抗咪唑啉酮类除草剂水稻种质的筛选鉴定

咪唑啉酮类除草剂是一类广谱高效除草剂,其作用靶标是乙酰乳酸合成酶(ALS,acetolactate synthase)。培育抗咪唑啉酮类除草剂水稻品种是防治直播稻田杂草危害的有效途径之一。本研究通过喷施咪唑啉酮类除草剂,从30570份水稻种质资源中,获得1份抗咪唑啉酮类除草剂的水稻新种质,该材料抗性性状稳定、抗性效应明显,序列分析表明其ALS基因编码区第1880位的G/A突变导致第627位氨基酸由丝氨酸改变为天冬酰胺,从而产生抗性。本研究发现的抗除草剂新材料,为选育抗除草剂水稻新品种奠定了种质基础。     

乙酰羟酸合成酶同工酶基因ilvBN、ilvGM和ilvIH的表达及对除草剂抗性的比较

乙酰羟酸合成酶(AHAS)是磺酰脲类和咪唑啉酮类等AHAS抑制剂类除草剂的作用靶标。获得抗此类除草剂的AHAS突变基因资源具有非常重要的理论和应用价值。本研究从抗甲磺隆菌株Klebsiella sp.HR11和甲磺隆敏感菌株Klebsiella pneumoniae MGH 78578中分别克隆到AHAS三种同工酶基因ilvBN、ilvGM和ilvIH。抗性菌株和敏感菌株AHAS三种同工酶基因在氨基酸水平上差异位点主要集中在ilvBN和ilvGM的大亚基上。将2株菌的ilvBN、ilvGM和ilvIH分别构建到表达载体pET29a(+)中,在Escherichia coli BL21(DE3)中进行表达,测得只有含菌株HR11 ilvBN和ilvGM的转化子细胞破碎液AHAS对各类AHAS抑制剂类除草剂具有较强的抗性,而含菌株HR11 ilvIH和菌株MGH78578 ilvBN、ilvGM和ilvIH的转化子细胞破碎液AHAS对各类AHAS抑制剂类除草剂敏感。     

美国培育耐除草剂水稻

AgBiotech Reporter 2000年10月号报道：BASF和RiceTech公司签署了一项协议。内容是利用Clear－field技术开发、生产和商品化杂交稻。公司认为协议的实行将导致开发出首个具有耐受咪唑啉酮类除草剂的杂交水稻品种,作为美国2003年的商业化候选品种。这种除草剂耐性不是通过转基因获得的。咪唑啉酮是一类用于防除杂草的植保产品。作用原理是影响植物中的一种酶,这种酶只存在于植物中,在哺乳动物、鸟类、鱼类或昆虫中没有。美国培育耐除草剂水稻@孙雷心     

拟南芥乙酰乳酸合成酶基因(Atals)多位点突变以及转基因过表达植株除草剂抗性分析

乙酰乳酸合成酶(ALS)是支链氨基酸、缬氨酸、亮氨酸和异亮氨酸生物合成途径中的关键酶,也是多种除草剂的靶点。为了研究als基因不同突变位点组合后其抗除草剂抗性的变化,并整合和增强植株对不同类型除草剂的抗性,本研究对已知抗性位点进行组合并进行了拟南芥转基因分析。我们通过重叠延伸PCR技术体外突变扩增四个已知位点突变的P197S/R199A/W574S/S653F拟南芥Atals,克隆到pCAMBIA1 300-GFP载体上,从而构建了四个位点突变的m4Atals-GFP融合蛋白过表达载体。然后用农杆菌介导法转化野生型拟南芥Col-0,获得转基因株系。采用潮霉素抗性筛选鉴定阳性转基因植株,并利用荧光体式显微镜观察过表达植株以及在蛋白水平检测GFP-m4Atals融合蛋白表达情况。对纯合转基因株系进行除草剂抗性分析。分析表明转基因拟南芥具有磺酰脲和咪唑啉酮两种除草剂的整合抗性。此研究有助于系统地分析als基因不同突变位点对抑制剂的抗性,有效避免和应对自然界als单一位点突变的杂草的困扰。     

抗草丁膦和抗草甘膦转基因油菜的抗性基因向野芥菜的流动

通过人工去雄授粉和田间隔行种植试验,研究了抗草丁膦和抗草甘膦转基因油菜(Brassica napus)中的bar基因和EPSPS基因向野芥菜(B. juncea var. gracilis)流动的可能性。结果表明在人工授粉的情况下,以野芥菜为母本,分别以两种转基因油菜为父本,亲和性指数都很高,达13以上,与野芥菜自交或开放授粉条件下的亲和性指数没有明显差异,说明两种转基因油菜和野芥菜的亲和性较好。经两次除草剂筛选,人工杂交获得的所有F₁对相应的除草剂都表现出了明显的抗性,且经PCR检测扩增出了各自的特异性条带,说明人工杂交获得的所有F₁都携带了相应的抗性基因。F₁的适合度研究表明,两种F₁种子萌发率和母本都没有明显差异,营养生长明显好于母本。但花粉活力和结实率明显下降,携带抗草丁膦基因F₁的花粉活力和每角果粒数分别是32.4%和0.59粒,携带抗草甘膦基因F₁的花粉活力和每角果粒数分别是35.1%和0.58粒。经两次除草剂筛选和PCR检测,表明野芥菜和抗草丁膦油菜或与抗草甘膦油菜田间隔行种植分别能产生0.02%和0.014%的携带抗性基因的F₁杂种。以上结果表明抗除草剂转基因油菜的抗性基因具有向野芥菜流动的可能性,且bar和EPSPS基因向野芥菜流动的可能性类似,但对其可能引起的环境后果需要做进一步地深入研究。     

抗甲磺隆假单胞菌的分离及其乙酰乳酸合酶的大小亚基ilvIH基因的克隆和表达

乙酰乳酸合酶(也称乙酰羟酸合酶acetohydroxyacid synthase,AHAS)是植物、真菌和细菌细胞内支链氨基酸Val、Leu、Ile生物合成过程中关键酶,是乙酰乳酸合酶抑制剂类除草剂如磺酰脲类、咪唑啉酮类、嘧啶水杨酸和磺酰氨类的作用靶标.[目的]获得抗甲磺隆的乙酰乳酸合酶基因,构建其表达载体,并分析基因中的位点突变与乙酰乳酸合酶对磺酰脲类除草剂抗性产生原因.[方法]从长期使用甲磺隆的土壤中分离到l株抗甲磺隆的菌株Lm10,利用PCR技术从Lm10总DNA中克隆到乙酰乳酸合酶的大小亚基基因ilvIH,对ilvIH氨基酸序列进行比对分析.分别将ilvI和ilvH分别连接到表达载体pET29a( )多克隆位点,转化大肠杆菌(Escherichia coli)获得转化子BL21(pET-I)和BL21(pET-H),并诱导表达.[结果]菌株Lm10鉴定为假单孢菌(Pseudomonas sp.),对甲磺隆的最高耐受浓度达到14000 μmol/L,且对各种乙酰乳酸合酶抑制剂类除草剂具有交叉抗性.Lm10与甲磺隆敏感菌株KT2440的小亚基氨基酸序列完全相同,而大亚基有6个氨基酸位点发生变异.转化子在IPTG诱导下,乙酰乳酸合酶的大小亚基的蛋白成功表达,粗酶液酶活试验结果表明Lm10的ilvI基因表达的乙酰乳酸合酶大亚基对甲磺隆有很强的抗性.[结论]发现菌株Lm10的乙酰乳酸合酶大亚基对甲磺隆有很强的抗性,抗甲磺隆菌株Lm10与敏感菌株KT2440的ilvI有6个氨基酸位点差异,这些位点突变可能是乙酰乳酸合酶对甲磺隆抗性产生的原因.     

除草剂抗性基因的研究进展

概述了除草剂抗性基因的种类,主要来源以及抗除草剂基因的应用。并对新的抗除草剂基因的发掘、既有的抗除草剂基因的改良、除草剂抗性机理研究以及新的抗除草剂作物的培育等方面的进一步研究进行了探讨。     

新型遗传工程菜籽油适合在英国消费

最近。英国政府接受了新型食品与加工顾问委员会(ACNFP)的建议,批准了从第四种遗传工程油菜生产菜籽油。这种油菜是经遗传改良具有除草剂草甘膦(GM)抗性的品种。 GM油菜生产是通过导入两种细菌基因即众所周知的Roundup-Read~(TM)基因获得的,两种基因共同作用使油菜产生对广谱除草剂草甘膦的抗性。这种除草荆通常用于防治和抑制油料作物生产中杂草的滋生。 根据食品安全性评估,委员会认为,这种GM油菜菜籽油的组分与用常规育种法培育的品种的油组分没     

以草甘膦为筛选标记的大豆转基因体系的建立及抗除草剂转基因大豆的培育

抗草甘膦转基因大豆能显著提高大豆生产效率,具有重大的应用前景.本实验室前期研究建立了农杆菌介导、草胺膦为筛选剂的大豆转基因体系,转化效率在4%以上.在此基础上,利用G6-EPSPS和G10-EPSPS 2个具有自主知识产权的草甘膦抗性基因,通过优化转化体系,成功建立了以草甘膦为筛选剂的大豆遗传转化体系,转化效率达1%以上.浓度梯度实验发现,当草甘膦的筛选浓度为100 mg/L时,虽然丛生芽的再生率下降了50%～60%,但最终转化效率不受影响.进一步通过基因表达分析、Western blot、Southern blot和除草剂抗性鉴定等方法对转基因大豆进行了分子检测和验证,最后获得了分子特征明确、对草甘膦抗性稳定的抗草甘膦转基因大豆后代.结果对国内抗草甘膦转基因大豆转基因方法研究及抗除草剂新品种选育具有意义.     

Cyanamid公司与Pioneer Hi-bred公司联合发展耐除草剂的玉米

美国Cyanamid公司已经求助于第一流的种子生产者Pioneer Hi-bred公司,以发展能够耐受该公司新的咪唑啉酮除草剂的商品化玉米。以前曾经报道过,Molecular Genetics公司与Cyanamid公司已经研制出了能够耐受这类除草剂的玉米。现在,Pioneer Hi-breed公司试图把取自这些玉米品系的耐除草剂基因结合到该公司的专利玉米自交系以及一些“大众化”的品系中。商业目的是获得能够高产而又不被Cyanamid公司专利的除草剂所伤害的     

New RNAi strategy for selective suppression of a mutant allele in polyglutamine disease

In gene therapy of dominantly inherited diseases with small interfering RNA (siRNA), mutant allele specific suppression may be necessary for diseases in which the defective gene normally has an important role. It is difficult, however, to design a mutant allele-specific siRNA for trinucleotide repeat diseases in which the difference of sequences is only repeat length. To overcome this problem, we use a new RNA interference (RNAi) strategy for selective suppression of mutant alleles. Both mutant and wild-type alleles are inhibited by the most effective siRNA, and wild-type protein is restored using the wild-type mRNA modified to be resistant to the siRNA. Here, we applied this method to spinocerebellar ataxia type 6 (SCA6). We discuss its feasibility and problems for future gene therapy.     

荧光假单胞菌Tn5转座诱变及对青枯病生防特性

目的：利用Tn5转座诱变荧光假单胞菌PF20001,研究所获得的突变株对青枯病的生防效果。方法：利用三亲本杂交方式,将带有转座子Tn5的Tn5-102（含luxAB）的质粒pTR102成功地转入PF20001,利用平板相互拮抗法分析突变株对青枯病致病菌的拮抗作用。结果：通过诱导Tn5转座,得到荧光假单胞菌PF20001的Tn5插入突变库。经平板相互拮抗实验发现,菌株PF20001-lux-48拮抗圈明显大于野生型（半径达0.35cm）。用Tn5-lux特异引物进行PCR扩增,结果显示只有以该突变株的DNA为模板才能得到300bp的扩增产物,证实该菌株基因组中有Tn5插入。结论：Tn5的插入使菌株PF20001对青枯病生物防治能力增强。     

Overexpression of the D-alanine racemase gene confers resistance to D-cycloserine in Mycobacterium smegmatis.

D-Cycloserine is an effective second-line drug against Mycobacterium avium and Mycobacterium tuberculosis. To analyze the genetic determinants of D-cycloserine resistance in mycobacteria, a library of a resistant Mycobacterium smegmatis mutant was constructed. A resistant clone harboring a recombinant plasmid with a 3.1-kb insert that contained the glutamate decarboxylase (gadA) and D-alanine racemase (alrA) genes was identified. Subcloning experiments demonstrated that alrA was necessary and sufficient to confer a D-cycloserine resistance phenotype. The D-alanine racemase activities of wild-type and recombinant M. smegmatis strains were inhibited by D-cycloserine in a concentration-dependent manner. The D-cycloserine resistance phenotype in the recombinant clone was due to the overexpression of the wild-type alrA gene in a multicopy vector. Analysis of a spontaneous resistant mutant also demonstrated overproduction of wild-type AlrA enzyme. Nucleotide sequence analysis of the overproducing mutant revealed a single transversion (G-->T) at the alrA promoter, which resulted in elevated beta-galactosidase reporter gene expression. Furthermore, transformants of Mycobacterium intracellulare and Mycobacterium bovis BCG carrying the M. smegmatis wild-type alrA gene in a multicopy vector were resistant to D-cycloserine, suggesting that AlrA overproduction is a potential mechanism of D-cycloserine resistance in clinical isolates of M. tuberculosis and other pathogenic mycobacteria. In conclusion, these results show that one of the mechanisms of D-cycloserine resistance in M. smegmatis involves the overexpression of the alrA gene due to a promoter-up mutation.     

Phenylalanine ammonia lyase production by gamma irradiated and analog-resistant mutants of Rhodotorula glutinis

Mutants resistant to phenylalanine analogs (L-tyrosine, p-fluoro-D, L-phenylalanine (PFP) and trans-cinnamic acid) were isolated from a wild type strain of Rhodotorula glutinis A-97 by mutagenic treatment with gamma radiation and screened for phenylalanine ammonia lyase (PAL) production. One such mutant, gammaT11 (resistant to L-tyrosine), exhibited four times the PAL activity of the parent wild strain A-97. Mutant isolate gammaTFP5.6 which was selected as L-tyrosine and PFP resistant isolate, produced inducible PAL activity at levels 5.94-fold higher than the wild-type A-97 and 2.66-fold higher than its parent mutant isolate gammaT5 which was resistant to L-tyrosine. The mutant isolate gammaTC5d which was resistant to L-tyrosine and trans-cinnamic acid, exhibited 3.48 and 1.56-fold increase in PAL activity compared to the parent wild strain A-97 and its parent mutant isolate gammaT5, respectively. Different media have been examined for the induction of PAL.     

Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents.

Three independently isolated mutants of human cytomegalovirus strain AD169 were found to be resistant to ganciclovir at a 50% effective dose of 200 microM. Phosphorylation of ganciclovir was reduced 10-fold in mutant-infected cells compared with AD169-infected cells. All three mutants were also determined to be resistant to the nucleotide analogs (S)-1-[(3-hydroxy-2- phosphonylmethoxy)propyl]adenine (HPMPA) and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) and hypersensitive to thymine-1-D-arabinofuranoside (AraT). Single base changes resulting in amino acid substitutions were demonstrated in the nucleotide sequence of the DNA polymerase gene of each mutant. The polymerase mutation contained in one of the mutants was transferred to the wild-type AD169 background. Ganciclovir phosphorylation in cells infected with the recombinant virus produced by this transfer was found to be equivalent to that of AD169-infected cells. The ganciclovir resistance of the recombinant was reduced fourfold compared with that of the parental mutant; however, the recombinant remained resistant to HPMPA and HPMPC and hypersensitive to AraT. The ganciclovir resistance of the mutants therefore appears to result from mutations in two genes: (i) a kinase which phosphorylates ganciclovir and (ii) the viral DNA polymerase.     

Isolation and Characterization of a Phosphonomycin-Resistant Mutant of Escherichia coli K-12

A mutant was isolated from Escherichia coli K-12 which showed increased resistance towards phosphonomycin, a new bactericidal antibiotic recently isolated from strains of Streptomyces. Evidence is presented which suggests that this mutant is resistant to lysis by phosphonomycin because of a lower affinity of phosphoenolpyruvate: uridine diphospho-N-acetylglucosamine enolpyruvyl transferase for this antibiotic. This mutant was also found to be temperature-sensitive in growth. At 42 C mutant cells grew poorly, and the rate of incorporation of (3)H-diaminopimelic acid into trichloroacetic acid-insoluble material was also greatly reduced. Genetic studies indicate that the increased resistance toward phosphonomycin and temperature sensitivity in growth of this mutant are probably the consequences of a single mutation.     

Coexpression of mutant and wild type protein kinase in lymphoma cells resistant to dibutyryl cyclic AMP.

A mutant clone resistant to dibutyryl cyclic AMP was isolated from S49 mouse lymphoma cells. The mutant expressed a form of cyclic AMP-dependent protein kinase distinguishable from wild type kinase by its decreased sensitivity to activation by cyclic AMP and its increased thermal lability. Hybrids formed between mutant and wild type cells were resistant to dibutyryl cyclic AMP and expressed both mutant and wild type activities in about equal amount. The parent mutant cells also appeared to express wild type kinase activity, but at a lower level. We conclude that wild type S49 cells have and express two identical alleles for the regulatory subunit of protein kinase, one of which has undergone mutation in the mutant cells.     

Extranuclear mutant ofSchizophyllum commune

A mutant of the hymenomyceteSchizophyllum commune was isolated which, owing to an extranuclear mutation, did not utilize acetate as the sole carbon source for growth. The growth of the mutant on glucose minimal medium was completely inhibited by sodium azide but was resistant to the effect of 2,4-dinitrophenol or oligomycin. Its endogenous respiration was cyanide-sensitive and was stimulated by 2,4-dinitrophenol to a considerably smaller degree than that of the wild-type strain. The experimental results obtained with this mutant suggest a defect in aerobic phosphorylation.     

Increase in arginine and citrulline production by 6-azauracil-resistant mutants of Bacillus subtilis.

In the arginine producer AHr-5, an L-arginine hydroxamate-resistant mutant of Bacillus subtilis, accumulation of N8-acetyl-L-ornithine increased as the level of L-arginine accumulation increased in the medium containing L-glutamic acid. Ornithine carbamoyltransferase of this strain was genetically derepressed. These results suggested that carbamoylphosphate might be deficient in vivo. With the intention to increase endogenous carbamoylphosphate, pyrimidine analogs inhibiting growth were selected and the mutants resistant to these compounds were derived from the AHr-5 mutant. Of the resistant mutants derived, the 6-azauracil-resistant mutant AAr-9 produced 28 mg of L-arginine per ml, which corresponded to more than twofold the amount produced by the parent strain. Derivation of an arginine-requiring mutant from the double-resistant mutant AAr-9 provides a new advantageous method for the production of L-citrulline. The increase in arginine and citrulline production is discussed.