Soil‐less bioassays for early screening for resistance to imazapyr in sunflower (Helianthus annuus L.)

BACKGROUND: Rapid and efficient diagnostic tests for early screening of herbicide resistance are convenient alternatives to field screening methods. There is a need for a quick, reliable and cost‐effective method for rapid diagnosis of imidazolinone resistance in sunflower (Helianthus annuus L.). RESULTS: Two seed germination bioassays were developed. Seeds from three sunflower inbred lines differing in resistance to imidazolinones were germinated either on solid culture medium or placed in plastic pots filled with commercial perlite. After 8 days incubation under controlled conditions, both assays successfully distinguished susceptible genotype from the resistant and intermediate ones. The susceptible genotype showed arrested root growth at all herbicide treatments (root length < 1 cm). The resistant genotype developed a complete root system even when exposed to the highest dose of herbicide. However, no definite differences were observed for the intermediate and resistant genotypes with respect to root growth under the different herbicide treatments. CONCLUSION: The simple and rapid screening assays described in the present study were useful in discriminating imidazolinone resistance at the seedling stage. Therefore, these bioassays could be potential tools for early screening of imidazolinone resistance genes from large sunflower populations. Copyright © 2009 Society of Chemical Industry     

Detection of resistance to acetolactate synthase inhibitors in weeds with emphasis on DNA-based techniques: a review

Resistance to herbicides inhibiting acetolactate synthase (ALS) has been increasing at a faster rate than in any other herbicide group. The great majority of these cases are due to various single-nucleotide polymorphisms in the ALS gene endowing target site resistance. Many diagnostic techniques have been devised in order to confirm resistance and help producers to adopt the best management strategies. Recent advances in DNA technologies coupled with the knowledge of sequence information have allowed the development of accurate and rapid diagnostic tests. While whole plant-based diagnostic techniques such as seedling bioassays or enzyme-based in vitro bioassays provide accurate results, they tend to be labour- and/or space-intensive and will only respond to the particular herbicides tested, making resolution of cross-resistance patterns more difficult. Successful DNA-based diagnosis of ALS inhibitor resistance has been achieved with three main techniques, (1) restriction fragment length polymorphism, (2) polymerase chain reaction amplification of specific alleles and (3) denaturing high-performance liquid chromatography. All DNA-based techniques are relatively rapid and provide clear identification of the mutations causing resistance. Resistance based on non-target mechanisms is not identified by these DNA-based methods; however, given the prevalence of target site-based ALS inhibitor resistance, this is a minor inconvenience.     

副溶血弧菌对斜带石斑鱼血清中一氧化氮合酶活力的影响

为了研究感染副溶血弧菌对斜带石斑鱼体内一氧化氮合酶活力的影响 ,分别对斜带石斑鱼腹腔注射浓度为 5× 10 8cfu/ml、5× 10 7cfu/ml、5× 10 6cfu/ml的副溶血弧菌悬浮液 ,在注射后 2 4h、4 8h、72h、12 0h和 192h尾部取血 ,测定其血清中NOS活力的变化情况。结果表明 ,注射副溶血弧菌后斜带石斑鱼血清中NOS活力显著高于对照组 ,并且高浓度组NOS活力显著高于低浓度组 ,证明注射副溶血弧菌可以诱导斜带石斑鱼体内NOS活力的升高。     

苹果α-法尼烯合酶基因组结构和序列的多态性分析

通过设计引物进行PCR扩增、测序、拼接序列, 以及与GenBank中的cDNA序列(登录号AY182241) 进行比对, 获得α - 法尼烯合酶基因(AFS ) 的基因组序列和内含子/外显子结构。获得的AFS基因序列已在GenBank注册(登录号DQ901739) , 它有6个内含子和7个外显子, 编码一个大小为576个氨基酸的蛋白质。间隔外显子的6个内含子相位分别是0、1、2、2、0、0, 其大小分别是108、113、>1 000、125、220和88 bp, 7个外显子的推导氨基酸序列大小分别是57、89、127、73、48、83和99。编码的蛋白质中有3个序列模式(motif) , RR (X8) W 模式的编码基因序列在基因5′端的外显子1上, RxR 模式和DDxxD 模式的编码基因序列在外显子4上。将获得的AFS基因序列与GenBank中的cDNA序列(登录号AY523409) 进行比对, 结果发现两序列间有6个单核苷酸多态性, 其中4个引起氨基酸突变。有意义的是, 1个氨基酸突变(291R→G) 发生在RxR模式上, 此氨基酸突变以及其它突变是否与苹果α - 法尼烯合成能力和虎皮病发生能力有关, 值得进一步探讨。     

Imazethapyr inhibition of acetolactate synthase in Rhizobium and its symbiosis with pea

Acetolactate synthase (ALS) activity extracted from Rhizobium leguminosarum biovar. viciae has been characterized. The optimum pH for extraction was 7·6 and for the assay 7·0. The K_m for pyruvate was 7·2 mM , and the enzyme was saturated at 40 mM . An obligatory requirement of TPP and Mg²⁺ for full ALS activity was observed. Valine was the only branched-chain amino acid that caused ALS feedback inhibition. The specific activity of Rhizobium ALS was nearly 20 times the activity found in pea (Pisum sativum) leaves. Bacteroids from pea nodules also showed high ALS activity, and the nodule plant fraction had higher ALS activity than other plant tissues. ALS sensitivity to imazethapyr was also dependent on the source: ALS activity of free-living Rhizobium and bacteroids was slightly more tolerant than that of other pea tissues, but the differences were less than those found in rates of specific activity. It is proposed that the high ALS activity expressed by Rhizobium, both as free-living bacteria and as bacteroids, is related to the growth tolerance of rhizobia to imazethapyr and is also related to the relative tolerance of symbiotic pea plants. © 1998 SCI     

白藜芦醇合酶基因表达载体构建及对马铃薯的遗传转化

利用从葡萄中克隆到的白藜芦醇合酶基因(Resveratrol Synthase,简称RS)构建了含有35S组成型启动子的植物表达载体P35s-2300-RS,应用农杆菌介导方法对马铃薯品种进行遗传转化.通过对抗性芽、生根、再生植株的筛选,得到5株再生小苗.经PCR、Southern检测证实,有3株为真正的转基因植株.     

Dimethoxypyrimidines as Novel Herbicides. Part 2. Synthesis and Herbicidal Activity of O-Pyrimidinylsalicylates and Analogues

A new series of the O-pyrimidinylsalicylates was synthesized and their herbicidal activity was examined. Some of these compounds showed very strong herbicidal activity under pre- and post-emergent treatment conditions against various kinds of grass and broadleaf weeds. Among these compounds, O-(4, 6-dimethoxypyrimidin-2-yl) salicylic acid and its methyl ester were found to exhibit the highest activity. The herbicidal symptoms observed after the treatments included early cessation of plant growth followed by chlorosis, necrosis and plant death. The symptoms were similar to those caused by sulfonylureas and imidazolinones, which inhibit branched-chain amino acid biosynthesis.     

富有柿果实ACC合成酶3′末端的cDNA克隆

利用3’RACE的方法对柿果实ACC合成酶基因的3’末端进行扩增,扩增产物克隆到pMD18－T Vector载体上,转化到大肠杆菌感受态细胞DH5a,蓝白斑筛选重组质粒,并对插入片段进行序列测定。结果表明,3’RACE产物长912bp,开放阅读框内核苷酸序列编码251个氨基酸,此氨基酸序列含有ACC合成酶中3个高度保守区,GenBank中登录的DK－ACS1（登录号AB073005）序列只有一个的氨基酸不同。终止子后面的3’非编码区的长度为155bp,其中只有一个核苷酸与DK—ACS1的3’序列非编码区不同。序列分析结果显示,克隆的片段含有5’端GSP Inner Primer和3’端为RACE Inner Primer的引物。     

瓯柑NIN和Sus基因克隆、序列分析与表达

以瓯柑果肉为材料,分离瓯柑中性转化酶（NIN）和蔗糖合成酶基因（Sus）,了解其序列特征,并对瓯柑NIN,Sus在不同组织的表达进行分析。结果显示：该研究首次从瓯柑果实中克隆获得NIN和Sus基因CDS全长序列,分别为1 932 bp和2 418 bp,预测分别编码643和805个氨基酸。序列比对结果显示：3个基因均高度保守,与NCBI网站上其他物种的序列同源性均高于70%,与柑橘类物种的同源性接近100%。NIN和Sus蛋白的相对分子量分别为72.18和92.19 ku,理论等电点（pI）分别为6.882和6.051。系统进化树结果显示：瓯柑NIN属于α组,推测可能定位于质体;瓯柑Sus属于Sus Ⅰ组。实时荧光定量PCR结果显示：瓯柑NIN,Sus基因在茎中表达量均最高,叶其次,果肉中表达量最低;NIN在果皮中的表达量比果肉高,Sus则处于相同水平。在果实不同发育阶段,NIN,Sus基因均在花后120 d表达最强,随后下降,接近成熟表达维持在较低的水平。结果表明：2个基因在瓯柑不同组织间的表达具有不同程度的差异性,在果实发育前期表达水平略高,随着成熟表达水平降低。     

Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes

Considering that 70% of our planet’s surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs) and polyketides (PKs) are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes) and type-I polyketide synthases (PKSes-I), respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS) technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds.