Triosephosphate isomerase, a cytosolic glycolytic enzyme, plays an important role in XA3/XA26-mediated innate immunity by modulating the reactive oxygen species level in rice. Bacterial blight caused by Xanthomonas oryzae pv oryzae (Xoo) causes severe damage to rice (Oryza sativa) production worldwide. The major disease resistance gene, Xa3/Xa26, confers broad-spectrum and durable resistance to Xoo at both seedling and adult stages. However, the molecular mechanism of the Xa3/Xa26-initiated defense pathway against Xoo is still largely unknown. Here, we show that a triosephosphate isomerase (TPI), OsTPI1.1, is a key component in XA3/XA26-mediated resistance to Xoo. OsTPI1.1 is a glycolytic enzyme that catalyzes the reversible interconversion of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate. Transcriptional suppression of OsTPI1.1 in plants harboring Xa3/Xa26 largely impaired the XA3/XA26-mediated resistance to Xoo, and constitutive overexpression of OsTPI1.1 in susceptible rice plants without Xa3/Xa26 only slightly decreased the susceptibility to Xoo. Therefore, both XA3/XA26 and OsTPI1.1 are required in XA3/XA26-mediated resistance. We show that OsTPI1.1 participates in the resistance through its enzymatic activity, which was enhanced significantly by its binding with XA3/XA26. Reactive oxygen species (ROS), especially hydrogen peroxide, accumulated in the OsTPI1.1-overexpressing plants, and suppression of OsTPI1.1 decreased ROS accumulation. The changes in ROS are associated with the reduction of NADP+ to NADPH, which may act as a redox cofactor to scavenge ROS, leading to reduced resistance to Xoo. These results suggest that OsTPI1.1 modulates ROS production as a resistance mechanism against Xoo.

Bacterial blight caused by Xanthomonas oryzae pv oryzae (Xoo) causes severe damage to rice (Oryza sativa) production worldwide. The major disease resistance gene, Xa3/Xa26, confers broad-spectrum and durable resistance to Xoo at both seedling and adult stages. However, the molecular mechanism of the Xa3/Xa26-initiated defense pathway against Xoo is still largely unknown. Here, we show that a triosephosphate isomerase (TPI), OsTPI1.1, is a key component in XA3/XA26-mediated resistance to Xoo. OsTPI1.1 is a glycolytic enzyme that catalyzes the reversible interconversion of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate. Transcriptional suppression of OsTPI1.1 in plants harboring Xa3/Xa26 largely impaired the XA3/XA26-mediated resistance to Xoo, and constitutive overexpression of OsTPI1.1 in susceptible rice plants without Xa3/Xa26 only slightly decreased the susceptibility to Xoo. Therefore, both XA3/XA26 and OsTPI1.1 are required in XA3/XA26-mediated resistance. We show that OsTPI1.1 participates in the resistance through its enzymatic activity, which was enhanced significantly by its binding with XA3/XA26. Reactive oxygen species (ROS), especially hydrogen peroxide, accumulated in the OsTPI1.1-overexpressing plants, and suppression of OsTPI1.1 decreased ROS accumulation. The changes in ROS are associated with the reduction of NADP+ to NADPH, which may act as a redox cofactor to scavenge ROS, leading to reduced resistance to Xoo. These results suggest that OsTPI1.1 modulates ROS production as a resistance mechanism against Xoo.

BACKGROUND:As event-specific sequence information for most unauthorised genetically modified organisms (GMOs) is currently still unavailable, detecting unauthorised GMOs remains challenging. Here, we used insect-resistant rice TT51-1 as an example to develop a novel approach via detecting GMOs by RNA-seq (sequencing) and PCR. RNA-seq of TT51-1 generated 4.8 million (M) 21-nt cDNA tags. Alignment to the Oryza sativa subsp. japonica reference genome revealed 24 098 unmapped tags. Foreign tags from the nopaline synthetic enzyme gene (NOS) terminator and insect-resistant genes were then identified by searching against the NCBI VecScreen and NT databases.RESULTS:To further isolate foreign DNA sequences, putative NOS terminator and insect-resistant gene tags were combined and used directly as primer pairs for long-range PCR, producing a 5016-bp fragment. The inserted DNA sequence of TT51-1 has been submitted to a database, and thus, similarity analysis using the database could identify a test sample.CONCLUSION:The novel approach has a great potential for application to the detection and identification of unauthorised GMOs in food and feed products. © 2016 Society of Chemical Industry.

The major disease resistance gene Xa4 confers race-specific durable resistance against Xanthomonas oryzae pv. oryzae, which causes the most damaging bacterial disease in rice worldwide. Although Xa4 has been one of the most widely exploited resistance genes in rice production worldwide, its molecular nature remains unknown. Here we show that Xa4, encoding a cell wall-associated kinase, improves multiple traits of agronomic importance without compromising grain yield by strengthening the cell wall via promoting cellulose synthesis and suppressing cell wall loosening. Strengthening of the cell wall by Xa4 enhances resistance to bacterial infection, and also increases mechanical strength of the culm with slightly reduced plant height, which may improve lodging resistance of the rice plant. The simultaneous improvement of multiple agronomic traits conferred by Xa4 may account for its widespread and lasting utilization in rice breeding programmes globally.

在利用实时荧光定量PCR（Real-time quantitative PCR,qRT-PCR）检测转基因成分的含量时,数据分析的规范化与标准化对保证实验数据的准确性及实验室间数据的可比性具有重要意义.本研究以转基因玉米（Zea mays）NK603为研究材料,分析了分别以qRT-PCR中3次平行反应的单个Ct值和3次平行反应Ct值的平均值所绘制标准曲线的差异;分析了分别以盲样3次平行反应的Ct平均值和3次平行反应的拷贝数平均值（算术平均值或几何平均值）计算转基因成分含量的差异;并研究了结果准确性判定的方法.结果表明,标准曲线应基于单个Ct值;在计算转基因成分的含量时,Ct值应取算术平均值,拷贝数应取几何平均值;最后通过比较样品的测量结果与标准值之间的差异是否显著（U△＞△m,表明测量结果的平均值与样品的标准值无显著差别）来判断实验结果的可靠性.本研究为转基因产品检测中qRT-PCR实验数据处理的标准化提供了参考资料.

Accurate measurement of DNA concentration is important for DNA-based biological applications. DNA concentration is usually determined by the ultraviolet (UV) absorption, fluorescence staining, and diphenylamine reaction methods. However, the best method for quality assurance of measurements is unknown. Here, we comprehensively compared these methods using different types of samples. We found that all three methods accurately determined the concentrations of high-purity DNA solutions. After digestion of DNA samples, concentration measurements revealed that the PicoGreen dye method was very sensitive to the degradation of DNA. The three methods displayed different anti-jamming ability when contaminants such as transfer RNA (tRNA), protein, and organic chemicals were included in DNA solutions. The diphenylamine reaction method gave the highest accuracy, with an average error of approximately 10% between measured and true values. The PicoGreen dye method was influenced by tRNA and protein, and the UV absorption method was susceptible to all kinds of impurities. Overall, the diphenylamine reaction method gave the most accurate results when DNA was mixed with contaminants, the PicoGreen dye method was most suitable for degraded DNA samples or DNA extracted from processed products, and the UV absorbance method was best for evaluating the impurities in DNA solutions.

Plasmid calibrators are increasingly applied for polymerase chain reaction (PCR) analysis of genetically modified organisms (GMOs). To evaluate the commutability between plasmid DNA (pDNA) and genomic DNA (gDNA) as calibrators, a plasmid molecule, pBSTopas, was constructed, harboring a Topas 19/2 event-specific sequence and a partial sequence of the rapeseed reference gene CruA. Assays of the pDNA showed similar limits of detection (five copies for Topas 19/2 and CruA) and quantification (40 copies for Topas 19/2 and 20 for CruA) as those for the gDNA. Comparisons of plasmid and genomic standard curves indicated that the slopes, intercepts, and PCR efficiency for pBSTopas were significantly different from CRM Topas 19/2 gDNA for quantitative analysis of GMOs. Three correction methods were used to calibrate the quantitative analysis of control samples using pDNA as calibrators: model a, or coefficient value a (Cva); model b, or coefficient value b (Cvb); and the novel model c or coefficient formula (Cf). Cva and Cvb gave similar estimated values for the control samples, and the quantitative bias of the low concentration sample exceeded the acceptable range within ±25 % in two of the four repeats. Using Cfs to normalize the Ct values of test samples, the estimated values were very close to the reference values (bias −13.27 to 13.05 %). In the validation of control samples, model c was more appropriate than Cva or Cvb. The application of Cf allowed pBSTopas to substitute for Topas 19/2 gDNA as a calibrator to accurately quantify the GMO.

A new thermo-sensitive dominant genic male sterility (TSDGMS) line of Brassica napus was found and mapped in this paper. Our result will greatly accelerate the map-based cloning of the BntsMs gene.

对转基因玉米T25转化体特异性荧光定量PCR方法的关键参数进行了方法验证,选择了7家实验室采用经过验证的方法对3个水平的转基因玉米T25基体标准物质进行协同实验研究.通过比较分析各参与实验室的实验条件、标准曲线的参数,进一步证明该方法室间重复性好.经统计学分析各参与实验室的数据等精度,因此计算各实验室的总平均值做为标准物质的标准值.3个水平的基体标准物质的标准值和扩展不确定度(k=2)分别为1.17％±0.22％、2.17％±0.38％和9.81％±2.30％.

Plant non-specific lipid transfer proteins (nsLTPs) constitute large multigene families that possess complex physiological functions, many of which remain unclear. This study isolated and characterized the function of a lipid transfer protein gene, BraLTP1 from Brassica rapa, in the important oilseed crops Brassica napus. BraLTP1 encodes a predicted secretory protein, in the little known VI Class of nsLTP families. Overexpression of BnaLTP1 in B. napus caused abnormal green coloration and reduced wax deposition on leaves and detailed wax analysis revealed 17-80% reduction in various major wax components, which resulted in significant water-loss relative to wild type. BnaLTP1 overexpressing leaves exhibited morphological disfiguration and abaxially curled leaf edges, and leaf cross-sections revealed cell overproliferation that was correlated to increased cytokinin levels (tZ, tZR, iP, and iPR) in leaves and high expression of the cytokinin biosynthsis gene IPT3. BnaLTP1-overexpressing plants also displayed morphological disfiguration of flowers, with early-onset and elongated carpel development and outwardly curled stamen. This was consistent with altered expression of a a number of ABC model genes related to flower development. Together, these results suggest that BraLTP1 is a new nsLTP gene involved in wax production or deposition, with additional direct or indirect effects on cell division and flower development.

Five targets,including the CaMV35S promoter,NOS terminator,Bar gene,HPT gene and NPT Ⅱ gene were selected to be detecting targets of transgenic rice screening,based on the application frequency of the regulation elements and function genes used in rice(Oryza sativa)genetic engineering research.A rice reference geneSPS,five screening targets(CaMV35S promoter,NOS terminator,Bargene,HPTgene andNPT Ⅱgene)and three event-specific detection fragments(TT51-1,KF-6,KMD1)were fused using a overlap extension PCR technique and cloned into a plasmid vector pBluescript II SK+ to construct pBS Rice.The results showed that the plasmid molecules pBS Rice could be applied not only for the screening of genetically modified rice,but also in the event-specific detections of insect-resistant rice TT51-1,KF-6 and KMD1.This study provides a raw materials independent positive control with a coverage rate of 100% for the worldwide approved rice events,meeting the needs of the transgenic rice security supervision.

In this study, a collaborative trial of validating a real-time PCR method for the TT51-1 rice event was organized, including six participating laboratories. In this validation, serially diluted solutions from homogeneous genomic DNA of the TT51-1 event were used to construct standard curves of the TT51-1 event and phospholipase D (PLD) assays. The PCR efficiency was 95%, and the R-2 coefficient was 0.99 for the TT51-1 system. The mean quantitative values for blind samples containing 0.1%, 0.5% 1%, 5%, and 10% (w/w) TT51-1 corresponded to 0.1%, 0.51%, 1.06%, 4.83%, and 9.62%, respectively, with a bias (%) ranging from -3.77% to 5.87%. The repeatability and reproducibility were all below 25% across the entire dynamic range. Furthermore, the measurement uncertainties of the quantitative results were estimated to be 0.10%, 0.20%, 0.40%, 1.76%, and 3.52% (w/w) for the tested samples. Both the LOD and LOQ were calculated to be 0.22%. This collaborative trial demonstrated that the TT51-1 method produces reliable, comparable, and reproducible results for a given sample set and can be adopted as a detection standard for testing laboratories.

KCS10 gene is one of the members of the β-ketoacyl-CoA synthase(β-ketoacyl coa synthase,KCS) gene family.To understand the function of KCS10 gene,genome walking and RACE techniques were used to clone BnKCS10 gene from B.napus cv.Zhongshuang 9.The BnKCS10 gene has a 2 344bp-coding region containing 2 introns spanning 527bp and 167bp respectively.BnKCS10 gene expressed at significantly higher levels in flower buds,and also in stigma and 30d developing seeds than in the vegetative organs.The yeast cells tranformed with BnKCS10 gene produced KCS10 protein normally,but no synthesis of very long chain fatty acids was detected.

By the feature of easy acquisition,low cost and short cycle,plasmid reference materials could be used for GMO quantifications.In this experiment,real-time quantitative PCR was used to quantify GMO rapeseed TOPAS 19/2 collaboratively with 7 laboratories.The genome substitutability,collaborative experiment and uncertainty evaluation were carried out on transgenic rapeseed TOPAS19/2 plasmid molecule.T test showed that there were no significant differences between the slope of standard curve and linear correlation coefficient produced by endogenous and exogenous genes of two standards.The statistical analysis conducted on several laboratories′ fixed data showed that the quantity value of Rapeseed TOPAS 19/2 was 0.910 with an uncertainty of 0.013(K=2).

To standardize the rice-specific PCR detection methods, five previously reported rice (Oryza sativa) taxon-specific genes were compared and evaluated. The investigated genes included the rice root-specific gene (gos9), the ppi phosphofructokinase gene (ppi-PPF), the phospholipase D gene (PLD), the starch branching enzyme gene (RBE4) and the sucrose phosphate synthase gene (SPS). Sequencing analyses revealed that among the tested rice cultivars, single-nucleotide polymorphisms (SNPs) existed in the gos9, PLD, ppi-PPF and SPS amplicons, though no statistically significant effect on their Ct values was found. The ppi-PPF and PLD systems were found to produce amplicons in non-rice species, such as sugarcane and broomcorn. The quantitative real-time PCR results revealed that this cross-reaction led to an underestimate of the GM (genetically modified) rice content. With the exception of the aforementioned shortcomings, these five endogenous reference genes all have acceptable amplification efficiencies, which ranged from 98 to 108 %, and high sensitivity within the limit of detection (LOD) values, which ranged from 5 to 10 copies of the haploid genome. In estimating the GM content in blinded rice samples, these five systems produce relatively accurate quantitative results with deviations less than 15 %, but the RBE4 system produced the most accurate quantitative results. Therefore, we have determined that the RBE4 gene is the most suitable rice reference gene, and the gos9 and SPS genes can also be used as rice reference genes because they have good commutability with the RBE4 gene. Care should be taken when interpreting results based on the PLD and ppi-PPF genes owing to their cross-reaction with other species.

Based on inserted gene elements information for commercial domestic and foreign events in transgenic rapeseed,a matrix of molecular characteristics from transgenic rapeseed was established to investigate the gene frequencies.A screening strategy was developed for detecting complex molecular structure in transgenic rapeseed events based on element frequencies.Results showed that the detection rate could reach 100% for existing transgenic rapeseed events using the combination of P-CaMV35S,P-FMV35S and bar.When using element set of P-CaMV35S,P-FMV35S,bar,CP4-EPSPS,PAT and T-NOS,every transgenic event was detected more than once.Sequence alignments of the synonymous elements used in transgenic events were also analysed which improved the reliability of the matrix screening detection method.

GenomeWalking together with RACE techniques were used to clone KCS6 genes from B.napus cv.Zhongshuang 9.Two copies of KCS6 gene were found in B.napus,depending on their ancestral origin.One was BnKCS6c,showing 99% identity to corresponding B.oleracea mRNA.The other was BnKCS6a showing 99% identity to corresponding B.rapa mRNA.Both genes had a 1 494bp-coding region composed by 2 exons spanning 1 045bp and 449bp respectively.In early stage of seed development,KCS6 expression level was high in silique shell and seeds,but rapidly reduced when seeds matured.Both transgenic KCS6 genes could be normally translated into proteins in yeast cells,but no synthesis of very long chain fatty acids was observed.It indicated that the heterologous expressed KCS6 protein has no catalytic activity,or KCS6 protein is not able to utilize the yeast fatty acid as catalytic substrate to synthesize very long chain fatty acid.