. Nipponbare), and the CYP2B6-expressing rice plants became more tolerant to various herbicides than nontransgenic Nipponbare rice plants. In particular, CYP2B6 rice plants grown in soil showed tolerance to the chloroacetanilide herbicides alachlor and metolachlor. We evaluated the degradation of metolachlor by CYP2B6 rice plants to confirm the metabolic activity of the introduced CYP2B6. Although both CYP2B6 and nontransgenic Nipponbare rice plants could decrease the amount of metolachlor in plant tissue and culture medium, CYP2B6 rice plants could remove much greater amounts. In a greenhouse, the ability of CYP2B6 rice plants to remove metolachlor was confirmed in large-scale experiments, in which these plants appeared able to decrease residual quantities of metolachlor in water and soil.  相似文献   

12.
The human cytochrome P450 CYP1A1 gene was introduced into rice plants (Oryza sativa cv. Nipponbare). One-month-old CYP1A1 plants grown in soil clearly showed a healthy growth and tolerance to 8.8 microM atrazine and 50 microM simazine, but nontransgenic plants were completely killed by the herbicides. Although transgenic and nontransgenic plants metabolized the two herbicides into the same sets of compounds, CYP1A1 plants metabolized atrazine and simazine more rapidly than did control plants. In small-scale experiments, residual amounts of atrazine and simazine in the culture medium of CYP1A1 plants were 43.4 and 12.3% of those in control medium; those of nontransgenic Nipponbare were 68.3 and 57.2%, respectively. When cultivated in soil with 2.95 microM atrazine and 3.15 microM simazine for 25 days, CYP1A1 plants eliminated 1.3 times more atrazine and 1.4 times more simazine from the soil than did control plants. Thus, CYP1A1 rice plants make it possible to remove atrazine and simazine more rapidly from the culture medium and soil than can nontransgenic Nipponbare.  相似文献   

13.
This study evaluated the expression of human cytochrome P450 genes CYP1A1, CYP2B6, and CYP2C19 in rice plants (Oryza sativa cv. Nipponbare) introduced using the plasmid pIKBACH. The transgenic rice plants (pIKBACH rice plants) became more tolerant toward various herbicides than nontransgenic Nipponbare rice plants. Rice plants expressing pIKBACH grown in soil showed tolerance to the herbicides atrazine, metolachlor, and norflurazon and to a mixture of the three herbicides. The degradation of atrazine and metolachlor by pIKBACH rice plants was evaluated to confirm the metabolic activity of the introduced P450s. Although both pIKBACH and nontransgenic Nipponbare rice plants could decrease the amounts of the herbicides in plant tissue and culture medium, pIKBACH rice plants removed greater amounts in greenhouse experiments. The ability of pIKBACH rice plants to remove atrazine and metolachlor from soil was confirmed in large-scale experiments. The metabolism of herbicides by pIKBACH rice plants was enhanced by the introduced P450 species. Assuming that public and commercial acceptance is forthcoming, pIKBACH rice plants may become useful tools for the breeding of herbicide-tolerant crops and for phytoremediation of environmental pollution by organic chemicals.  相似文献   

14.
The human gene for CYP2B6, a cytochrome P450 monooxygenase that inactivates xenobiotic chemicals, was introduced into Oryza sativa cv. Nipponbare by Agrobacterium-mediated transformation. At germination, R(1) seeds of transgenic rice plants expressing CYP2B6 (CYP2B6 rice) showed a high tolerance to 5 microM metolachlor, a preemergence herbicide that is degraded by CYP2B6. Thin-layer chromatography after culture with (14)C-labeled metolachlor revealed that the amounts of residual metolachlor decreased in plant tissues and the medium of CYP2B6 rice faster than those of untransformed Nipponbare. CYP2B6 rice plants were able to grow in the presence of 13 out of 17 herbicides: five chloroacetamides and mefenacet, pyributicarb, amiprofos-methyl, trifluralin, pendimethalin, norflurazon, and chlorotoluron. These herbicides differ in their modes of action and chemical structures. Transgenic rice expressing a xenobiotic-degrading human CYP2B6, which has broad substrate specificity, should be good not only for developing herbicide tolerant rice but also for reducing the environmental impact of agrochemicals.  相似文献   

15.
构建了两个含西红柿原系统素基因的双元载体pNAR304（UbiI5’+Prosystemin+NOS3’）和pNAR305 (UbiI5’+Prosystemin+NOS3’+ PinⅡ5’+PinⅡ+PinⅡ3’)，并用农杆菌介导方法将其转入水稻品种秀水63、合江19和日本晴。经潮霉素抗性、PCR和Southern blot确证，共获得转基因水稻14株。Northern blot检测表明，原系统素基因(Prosystemin)和马铃薯蛋白酶抑制剂基因(PinⅡ)在这些转基因植株中都能转录表达。然而，植株的二化螟和褐飞虱抗虫性鉴定表明：单独转入Prosystemin（pNAR304）不能提高转基因水稻的抗虫能力；Prosystemin和PinⅡ双价（pNAR305）转基因植株能明显提高水稻的抗虫性，但其抗性水平与PinⅡ单个基因的转基因水稻植株间并无显著差异。 这表明，Prosystemin基因转入水稻并不能有效调节转基因水稻PinII基因的表达量。据此试验结果推测，水稻中可能不存在类似于西红柿系统素的信号途径，很可能水稻的伤害信号转导是经由与双子叶植物系统素体系不同的其它途径来实现的。  相似文献   

16.
铝毒是酸性土壤中限制植物生长的主要因子之一,探讨植物耐铝特性与机理对提高酸性土壤植物生产力具有重要意义。通过一系列生理和分子生物学试验,主要探究了Nipponbare和Kasalath两个水稻品种的铝耐性差异。结果发现,与Kasalath相比,Nipponbare在铝胁迫下不仅拥有较长的根伸长,且根尖铝含量较少,表明Nipponbare是水稻铝耐性品种,而Kasalath为铝敏感品种。进一步研究发现,水稻根尖控制铝吸收的NRAT1基因在Kasalath中表达量更高,表明,NRAT1基因的高表达量可能是导致铝敏感品种Kasalath根尖铝含量较高的主要原因。此外,铝胁迫下,Nipponbare根系柠檬酸的分泌量显著高于Kasalath。而且,水稻中控制柠檬酸分泌的OsFRDL4基因在铝耐性品种Nipponbare中的表达量显著高于Kasalath,由此证明,柠檬酸在这两个品种水稻耐铝途径中可能起着重要的作用,并且,该品种水稻可以通过调控OsFRDL4基因的表达量来控制柠檬酸的分泌量。本研究中还分析了其他4个与铝胁迫相关的基因,但并未发现明显的相关性,值得进一步的探究。  相似文献   

17.
利用氮离子束和γ射线辐照处理粳稻日本晴和籼稻"9311",经过M1代损伤鉴定、M2代筛选和M3代重复鉴定,分别得到了740份和666份日本晴突变体,571份和781份"9311"突变体。获得的突变体发生了叶片、茎秆、穗部和籽粒、生理等性状的突变。在氮离子束辐照处理中,日本晴和"9311"分别在5.0×1016N+/cm2和2.5×1016N+/cm2剂量辐照时获得高突变率,为6.44%和6.38%;γ射线辐照处理中,2种材料均在150Gy剂量辐照时获得高突变率,分别为5.68%和9.44%。在本试验中,各高突变率群体均为辐照当代损伤最低的群体。新构建的突变体库将为水稻功能基因组学研究提供较好的基础材料。  相似文献   

18.
为研究水稻种胚脂肪氧化酶Lox1的遗传规律及分子机制,以Lox1缺失突变体1297分别与Lox1活性正常的9311、日本晴杂交,构建2个F2群体,对2个F2群体种子的种胚Lox1进行定量测定和遗传学分析,结果表明低Lox1是受1对单基因控制的隐性性状。以1297与日本晴组配的F2分离群体300个单株为定位群体,将水稻种胚低Lox1基因定位于水稻第3染色体的RM4512和RM282之间,距两侧标记的遗传距离分别为13.0cM和9.1cM,为进一步的分子标记辅助育种和图位克隆打下了基础。通过人工加速老化对种子进行了耐储性评价,表明水稻种胚Lox1与种子储藏特性关系密切。  相似文献   

19.
ABSTRACT

Microbial siderophore-chelated Fe(III) is suggested to be an important source of Fe for plants, although it is hardly reduced by plant roots. Here, we investigated the efficacy of the easily reducible artificial microbial siderophore tris[2-{(N-acetyl-N-hydroxy)glycylamino}ethyl]amine (TAGE)-Fe(III) as an alternative Fe source to correct Fe deficiency in rice plants, and compared it to that of the natural siderophore deferoxamine B (DFOB)-Fe(III). We also evaluated the absorption of Fe from TAGE-Fe(III) by the Strategy I-like system of gramineous plants using nicotianamine aminotransferase 1 (naat1) mutant rice, which does not synthesize phytosiderophores. Fe(III)-siderophores were synthesized in vitro. Nipponbare rice and its naat1 mutant were reared in soil and gel cultures to determine Fe availability. Hydroponically grown naat1 mutant seedlings were used for reducibility assays to determine the ability of rice roots to reduce Fe(III) chelated by TAGE or DFOB. The expression of a Fe-deficiency inducible gene was also determined, as well as chlorophyll and Fe concentrations. Reduci bility assays on naat1 mutant seedlings revealed that the reduction level of TAGE-Fe(III) was approximately three times higher than that of DFOB-Fe(III). Application of TAGE-Fe(III) to both culture medium and alkaline soil improved Fe chlorosis, growth, and Fe concentration in both naat1 and wild type plants, whereas application of DFOB-Fe(III) only did so in wild type plants. Easily reducible Fe(III)-chelates such as TAGE-Fe(III) can be a better source of Fe for rice plants than most natural microbial siderophores-Fe(III). Our study also demonstrated that rice plants have the ability to utilize microbial siderophores-Fe(III) as the Fe source through the Strategy I-like Fe acquisition system.  相似文献   

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