A novel potential primary method for quantification of enantiomers by high performance liquid chromatography-circular dichroism.

Primary methods play an important role in metrology. They can be used for the value assignment of certified reference materials, enabling the accuracy and comparability of the measurement. A novel potential primary method for enantiomer quantitation based on high-performance liquid chromatography-circular dichroism is described using L-phenylalanine as an example. The optimal quantitation range of L-Phe was from 0.1 mg/g to 1.2 mg/g, where both the relative bias and method variance were lower than 1%. The LOD and LOQ were 4 µg/g and 30 µg/g, respectively. The proposed method was also applied to the determination of the mass fraction of pure porcine insulin in solid. The average mass fraction obtained was 0.922 g/g with a RSD of 1.5%, and the associated relative uncertainty is 3.8% (k = 2), which agreed well with that obtained from the traditional isotope dilution mass spectrometry method. The LOD and LOQ for insulin quantitation were found to be 0.12 mg/g and 0.44 mg/g, respectively. The proposed method can be entirely described and understood by equations and a complete uncertainty statement can be defined in SI units.Therefore, it may be a potential primary method useful for the quantification of chiral compounds and proteins, and a supplementary method to the traditional isotope dilution mass spectrometry approach.

SI-traceable calibration-free analysis for the active concentration of G2-EPSPS protein using surface plasmon resonance.

Active proteins play important roles in the function regulation of human bodies and attract much interest for use in pharmaceuticals and clinical diagnostics. However, the lack of primary methods to analyze active proteins means there is currently no metrology standard for active protein measurement. In recent years, calibration-free concentration analysis (CFCA), which is based on surface plasmon resonance (SPR) technology, has been proposed to determine the active concentration of proteins that have specific binding activity with a binding partner without any higher order standards. The CFCA experiment observes the changes of binding rates at totally different two flow rates and uses the known diffusion coefficient of an analyte to calculate the active concentration of proteins, theoretically required, the binding process have to be under diffusion-limited conditions. Measuring the active concentration of G2-EPSPS protein by CFCA was proposed in this study. This method involves optimization of the regeneration buffer and preparation of chip surfaces for appropriate reaction conditions by immobilizing ligands (G2-EPSPS antibodies) on sensor chips (CM5) via amine coupling. The active concentration of G2-EPSPS was then determined by injection of G2-EPSPS protein samples and running buffer over immobilized and reference chip surfaces at two different flow rates (5 and 100µLmin-1). The active concentration of G2-EPSPS was obtained after analyzing these sensorgrams with the 1:1 model. Using the determined active concentration of G2-EPSPS, the association, dissociation, and equilibrium constants of G2-EPSPS and its antibody were determined to be 2.18 ± 0.03 × 106M-1s-1, 5.79 ± 0.06 ×10-3s-1, and 2.65 ± 0.06 × 10-9M, respectively. The performance of the proposed method was evaluated. The within-day precisions were from 3.26% to 4.59%, and the between-day precision was 8.36%. The recovery rate of the method was from 97.46% to 104.34% in the concentration range of 1.5-8nM. The appropriate concentration range of G2-EPSPS in the proposed method was determined to be 1.5-8nM. The active G2-EPSPS protein concentration determined by our method was only 17.82% of that obtained by isotope dilution mass spectrometry, showing the active protein was only a small part of the total G2-EPSPS protein. The measurement principle of the proposed method can be clearly described by equations and the measurement result can be expressed in SI units. Therefore, the proposed method shows promise to become a primary method for active protein concentration measurement, which can benefit the development of certified reference materials for active proteins.

A Gaijin-like miniature inverted repeat transposable element is mobilized in rice during cell differentiation.

BACKGROUND: Miniature inverted repeat transposable element (MITE) is one type of transposable element (TE), which is largely found in eukaryotic genomes and involved in a wide variety of biological events. However, only few MITEs were proved to be currently active and their physiological function remains largely unknown. RESULTS: We found that the amplicon discrepancy of a gene locus LOC_Os01g0420 in different rice cultivar genomes was resulted from the existence of a member of Gaijin-like MITEs (mGing). This result indicated that mGing transposition was occurred at this gene locus. By using a modified transposon display (TD) analysis, the active transpositions of mGing were detected in rice Jiahua No. 1 genome under three conditions: in seedlings germinated from the seeds received a high dose γ-ray irradiation, in plantlets regenerated from anther-derived calli and from scutellum-derived calli, and were confirmed by PCR validation and sequencing. Sequence analysis revealed that single nucleotide polymorphisms (SNPs) or short additional DNA sequences at transposition sites post mGing transposition. It suggested that sequence modification was possibly taken place during mGing transposition. Furthermore, cell re-differentiation experiment showed that active transpositions of both mGing and mPing (another well studied MITE) were identified only in regenerated plantlets. CONCLUSIONS: It is for the first time that mGing active transposition was demonstrated under γ-ray irradiation or in cell re-differentiation process in rice. This newly identified active MITE will provide a foundation for further analysis of the roles of MITEs in biological process.

Isolation and characterization of defense response genes involved in neck blast resistance of rice.

Two cDNA libraries enriched for transcripts differentially expressed in plants of two rice lines with similar genetic backgrounds and same leaf blast resistance but different responses to neck blast using suppression subtractive hybridization (SSH). After differential screening and sequence analysis of the selected clones, 90 unique cDNA clones were found, of which 74 clones were with known functions according to the putative functions of their homologous genes in the database. They may be involved in pathogen response, signal transduction, transcription, etc. Expression differences of 17 out of the 26 selected cDNA clones in resistant and susceptible lines were confirmed by RT-PCR. Expression profilings of the 26 cDNA clones at the early stages after inoculation were also revealed by RT-PCR. This is the first report on the rice neck blast resistance at mRNA level and will facilitate the further study of genetic mechanism of neck blast resistance.

[Genetic analysis of starch branching enzyme activity in rice grain].

To make an genetic analysis of starch branching enzyme (Q enzyme) inheritance, which is important in catalyzing the formation of amylopectin and thereby raising the amylopectin/amylose ratio of starch in rice grains, a linkage map of 207 DNA markers were constructed by using 247 recombinant inbred lines derived from an indica-indica rice cross Zhenshan 97B/Milyang46. In 2002 and 2003, the activities of starch branching enzyme of the parents and 247 RILs were measured 10 d and 20 d respectively after flowering (Table 1). A total of 3 quantitative trait loci (QTLs) were detected to have significant additive effects on Q enzyme activities 10 d after flowering with 10% phenotypic variations explained for the three QTLs (Table 2, Fig.1). Meanwhile, qQ10-6 with significant QTL x environment interaction was detected. Epistasis analysis detected 5 and 2 significant additive-by-additive interactions for Q enzyme 10 d and 20 d respectively after flowering (Table 3), and three pairs of QTLs 10 d after flowering with significant epistasis x environment interactions were detected, explaining 3% to 12% of the phenotypic variation. The results showed that the environmental factors had obvious effect on the gene expression of Q enzyme activity in rice grain. Meanwhile, the location of qQ10-6 in relation to that of QTLs for gelatinization temperature, gene loci involved in ADPG pyrophosphorylase and soluble starch synthase were discussed with the help of a genetic map.

Genetic dissections of partial resistances to leaf and neck blast in rice (Oryza sativa L.).

In a recombinant inbred line (RIL) population of indica rice, two subpopulations composed of susceptible lines were selected for mapping of partial resistance to leaf blast with two isolates of the pathogen. A third subpopulation composed of susceptible lines with similar heading time was used for mapping of partial resistance to neck blast with a third isolate. The traits measured for partial resistance included diseased leaf area (DLA), lesion size (LS) and lesion number (LN) for leaf blast and lesion length (LL) and conidium amount (CA) for neck blast. A linkage map consisting of 168 DNA markers was constructed by using the whole RIL population. Quantitative trait loci (QTLs) conditioning these traits were determined at one-locus and two-locus levels. Eleven main-effect QTLs and 28 digenic interactions were detected by QTLMapper 1.01 b. Only three QTLs showing main effects were also involved in digenic interactions for the same trait. General contributions of epistatic QTLs of each trait ranged from 16.0% to 51.7%, while those of main-effect QTLs of each trait ranged from 4.7% to 38.8%. The general contributions of main-effect QTLs of most traits were smaller than those of epistatic QTLs, confirming the importance of epistasis as the genetic basis for complex traits. The general contributions of the main and epistatic effects of all QTLs detected for the two traits LL and CA of the partial resistance to neck blast reached 70.6% and 82.6% respectively, which obviously represented a major part of the genetic basis controlling partial resistance to neck blast. The results indicated the necessity for partial resistance mapping to use susceptible subpopulations where the interference of major resistance genes is avoided.

[Analysis of additive and AE interaction effects of QTLs controlling plant height, heading date and panicle number in rice (Oryza sativa L.)].

Plant height (PH), heading date (HD) and productive panicle number (PN) are important agronomic trait in rice. Appropriate plant height, heading date and panicle number are prerequisites for the desired high and stable yield level in rice breeding programs. A recombinant inbred line (RIL) population consisting of 304 individuals was derived from a cross between indica varieties Zhong156 and Gumei2, from which a linkage map consisting of 168 RFLP, SSLP, RAPD and RGA markers that distribute on all the 12 rice chromosomes was constructed, and covers 1447.9 cM of the rice genome. The parents and 304 F9 lines were grown in the paddy field in China National Rice Research Institute (CNRRI), Hangzhou, China in 2001. The experiments were carried out in two seasons followed a randomized complete block design. QTLMapper 1.01 was applied to detect QTLs and QTL x environment (QE) interaction for HD (heading data), PH (plant height) and PN (panicle number), and conditional mapping for PH and PN was performed as well. A total of 15 QTLs with significant additive effects were detected, among which 4 QTLs had significant QE interaction. Ten QTLs with additive x additive epistatic effects for PH, HD and PN were detected, among which none showed significant epistatisis x environment interaction. These QTLs explained 12.12%, 1.38% and 5.00% of the total phenotypic variance for PH, HD and PN, respectively, and contributions were generally lower due to the strong epistatic effects. In conditional QTL analysis, the numbers of QTLs showing significant additive and epistatic effects were 7 and 6 for PH, and 3 and 3 for PN, respectively. Among the QTLs having significant additive effects for PH, qPH7-2 showed both additive effects and QE interaction, qPH7-1 and qPH10 showed QE interaction only, and the remaining 4 QTLs showed additive effects only. Each of the 3 QTLs having significant additive effects for PN did not display significant QE interaction. No epistatic QE interactions was detected. In addition, conditional QTL analysis indicated that the expression of QTLs for PH and PN may vary depending on the QTLs for HD.

[Molecular biological study on the action mechanism of rice allelochemicals against weeds].

Rice allelopathy is implemented through its release of allelochemicals to environment. Many researchers considered that rice allelochemicals were phenolics. The action mechanisms of rice against weeds allelochemicals included the inhibition of seed germination and emergence, the effect on the balance of hormones, the damage on the integrity of cell membrane systems, the effect on photosynthesis and respiration, the disturbance of nutrient and water uptake and the effect on the protein synthesis and gene expression. Rice allelopathy is controlled by polygenes, and inherited quantitatively. Several QTLs were identified by the methods of molecular biological techniques and allelopathic bioassay. It might be the important research work to locate the QTLs accurately and to clone the allelopathic genes with the method of marker-assisted selection and the near isogenic lines.

[QTLs mapping of leaf traits and root vitality in a recombinant inbred line population of rice].

By employing a recombinant inbred line (RIL) population of 247 lines derived from an inidca-indica cross Zhenshan 97B x Milyang 46, a linkage map consisting of 158 DNA markers was constructed and used for the determination of QTLs conditioning five leaf traits and root exudates. The leaf traits analyzed were leaf area, leaf length, leaf width, leaf perimeter and leaf length/width ratio measured on top first leaf, top second and top third leaves. The RIL population showed transgressive segregation on each trait, and highly significant or significant positive correlations were observed between all traits except between leaf length and width, and between leaf perimeter and length/width ratio. A total of 24 QTLs located in 9 intervals were detected to have significant additive effects for leaf traits analyzed, with LOD scores ranging 2.9-11.8 and 4.0%-32.5% phenotypic variation explained for a single QTL. Clustering of QTLs for leaf traits was evident. In interval RM197-RZ516 on chromosome 6, 2 QTLs for leaf length, 2 QTLs for leaf width and 3 QTLs for leaf length/width ratio of different leaves were detected, among which the alleles for increasing trait values were from Zhenshan 97B for leaf length and leaf length/width ratio, and from Milyang 46 for leaf width. In interval RM1-RG532 on chromosome 1,2 QTLs for leaf length and 2 QTLs for leaf perimeter were detected on top first leaf and top second leaf, respectively, and all the alleles for increasing trait values were from Milyang 46. In this interval, larger additive effects were observed for QTLs detected on top first leaf than at top second leaf. In interval RZ667-B10B on chromosome 6, a QTL for leaf perimeter of top second leaf was detected, accounting for 8.0% phenotypic variation. In intervals RZ66-RM264 and RG81-RM313 on chromosome 8 and 12, each QTL was detected for leaf length of top third leaf and explained for 9.0% and 15.3% phenotypic variation, respectively. Epistasis analysis detected 56 and 4 significant additive-by-additive interactions for leaf traits and root vitality, explaining 2.7% to 13.7% and 6.8% to 14.9% of the total phenotypic variation, respectively. On comparison with QTLs for yield traits detected in the same population previously, it was found that the majority of QTLs for leaf traits and root vitality and those for yield traits were located in similar intervals. Fine mapping of QTLs for both leaf and yield traits in these chromosomal regions would facilitate investigations of the source-sink relationship in rice.

[Mapping of fertility-restoring genes with main effects and epistatic effects for CMS-DA in rice].

A test-cross population was established for mapping genes conditioning fertility restoration for dwarf-wild-abortive cytoplasmic male sterility(CMS-DA) in rice. A recombinant inbred line(RIL) population consisting of 210 lines was derived from the cross Xieqingzao B/Miyang 46, in which Xieqingzao B is the maintainer line of CMS-DA Xieqingzao A, and Miyang 46 is the restorer line. Each of the RILs was crossed to Xieqingzao A, and the resulting F1s were used for phenotyping of the spikelet fertility in 1999 and 2000. A linkage map consisting of 129 RFLP and SSLP markers was constructed. QTLs having significant main effects and/or epistatic effects for spikelet fertility were determined with QTLMAPPER 1.0 of mixed linear model. Background genetic variation due to main and epistatic effects of important markers was controlled. Four QTLs having significant main effects for fertility restoration were detected. A major gene qRf-10-2 was located in interval RM258-RZ811 on the long arm of chromosome 10, and QTL qRf-1 displaying a moderate main effect was located in a position close to RG532 on chromosome 1. Two other QTLs were located on chromosome 5 (qRF-5) and the short arm of chromosome 10 (qRf-10-1), in which the main effect of qRf-5 bacame significant only in the absence of qRf-10-2. A significant digenic interaction was detected, which occurred between QTLs qRf-1 and qRf-5. No significant QTL by environmental interactions and epistasis by environmental interactions were detected. Analyses on the gene effects based on markers closest to the Rf genes were made, and multi-locus interactions were implied. The present results were also compared to a previous study on mapping fertility restoration genes in Milyang 46 for wild-abortive CMS Zhenshan 97A, and to other published reports. It was indicated that differences on the genetic control of fertility restoration among different rice populations were mainly attributed to variations on genes with minor main effects and epistatic effects.

[Analysis of gene expression profiles during host-Magnaporthe grisea interactions in a pair of near isogenic lines of rice].

A pair of near isogenic lines G205 and G71 were selected from recombinant inbred lines (RIL) of Zhong156 x Gumei2. On the resistance locus Pi-25(t), G205 had the resistant allele that was from Gumei 2 while G71 had the susceptible allele that was from Zhong156. For the genetic background, different alleles were detected on only 24 loci out of the 672 RFLP or SSLP loci surveyed. The expression profiles of G205 and G71 in response to Magnaporthe grisea were investigated using cDNA microarray containing 2200 Expression Sequence Tags (ESTs). The leaves were inoculated with the pathogen for 12 hours at 4-leaf stage and 998 genes were identified in total. Three genes were up-regulated significantly by the fungus in G205 only. The functions of two genes were known but that of the third gene were unknown. The two genes encoded casein kinase II alpha subunit and retrotransponson TOS17 insertion element respectively. Other thirty-five genes had similar expression patterns between NILs. Among them, 17 genes were up-regulated while 18 genes were down-regulated by the inoculation. The functions of 33 out of the 35 genes were known. BLAST analysis showed that all thirty-five. BLAST analysis showed that all thirty-five genes with known functions were relative to defense reactions, signal transduction, stress response, photosynthesis and sugar metabolism. Northern blot confirmed that four of five differentially displayed genes randomly selected had the same expression patterns as those detected in cDNA microarray. Two of them were up-regulated genes encoding casein kinase II alpha subunit and glycine-rich protein (Grp), and the other two down-regulated genes encoding nitrilase-associated protein and 18S small subnit ribosomal RNA gene respectively. Northern blot also revealed that the expression of Grp was consistently up-regulated from 0 to 36 h after the inoculation of the fungus. These results showed that cDNA microarray was a useful tool to study the molecular mechanisms of disease resistance in plants.