Rapid Generation of Selectable Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture

The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.

Construction of Marker-Free GFP Transgenic Tobacco by Cre/lox Site-Specific Recombination System

Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation. The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene retransformation. After removal of the Cre-NPT Ⅱ locus by genetic segregation through self-cross, plants that incorporated only the GFP transgene were obtained. Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained, which resisted herbicide Basta and GFP expressed well, then the Cre gene was subsequently introduced into 5 plants of them, respectively, by retransformation. The leaf disks from Cre transgenic plants were used to test the resistance to Basta on the medium with 8 mg L-1 of PPT. The results showed that few discs were able to regenerate normally, and the excision at 76-100% efficiency depended on individual retransformation events. Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basta sensitive plants. The result indicated that the excision event in the recombination sites was precise and conservative, without loss or alteration of any submarginal nucleotides of the recombination sites. Bar gene excised plants were selfpollinated to allow segregation of the GFP gene from the Cre-NPT Ⅱ locus. The progenies from self-pollinated plants were scored for Kan senstivity, then the segregation of GFP gene from Cre-NPT Ⅱ locus in the Kan senstive plants were confirmed by PCR analysis subsequently. Hence, constructing marker-free transgenic tobacco plants by Cre/lox sitespecific recombination system was reliable, and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.

Breeding of Selectable Marker-Free Transgenic Rice Lines Containing AP1 Gene with Enhanced Disease Resistance

In order to obtain marker-free transgenic rice with improved disease resistance, the AP1 gene of Capsicum annuum and hygromycin-resistance gene （HPT） were cloned into the two separate T-DNA regions of the binary vector pSB130, respectively, and introduced into the calli derived from the immature seeds of two elite japonica rice varieties, Guangling Xiangjing and Wuxiangjing 9, mediated by Agrobacterium-mediated transformation. Many cotransgenic rice lines containing both the AP1 gene and the marker gene were regenerated and the integration of both transgenes in the transgenic rice plants was confirmed by either PCR or Southern blotting technique. Several selectable marker-free transgenic rice plants were subsequently obtained from the progeny of the cotransformants, and confirmed by both PCR and Southern blotting analysis. These transgenic rice lines were tested in the field and their resistance to disease was carefully investigated, the results showed that after inoculation the resistance to either bacterial blight or sheath blight of the selected transgenic lines was improved when compared with those of wild type.

Genetic and agronomic traits stability of marker-free transgenic wheat plants generated from Agrobacterium-mediated co-transformation in T2 and T3 generations

Genetically modified wheat has not been commercially utilized in agriculture largely due to regulatory hurdles associated with traditional transformation methods. Development of marker-free transgenic wheat plants will help to facilitate biosafety evaluation and the eventual environmental release of transgenic wheat varieties. In this study, the marker-free transgenic wheat plants previously obtained by Agrobacterium-mediated co-transformation of double T-DNAs vector were identified by fluorescence in situ hybridization(FISH) in the T1 generation, and their genetic stability and agronomic traits were analyzed in T2 and T3 generations. FISH analysis indicated that the transgene often integrated into a position at the distal region of wheat chromosomes. Furthermore, we show that the GUS transgene was stably inherited in the marker-free transgenic plants in T1 to T3 generations. No significant differences in agronomic traits or grain characteristics were observed in T3 generation, with the exception of a small variation in spike length and grains per spike in a few lines. The selection marker of bar gene was not found in the transgenic plants through T1 to T3 generations. The results from this investigation lay a solid foundation for the potential application of the marker-free transgenic wheat plants achieved through the co-transformation of double T-DNAs vector by Agrobacterium in agriculture after biosafty evaluation.

Development of Marker-Free Transgenic Cry1Ab Rice with Lepidopteran Pest Resistance by Agrobacterium Mixture-Mediated Co-transformation

CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains （EHA105 and EHA101） contained Hpt and CrylAb genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and CrylAb genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T2 progenies were analyzed for CrylAb and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive （80.94%）, 62 plants were also CrylAb co-transformants （15.05% in total frequency）, and 42 plants among the 272 T2 progenies were CrylAb positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene Southern blot analysis of five independent marker-free T2 transgenic lines co-transformed from Zhejing 22 showed that CrylAb gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron.

Serum tumor markers (carcinoembryonic antigen, carbohydrate antigen 19-9, carbohydrate antigen 72-4, carbohydrate antigen 24-2, ferritin) and gastric cancer prognosis correlation

BACKGROUND Gastric cancer is a kind of malignant tumor which is prevalent all over the world.Although some progress has been made in the treatment of gastric cancer,its prognosis is still not optimistic,so it is of great significance to find reliable prog-nostic indicators to guide the treatment and management of patients with gastric cancer.AIM To explore the relationship between serum levels of five biomarkers[carcinoem-bryonic antigen(CEA),carbohydrate antigen(CA)19-9,CA72-4,CA24-2,and ferritin]and prognosis in patients with gastric cancer.METHODS This study included 200 patients with gastric adenocarcinoma,and conducted an in-depth analysis of their baseline characteristics,relationship between tumor markers and staging,and prognosis.The study found that CA19-9 has a signi-ficant correlation with tumor stage,the average levels of CA24-2,CEA,CA72-4 and ferritin were slightly increased disregarding the stage of tumor.Survival analysis showed that increases in CEA,CA19-9,CA24-2,and ferritin were all associated with shortened overall survival of patients.Further multivariate ana-lysis revealed that elevated serum CA72-4 levels were an inde-pendent adverse prognostic factor.RESULTS This study reveals that there is a significant correlation between the expression levels of serum tumor markers CEA,CA19-9,CA72-4,CA24-2 and ferritin in patients with gastric cancer and prognosis,and can be used as important indicators for prognostic evaluation of gastric cancer.In particular,markers that appear abnormally elevated initially may help identify gastric cancer patients with poor prognosis.CONCLUSION Serum CEA and CA19-9 play an important role in the prognosis assessment of gastric cancer,and are effective tools to guide clinical practice and optimize individualized treatment strategies for gastric cancer patients.

Development and drought tolerance assay of marker-free transgenic rice with OsAPX2 using biolistic particle-mediated co-transformation

Abiotic stresses such as drought, salinity, and low temperature cause–losses in rice production worldwide. The emergence of transgenic technology has enabled improvements in the drought resistance of rice plants and helped avert crop damage due to drought stress.Selectable marker genes conferring resistance to antibiotics or herbicides have been widely used to identify genetically modified plants. However, the use of such markers has limited the public acceptance of genetically modified organisms. Marker-free materials (i.e., those containing a single foreign gene) may be more easily accepted by the public and more likely to find common use. In the present study, we created marker-free drought-tolerant transgenic rice plants using particle bombardment. Overall, 842 T_0plants overexpressing the rice ascorbate peroxidase-coding gene OsAPX2 were generated. Eight independentmarker-free lines were identified from T_1 seedlings using the polymerase chain reaction.The molecular characteristics of these lines were examined, including the expression level,copy number, and flanking sequences of OsAPX2, in the T_2 progeny. A simulated drought test using polyethylene glycol and a drought-tolerance test of seedlings confirmed that the marker-free lines carrying OsAPX2 showed significantly improved drought tolerance in seedlings. In the field, the yield of the wild-type plant decreased by 60% under drought conditions compared with normal conditions. However, the transgenic line showed a yield loss of approximately 26%. The results demonstrated that marker-free transgenic lines significantly improved grain yield under drought-stressed conditions.

Efficiencies of Generating Selectable Marker-Free Transgenic Rice with Different Transformation Methods

To study the efficiency of generating selectable marker-free （SMF） transgenic rice, two transformation methods were employed for four rice varieties （Wuxiangjing 9, Longtefu, Xieqingzao and Zhenshan 97）. One method is by using a single twin T-DNA binary vector pYH592 in one Agrobacterium strain, which is composed of two separate T-DNA regions （one carrying an antisense Wx gene and the other carrying a HPTgene）. The other one, named as two-strain/two-vector system, is by using two separate binary vectors in two separate Agrobacterium cultures. The results indicated that the average co-transformation frequencies of the antisense Wx gene and the HPT gene were 10.1% and 45.0%, respectively, for the four rice varieties. And the SMF transgenic plants selected from the offsprings of co-transformants were 55.6% and 60.0% in the two-strain/two-vector and twin T-DNA vector binary systems, respectively.

Genetic Study on JS399-19 Resistance in Hyphal Fusion of Fusarium graminearum by Using Nitrate Nonutilizing Mutants as Genetic Markers

Twenty-two nitrate nonutilizing （nit） mutants were recovered from five wild-type isolates of Fusarium graminearum and fifty nit mutants were recovered from three JS399-19-resistant mutants of F. graminearum cultured on MMC medium. Some biological properties were compared between nit mutants and their parental isolates. The results showed that there were no significant differences in growth rate, cultural characters or pathogenicity between JS399-19-resistant nit mutants and their parental isolates. But the conidial production and the sexual reproduction ability changed to some extent. There was no cross resistance toward chlorate and JS399-19 in F. graminearum and the resistance could be stable through 20-time subcultures. Therefore, the nit could be used as a genetic marker for studying the genetics of JS399-19 resistance in E graminearum, which was used to study JS399-19 resistance transferability in hyphal fusion. Resistance in JS399-19 could not be transferred by hyphal fusion or could be transferred with low chance between two compatible isolates, which would delay the development of JS399-19 resistance in the field.

基于F-B NCP函数的可行QP-free算法

本文得到一种可行QP-free算法,引入ε-有效集策略使得每次迭代只需求解规模较小的线性方程组得到迭代方向,且方程组只包含工作集中的约束,其规模较原问题大大减小,同时不进行弧搜索,从而降低了运算量.

Process for improving the ladle free- opening rate for high aluminum steel in continuous casting

Steel 20Mn23AlV is a type of high aluminum steel with a very low ladle free-opening rate. The aluminum composition of 20Mn23AlV ranges from 1.6% to 2.45% ,which is significantly higher than other types of steel. According to the real condition of 40 t ladle in steel-making plant of Baosteel Special Steel Company, previous works show that the key factors affecting the ladle free-opening rate of high aluminum steel in continuous casting are：sand material, accessories baking, ladle nozzle cleaning, the process and amount of adding sand, and the rate of argon stirring during refining. Therefore, improving the ladle filler sand quality, baking all of the raw materials, controlling the addition of ladle filler sand, cleaning the ladle nozzle, and optimizing argon stirring during the refining process can resolve the problem of a low ladle free-opening rate of high aluminum steel caused by the long ladle time of liquid steel.

Development of Debris-free Laser Plasma Sources for EUV Lithography in CIOMP

We have been developing debris-free laser plasma sources for EUV lithography since 1996. Two types of debris-free sources, such as cryogenic target and gas-puff target laser plasma sources, were designed and built up in CIOMP. EUV radiation spectra of the sources with a variety of targets have been obtained by different ways.

基于Label-free技术的非小细胞肺癌蛋白质差异研究

目的 基于非小细胞肺癌(non-small-cell lung cancer,NSCLC)及与其配对的非癌性邻近组织(non-cancerous adjacent tissues,NATs)的蛋白质组学分析,寻找NSCLC诊断相关蛋白标志物。方法 应用非标记定量蛋白组学(LFP)技术,以NSCLC患者配对的NATs为对照,对5例NSCLC患者进行癌组织蛋白质组学分析。以差异倍数>1.5(上调)或<0.67(下调)且P<0.05为标准筛选差异蛋白。应用String网站和R语言对差异蛋白进行基因本体论(gene ontology,GO)分析、京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析,为进一步研究提供良好基础。结果 本研究发现差异蛋白共644个,其中339个蛋白表达上调,305个蛋白表达下调。PCA结果显示,差异蛋白可明显区分NSCLC与NATs。GO分析结果表明,差异蛋白大多以细胞外泌体的形式存在且主要富集于调节胞外分泌、胞外分泌、骨髓细胞激活参与免疫反应等的生物学过程以及钙粘着蛋白绑定、细胞外基质绑定等的分子功能。KEGG分析结果显示,差异蛋白主要富集在抗坏血酸和醛酸代谢、组氨酸代谢、蛋白质消化吸收、丙酮酸代谢等通路(P<0.05)。结论 NSCLC与NATs存在明显差异,可为发现NSCLC新型标志物提供线索。

Salicylic Acid Induced Self-excision of the Marker Gene npt Ⅱ Using CreloxP System from Transgenic Tobacco

Transgenic plants, despite a great deal of scientifi c evidences, have induced a number of environmental and consumer concerns for long time because various antibiotic resistance genes, according to common

Reliability of Passive Physical Activity Assessment for Inpatients Using Marker⁃Free Motion Tracking System

Evidence⁃based practices of public health will benefit from quantification of passive physical activity assessment.This study aims to investigate the reliability of marker⁃free system(MFS)such as Microsoft Kinect in measuring upper extremity motion from different angles.Ten healthy participants performed elbow and shoulder extension/flexion along frontal and median anatomical planes for ten pace⁃controlled repetitions,during which the spatiotemporal positions of upper extremity joints were concurrently recorded by two sensors from 0°and 45°viewing angles.Reliability between the two sensors were evaluated using Pearson correlation coefficient,intra⁃class correlation coefficients,and 95%limits of agreement and coefficient of variation.Worse reliability was observed when possibility of occlusion was higher.However,better reliability was found when longer observation interval(10 s)was used as elementary measuring unit than shorter observation interval(2 s).The overall angular reliability of activity as displacement or changes in angle was not satisfactory.The results are expected to inform the industry for the extension of MFS to clinic applications.

Electrochemical Enzyme Immunoassay of Tumor Marker CA15-3 with Capillary Electrophoresis

Tumor marker CA15-3 was determined by using capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED). The method can be used to detect CA15-3 with a limit of 0.024 U/mL.

Elevated soluble 4-1BB is associated with serum markers of hepatitis B virus in patients with chronic hepatitis B

BACKGROUND Previous studies have suggested that the costimulatory molecule 4-1BB plays pivotal roles in regulating immunity during chronic viral infection.However,up to now,there are few studies about 4-1BB in chronic hepatitis B(CHB).AIM To clarify this issue,we report our comprehensive study results on the expression levels of 4-1BB in patients with CHB.METHODS From September 2018 to June 2019,a total of 64 patients with CHB were recruited from the Department of Hepatology,The First Hospital of Jilin University.Peripheral blood samples were collected from 52 treatment-naïve and 12 entecavir-treated patients with CHB as well as 37 healthy donors(including 24 healthy adults and 13 healthy children).The levels of soluble 4-1BB(s4-1BB)in plasma were measured by ELISA.4-1BB mRNA expression in peripheral blood mononuclear cells was detected by real-time quantitative PCR.RESULTS The s4-1BB levels in the plasma of patients with CHB were significantly higher than those in healthy adults(94.390±7.393 ng/mL vs 8.875±0.914 ng/mL,P<0.001).In addition,the s4-1BB level in plasma was significantly increased in patients with a higher viral load and a disease flare up.However,there were no significant differences between treatment-naïve and entecavir-treated patients.Interestingly,among treatment-naïve patients with CHB,the levels of s4-1BB in plasma had a significant positive correlation with hepatitis B surface antigen,hepatitis B virus DNA,hepatitis B e antigen,and triglyceride levels(r=0.748,P<0.001;r=0.406,P=0.004;r=0.356,P=0.019 and r=-0.469,P=0.007,respectively).The 4-1BB mRNA expression was higher in the peripheral blood mononuclear cells of patients with CHB than in the peripheral blood mononuclear cells of healthy adults,but the difference was not statistically significant.CONCLUSION These results suggest that the levels of s4-1BB may be associated with pathogenesis of hepatitis B virus and therefore may be a promising biomarker for disease progression.

EXACT SOLUTION FOR RECTANGULAR SLAB WITH THREE EDGES SIMPLY-SUPPORTED AND OTHER FREE

In this paper,we give all-sided pastic analysis of the rectangular slab with three edges simply-supported and other free.Here we discuss the following four cases:(1)The uniformly distributedload over the area a slab.(2).A concentrated load act at midpoint of free edges slab.(3)A concen-trated load act at the center a slab.(4)The line load act along free edge of slab.

Influence of 10-(6-plastoquinonyl) decyltriphenylphosphonium on free-radical homeostasis in the heart and blood serum of rats with streptozotocin-induced hyperglycemia

BACKGROUND It is known that under conditions of tissue tolerance to insulin,observed during type 2 diabetes mellitus(DM2),there is an increased production of reactive oxygen species.Moreover,the free radicals can initiate lipid peroxidation(LPO)in lipoprotein particles.The concentration of LPO products can influence the state of insulin receptors,repressing their hormone connection activity,which is expressed as a reduction of the glucose consumption by cells.It is possible that reduction in glucose concentration during administration of 10-(6-plastoquinonyl)decyltriphenylphosphonium(SkQ1)to rats with DM2 may be related to the antioxidant properties of this substance.AIM To establish the influence of SkQ1 on free-radical homeostasis in the heart and blood serum of rats with streptozotocin-induced hyperglycemia.METHODS To induce hyperglycemia,rats were fed a high-fat diet for 1 mo and then administered two intra-abdominal injections of streptozotocin with a 7-d interval at a 30 mg/kg of animal weight dose with citrate buffer equal to pH 4.4.SkQ1 solution was administered intraperitoneally at a 1250 nmol/kg dose per day.Tissue samples were taken from control animals,animals with experimental hyperglycemia,rats with streptozotocin-induced glycemia that were administered SkQ1 solution,animals housed under standard vivarium conditions that were administered SkQ1,rats that were administered intraperitoneally citrate buffer equal to pH 4.4 once a week during 2 wk after 1-mo high-fat diet,and animals that were administered intraperitoneally with appropriate amount of solution without SkQ1(98%ethanol diluted eight times with normal saline solution).To determine the intensity of free radical oxidation and total antioxidant activity,we used the biochemiluminescence method.Aconitate hydratase(AH),superoxide dismutase,and catalase activities were estimated using the Hitachi U-1900 spectrophotometer supplied with software.The amount of citrate was determined by means of the Natelson method.Real-time polymerase chain reaction was carried out using an amplifier ANK-32.RESULTS It was found that the mitochondrial-directed antioxidant elicits decrease of biochemiluminescence parameter values that increase by pathology as well as the levels of primary products of LPO,such as diene conjugates and carbonyl compounds,which indicate intensity of free radical oxidation.At the same time,the activity of AH,considered a crucial target of free radicals,which decreased during experimental hyperglycemia,increased.Apparently,increasing activity of AH influenced the speed of citrate utilization,whose concentration decreased after administering SkQ1 by pathology.Moreover,the previously applied antioxidant during hyperglycemia influenced the rate of antioxidant system mobilization.Thus,superoxide dismutase and catalase activity,as well as the level of gene transcript under influence of SkQ1 at pathology,were changing to the direction of control groups values.CONCLUSION According to the results of performed research,SkQ1 can be considered a promising addition to be included in antioxidant therapy of DM2.

Serum tumor markers:Can they clinically implicate in type 2 diabetes mellitus?

“Serum tumor markers expression(CA19-9,CA242,and CEA)and its clinical implications in type 2 diabetes mellitus”authored by Meng and Shi presents an observational case-control study investigating the correlation between tumor markers and type 2 diabetes mellitus(T2DM).The study explores the diagnostic accuracy of tumor markers,particularly cancer antigen 19-9(CA19-9),CA242,and carcinoembryonic antigen,in poorly controlled T2DM patients with hemoglobin A1c levels exceeding 9%,employing receiver operating characteristic curve analysis.Though study offers valuable insights into the potential utility of tumor markers in clinical practice,caution is advised regarding routine tumor marker testing due to challenges such as limited availability and cost.Additionally,the study overlooks potential confounding factors like smoking and alcohol consumption.Variations in CA19-9 and CA242 expression underscore the complex interplay between tumor markers and systemic diseases,warranting further investigation into their diagnostic and prognostic implications.While Meng and Shi represent a significant contribution to the field,more extensive research is needed to fully elucidate the role of tumor markers in diabetes management and beyond.