Progress in the genetic manipulation of crops aimed at changing starch structure and increasing starch accumulation

The starch content and its composition have important consequences for the yield of the harvested crop and the materials extracted from it. The functional properties of the foods or other processed materials derived from these crops are also affected by the structure and composition of the starch. Recently, genetic engineering has been used to produce plants with an elevated starch content, achieved by transforming the plant with a mutated bacterial gene coding for an ADPglucose pyrophosphorylase that is active in the presence of metabolites which inhibit the plant enzyme. Besides the practical implications of these results, this experiment provided direct evidence for the regulatory role of the ADPglucose pyrophosphorylase in starch synthesis. Other bacterial enzymes, such as glycogen synthase and branching enzyme, could be introduced in order to modify starch structure. However, a more elegant (but longer-term) approach would be to learn enough about the structure-function relationships of the plant enzymes so that the product of their action could be changed. To achieve this objective, much more will have to be learned about the enzymes involved in the biosynthesis of starch than is presently known. Here, the basic properties of starch and the current research approaches to understanding its biosynthesis are described, together with a perspective of how genetic manipulation of starch structure may be achieved.Paper presented at the Bio/Environmentally Degradable Polymer Society—Third National Meeting, June 6–8, 1994, Boston, Massachusetts.     

一氧化氮合酶途径参与SO_2胁迫下蚕豆气孔运动调节

以蚕豆为材料,研究一氧化氮合酶(NOS)途径在SO2诱发气孔运动中的作用.研究发现:浓度7.5~200μmol·L-1的SO2衍生物处理后,蚕豆叶面气孔开度减小,气孔开度与SO2衍生物浓度呈负相关;SO2衍生物处理组叶组织中NOS活性增强;加入NO清除剂c-PTIO或NOS抑制剂L-NAME可抑制SO2衍生物诱发的气孔关闭;SO2衍生物处理组保卫细胞内NO和Ca2+水平升高,用c-PTIO降低胞内NO水平后Ca2+水平随之下降.结果表明,SO2衍生物胁迫可诱发保卫细胞内NO合成增加,NO通过调节胞内Ca2+水平升高,激活下游信号转导途径,调节气孔运动;NOS途径介导的NO合成参与了SO2胁迫下蚕豆气孔运动的调节.     

Cloning and expression analysis of geosmin synthase gene from Aphanizomenon gracile北大核心CSCD

Geosmin is a secondary metabolite responsible for earthy odors. The occurrence of geosmin has great impact on the quality of water environment. The gene essential for geosmin biosynthesis have been identified in several species. But little is known about the mechanism of geosmin synthesis in Aphanizomenon gracile. This study attempted to clone the gene involved in geosmin biosynthesis of Aphanizomenon gracile a nd a nalyze t he geosmin production u nder d ifferent e nvironments. T he high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) was used to amplify the full-length of geosmin synthase gene from Aphanizomenon gracile (WH-1). Real time PCR (RT-PCR) was applied to quantify the geosmin production in different light and temperature. As a result, geo, a geosmin synthase gene from Aphanizomenon gracile (WH-1) was cloned by hiTAIL-PCR. The full-length open reading frame (ORF) of geo was 2 262 bp, coding for a protein of 753 amino acids. Meanwhile, WH-1 was treated with different environment conditions and mRNA expression levels of geo were quantified by RT-PCR. It was found that low temperature (15 °C), high light intensity (35 μmol m-2 s-1) and continuous light illumination were beneficial to the expression of geo. The successful amplification of geosmin synthase gene verified that hiTAIL-PCR is an effective and simple procedure of low cost. The result provides fundamental knowledge on the monitoring and prevention for odorants.     

慢生型大豆根瘤菌(Bradyrhizobium japonicum)聚羟丁酸合成酶基因(phbC)突变体的构建及其特性

通过转座子Tn5诱变和同源重组，构建了Bradyrhizobium japonicum USDA110聚羟丁酸合成酶基因(phbC)突变体．序列测定确定了转座子插入的精确位置，所获得的4个转座子诱变的质粒其Tn5插在phbC基因内两个相距仅9bp的位点．被Southern和PCR证实的突变体菌株仍能产生相当于野生型菌株12．97％—25．10％的PHB，并且在突变体和野生型菌株总DNA杂交图上都呈现出一条约5kb的阳性带，推测在B.japonicum基因组中存在不止一个聚羟丁酸合成酶基因．图3表4参17     

Pseudomonas sp.L19抗除草剂AHAS基因的克隆、表达及特性

乙酰羟酸合酶(AHAS)是磺酰脲类、咪唑啉酮类、三唑嘧啶类、磺酰胺类和嘧啶水杨酸类等乙酰羟酸合酶抑制剂类除草剂的作用靶标,获得能抗此类除草剂的AHAS突变基因资源具有非常重要的理论和应用价值.从长期使用甲磺隆的农田土壤中分离到1株对乙酰羟酸合酶抑制剂类除草剂有广谱抗性的菌株L19,根据表型特征、生理生化特性和16S rDNA序列系统发育分析,将其鉴定为假单孢菌属(Pseudomonas sp.).利用PCR技术从Pseudomonas sp.L19的总DNA中克隆AHAS基因,氨基酸序列比对结果表明,L19与对除草剂敏感菌株P.putida KT2440的AHAS调节亚基氨基酸序列完全相同,而催化亚基有4个氨基酸位点不同:[Val 29→Ala(L19→KT2440),Pro93→Ser,Val 345→Ala,Pro 484→Arg].分别将菌株L19与KT2440的AHAS催化亚基克隆到pET29a(+)的多克隆位点,构建了表达载体pET-L19AHASc和pET-KT2440AHASc,通过镍柱亲和层析纯化得到带有组氨酸标签的乙酰羟酸合酶.抗性试验结果表明菌株L19的乙酰羟酸合酶对四大类乙酰羟酸合酶抑制剂类除草剂的抗性均要强于KT2440的乙酰羟酸合酶,对甲磺隆、咪唑乙烟酸、唑嘧磺草胺和嘧草醚的抗性倍数分别达到53.6、9.3、8.2和9.5倍.菌株L19的乙酰羟酸合酶比活力、对ThDP和Mg2+的Kc值相应地比KT2440乙酰羟酸合酶的要低;而对底物丙酮酸钠的米氏常数Km值要比KT2440乙酰羟酸合酶的要高近1倍.     

酸雨对水稻不同生育期叶片叶绿体ATP酶的影响

酸雨对植物生长发育具有显著影响。然而,不同生长阶段植物对逆境胁迫的响应存在差异。以模拟酸雨胁迫水稻(Oryza sativa)(孕穗期、灌浆期),利用植物生理生化方法测定了水稻叶肉细胞叶绿体Mg2+-ATPase活性及其基因表达量、光合磷酸化活性、ATP含量、植株相对生长速率(RGR)和叶片净光合速率(Pn),研究酸雨胁迫下孕穗期、灌浆期水稻叶绿体ATP酶的变化及其对植物光合作用影响的机理。结果表明,p H=4.5酸雨使孕穗期水稻RGR、Pn、叶片ATP质量比和光合磷酸化活性均显著提高,灌浆期上述指标变化不明显;p H=3.5和p H=2.5酸雨使上述指标均下降,且随酸雨p H值降低,变幅增加。p H=4.5酸雨使孕穗期、灌浆期水稻ATP合酶基因表达量及Mg2+-ATPase活性升高;p H=3.5和p H=2.5的酸雨使孕穗期、灌浆期水稻ATP酶基因表达量减少,造成Mg2+-ATPase活性降低,且随酸雨p H值降低变幅增加。可以认为,酸雨通过影响ATP酶转录水平,改变Mg2+-ATPase活性、光合磷酸化活性和叶片ATP质量比,进而影响Pn,并最终影响植物生长发育。酸雨胁迫对孕穗期水稻生长发育及光合作用有低促高抑的剂量效应,而低强度酸雨对灌浆期水稻影响不明显。与灌浆期相比,孕穗期对酸雨胁迫较敏感,表明植物不同生长发育阶段对酸雨胁迫响应存在差异,此发现应成为评价酸雨影响植物的一个因素。     

Microcystin-LR inhibited hippocampal long-term potential via regulation of the glycogen synthase kinase-3β pathway

We previously demonstrated that Cyanobacteria-derived microcystin-LR (MCLR) is able to induce cognitive dysfunction, but the mechanism is not understood. Long-term potential (LTP) in hippocampus is regarded as an important cellular mechanism of learning and memory. Here, the aim of this study was to evaluate the role of MCLR in LTP of hippocampal dentate gyrus (DG) by in vivo electrophysiological recording. We found that MCLR could suppress the induction of LTP in rat hippocampus, whereas simultaneous inhibition of glycogen synthase kinase-3β (GSK-3β) by LiCl or SB216763 attenuated the LTP impairments by MCLR. Furthermore, a decrease of the phosphorylated level at Ser9 of GSK-3β was observed by western blotting after intracerebroventricular (ICV) injection of MCLR, indicating GSK-3β was activated by MCLR. In addition, we showed that ICV administration of MCLR slightly stimulated activity of protein phosphatases (PPs) in the brain, which might activate GSK-3β via dephosphorylation of Ser9 site. Taken together, these findings demonstrated that GSK-3β plays a crucial role in regulating MCLR-induced cognitive deficit.     

The Probability of a Horizontal Gene Transfer from Roundup Ready® Soybean to Root Symbiotic Bacteria: A Risk Assessment Study on the GSF Lysimeter Station

The gene transfer from glyphosate tolerant soybean to Bradyrhizobium japonicum was evaluated in a free-air lysimeter experiment under natural conditions and increasing selection pressure, to monitor for the probability of horizontal gene transfer (HGT). A large volume lysimeter study that offers conditions comparable to normal farming was conducted in 2004 and 2005 with Roundup Ready® (RR) soybean and Roundup® application according to agricultural practice. Analysis of nodules showed, as expected, the presence of the transgenic 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). However, in bacteroids that were isolated from nodules and then cultivated for several rounds in the presence of high levels of glyphosate, the EPSPS gene could no longer be detected. This indicates no stable HGT transfer of the whole EPSPS gene under field conditions.     

Volatile components and their corresponding synthetic gene expression profile in the fruits of Actinidia chinensis北大核心CSCD

This study aimed to identify the difference in volatile aromatic components and the relationship with the expression pattern of their corresponding bio-synthesis genes in six kiwifruit (Actinidia chinensis) varieties (Cuiyu, Jintao, Jinyan, Chuhong, Donghong, and Xixuan). To provide a foundation for kiwifruit variety recognition, fruit quality evaluation, and molecular-assisted breeding, the volatile aromatic components in the ripe fruits of six kiwifruit varieties were evaluated by head space-solid phase micro extraction/gas chromatography-mass spectrometry. The aroma-synthesis-related genes, including acyltransferases (AcAT16), lipoxygenase (AcLox2), and terpene synthase genes (AcTPS1), were detected by the real time-quantitative polymerase chain reaction (qPCR) during the postharvest stage of fruits. Ninety-two aroma chemicals were identified in the tested kiwifruit cultivars. There were 35, 32, 30, 44, 28, and 17 of aromatic compounds in Cuiyu, Jintao, Jinyan, Chuhong, Donghong, and Xixuan, respectively. Esters were the main aroma components in Cuiyu, Jintao, and Jinyan. The major aromatic compounds of Chuhong, Donghong, and Xixuan were aldehydes and terpenoids. The expression level of AcATs16 and AcLox2 increased, and then decreased during the ripening of kiwifruit fruits. The expression of AcATs16 was significantly higher in Cuiyu and Jinyan than in other varieties. AcLox2 indicated significant abundance in Cuiyu and Chuhong. AcTPS1 was up-regulated in Jintao, Donghong, and Xixuan with fruit ripening; however, this was not observed in Cuiyu, Chuhong, and Jinyan. The difference in the composition and content of volatile aromatic components contributes to the difference in aroma in different kiwifruit cultivars. The diverse expression of AcATs16, AcLox2, and AcTPS1 might be closely related to the synthesis of ethyl butyrate, (E)-2-hexenal, and eucalyptol, respectively. © 2018 Science Press. All rights reserved.     

Enantioselective effects of herbicide imazapyr on Arabidopsis thaliana

The enantioselective toxicity of chiral herbicides in the environment is of increasing concern. To investigate the enantioselective effects of the chiral herbicide imazapyr on target organisms, we exposed Arabidopsis thaliana to imazapyr enantiomers and racemate. The results show that imazapyr was enantioselectively toxic to A. thaliana. The total chlorophyll content in A. thaliana was affected more by (+)-imazapyr than (±)-imazapyr and (?)-imazapyr. Concentrations of proline and malondialdehyde reflected a toxic effect in the order of (+)-imazapyr > (±)-imazapyr > (?)-imazapyr at every concentration. Acetolactate synthase (ALS) activity was inhibited more by (+)-imazapyr than (±)-imazapyr or (?)-imazapyr. At 100 mg L^?1 of imazapyr, ALS activity was 78%, 43%, and 19% with (?)-, (±)-, and (+)-imazapyr, respectively. The results suggest the significant enantioselective toxicity of imazapyr in A. thaliana for greater toxicity with (+)-imazapyr than (±)-imazapyr and (?)-imazapyr, which suggests that (+)-imazapyr has more herbicidal effect.