MicroRNA-139 modulates Alzheimer's-associated pathogenesis in SAMP8 mice by targeting cannabinoid receptor type 2.

Alzheimer's disease (AD) is a neurodegenerative disorder, and is the most common type of dementia in the elderly population. Growing evidence indicates that microRNAs (miRNAs) play a crucial role in neuroinflammation associated with AD progression. In this study, we analyzed the expression of microRNA-139 (miR-139) as well as the learning and memory function in AD. We observed that the miR-139 expression was significantly higher in the hippocampus of aged senescence accelerated mouse prone 8 (SAMP8) mice (2.92 ± 0.13) than in the control mice (1.49 ± 0.08). Likewise, the overexpression of miR-139 by means of hippocampal injection impaired the hippocampus-dependent learning and memory formation. In contrast, the downregulation of miR-139 in mice improved learning and memory function in the mice. The level of cannabinoid receptor type 2 (CB2), a potential target gene of miR-139, was inversely correlated with the miR-139 expression in primary hippocampal cells. Furthermore, we demonstrated that miR-139 inversely modulated the responses to proinflammatory stimuli. Together, our findings demonstrate that miR-139 exerts a pathogenic effect in AD by modulating CB2-meditated neuroinflammatory processes.

QTLs for rice flag leaf traits in doubled haploid populations in different environments.

Two rice doubled haploid (DH) populations derived from the crosses of ZYQ8/JX17 and CJ06/TN1 were used to detect quantitative trait loci (QTLs) for flag leaf length (FLL), width (FLW), and angle (FLA) under long-day conditions in Hangzhou (subtropical zone) and short-day conditions in Hainan (tropical zone), China. The four parents differed significantly in all 3 traits. FLL was found to be positively correlated with FLW in the 2 populations. A total of 30 QTLs were identified for flag leaf traits, with a contribution to the phenotypic variation of each QTL from 4.49 to 26.30%. Among these, qFLL-4b, qFLW-12, and qFLA-2a showed larger additive effects on the phenotype and explained more variations compared to the other QTLs. qFLL-1a and qFLL-8 were detected in both environments, while qFLL-2, qFLL-3, qFLL-10, qFLL-12, qFLW11, qFLW2, and qFLA8 were novel QTLs, which may be beneficial to rice ideal-type breeding.

Microsatellites, single nucleotide polymorphisms and a sequence tagged site in starch-synthesizing genes in relation to starch physicochemical properties in nonwaxy rice (Oryza sativa L.).

Starch characteristics determine the quality of various products of rice, e.g., eating, cooking and processing qualities. Our previous study indicated that molecular markers inside or close to starch synthesizing genes can differentiate the starch properties of 56 waxy rices. Here we report microsatellite (or simple sequence repeat, SSR) polymorphism in the Waxy (Wx) gene, soluble starch synthase I gene (SS1) and starch branching enzyme 1 gene (SBE1), single nucleotide polymorphism (SNP) in Wx and starch branching enzyme 3 gene (SBE3), and a sequence tagged site (STS) in starch branching enzyme 1 gene (SBE1) among 499 nonwaxy rice samples and their relationships with starch physicochemical properties. The nonwaxy rice samples consist of landraces (n = 172) obtained from germplasm centers and cultivars and breeding lines (n = 327) obtained from various breeding programs. Ten (CT)( n ) microsatellite alleles, (CT)(8), (CT)(10), (CT)(11), (CT)(12, )(CT)(17), (CT)(18), (CT)(19), (CT)(20), (CT)(21), and (CT)(22), were found at the Wx locus, of which (CT)(11) was the most frequent, and (CT)(12), (CT)(21) and (CT)(22) were identified for the first time. Four (CT)( n ) microsatellite alleles were found at the SBE1 locus, (CT)(8), (CT)(9), and (CT)(10 )together with an insertion sequence of CTCTCGGGCGA, and (CT)(8) alone without the insertion, of which (CT)(9) and the insertion was a new allele identified in only one rice, IR1552. Multiple microsatellites clustered at the SS1 locus, and in addition to the three alleles previously detected (SSS-A = (AC)(2)...TCC(TC)(11)...(TC)(5)C(ACC)(11), SSS-B = (AC)(3)...TCT(TC)(6)...(TC)(4)C(ACC)(9), and SSS-C = (AC)(3)...TCT(TC)(6)...(TC)(4)C(ACC)(8)), one new allele (SSS-D = (AC)(2)...TCC(TC)(10)...(TC)(4)C(ACC)(9)) was found. Analysis of the starch physicochemical properties of the samples with different microsatellites, SNPs and STS groups indicated that these molecular markers can differentiate almost all the physicochemical properties examined, e.g., apparent amylose content (AAC), pasting viscosity characteristics, and gel textural properties. Wx SSR and Wx SNP alone explained more variations for all physicochemical properties than the other molecular markers. The total six markers could explain 92.2, 81 and 86% of total variation of AAC, gel hardness (HD), and gel cohesiveness (COH), respectively, and they could explain more than 40% of the total variation of hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BD), setback viscosity (SB) and gel adhesiveness (ADH). However, only 29% of the total variation of peak viscosity (PV) and 37% of pasting temperature (PT) could be explained by all the molecular markers. Some of these markers can differentiate the starch physicochemical properties among the rice samples with the same Wx allele, indicating that the variation within Wx allele classes can be explained by other starch synthesizing genes. These SSRs, SNPs and STS are useful in marker-assisted breeding for the improvement of starch quality of rice.

Nucleotide diversity in starch synthase IIa and validation of single nucleotide polymorphisms in relation to starch gelatinization temperature and other physicochemical properties in rice (Oryza sativa L.).

The characteristics of starch, such as gelatinization temperature (GT), apparent amylose content (AAC), pasting temperature (PT) and other physicochemical properties, determine the quality of various products of rice, e.g., eating, cooking and processing qualities. The GT of rice flour is controlled by the alk locus, which has been co-mapped to the starch synthase IIa (SSIIa) locus. In this study, we sequenced a 2,051 bp DNA fragment spanning part of intron 6, exon 7, intron 7, exon 8 and part of 3' untranslated region of SSIIa for 30 rice varieties with diverse geographical distribution and variation in starch physicochemical properties. A total of 24 single nucleotide polymorphisms (SNPs) and one insertion/deletion (InDel) were identified, which could be classified into nine haplotypes. The mean pairwise nucleotide diversity pi was 0.00292, and Watterson's estimator theta was 0.00296 in this collection of rice germplasm. Tajima's D test for selection showed no significant deviation from the neutral expectation (D = - 0.04612, P > 0.10). However, significant associations were found between seven of the SNPs and peak GT (T (p)) at P < 0.05, of which two contiguous SNPs (GC/TT) showed a very strong association with T (p) (P < 0.0001). With some rare exception, this GC/TT polymorphism alone can differentiate rice varieties with high or intermediate GT (possessing the GC allele) from those with low GT (possessing the TT allele). In contrast, none of these SNPs or InDel was significantly associated with amylose content. A further 509 rice varieties with known physicochemical properties (e.g., AAC and PT) and known alleles of other starch synthesizing genes were genotyped for the SSIIa GC/TT alleles. Association analysis indicated that 82% of the total variation of AAC in these samples could be explained by a (CT)n simple sequence repeat (SSR) and a G/T SNP of Waxy gene (Wx), and 62.4% of the total variation of PT could be explained by the GC/TT polymorphism. An additional association analysis was performed between these molecular markers and the thermal and retrogradation properties for a subset of 245 samples from the 509 rice varieties. The SSIIa GC/TT polymorphism explained more than 60% of the total variation in thermal properties, whereas the SSR and SNP of Wx gene explained as much as the SSIIa GC/TT of the total variation in retrogradation properties. Our study provides further support for the utilization of the GC/TT polymorphism in SSIIa. As shown in our study of 509 rice varieties, the GC/TT SNP could differentiate rice with high or intermediate GT from those with low GT in about 90% of cases. Using four primers in a single PCR reaction, the GC/TT polymorphism can be surveyed on a large scale. Thus, this SNP polymorphism can be very useful in marker-assisted selection for the improvement of GT and other physicochemical properties of rice.

Analysis of the genetic behavior of some starch properties in indica rice ( Oryza sativa L.): thermal properties, gel texture, swelling volume.

Starch comprises about 90% of milled rice, so that the eating and cooking quality of rice is mainly affected by the starch properties. In the present paper, we analyzed the genetical behavior of gelatinization temperature tested by differential scanning calorimetry (DSC), gel texture, and the swelling volume (SV) of indica rice with an incomplete cross of 4x8 parents. A genetic model which can dissect the effects of triploid seed, the cytoplasm, and the maternal plant on the endosperm traits was used. The results indicated that peak temperature (T(p)), conclusion temperature (T(c)) and enthalpy (DeltaH) were controlled by three types of genetic effects: seed direct (endosperm) effects, cytoplasmic effects and maternal effects. No cytoplasmic effects for the onset temperature (T(o)), hardness and SV, and no maternal effects for cohesiveness were found. The additive variances ( V (A)+ V (Am)) were larger than the dominance variances ( V (D)+ V (Dm)) for all the traits except for T(c), which suggested that selection could be applied for the starch properties in early generations. The total narrow-sense heritability for each parameter was over 60%, indicating that selection advances were predictable in the early generations for these traits.

Properties of hybrid enzymes between Synechococcus large subunits and higher plant small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase in Escherichia coli.

To explore the function of small subunits of Rubisco, three hybrid enzymes were synthesized in Escherichia coli by construction of a transcriptionally coupled expression system in which the synthetic small subunit gene of rice, tobacco, and wheat, respectively, was cloned downstream from the large subunit gene of Synechococcus sp. PCC6301. These coexpression products were detected by utilizing SDS-PAGE and confirmed by immunoblotting. The amount of carboxylase activity from the intact cells revealed that each higher plant small subunit was able to assemble with the Synechococcus large subunit octamer core to form an active heterologous enzyme in E. coli. However, in these heterologous enzymes, the interaction between large subunits and small subunits was very weak, the small subunit readily dissociated from the large subunit octamer core. A detailed kinetic assay was carried out with the partially purified hybrid enzymes. Compared to Synechococcus Rubisco, the activity of rice, tobacco, and wheat hybrid Rubisco decreased to 37, 61, and 37% of the original activity, respectively. These hybrid enzymes showed a greater affinity for CO2 and RuBP than Synechococcus Rubisco. The specificity factor of the three hybrid Rubiscos was 98, 84, and 76%, respectively, of the original. These results indicate for the first time that the small subunit contributes to the stability, catalytic efficiency, and CO2/O2 specificity of Rubisco together, which suggests that small subunits may be fruitful targets for engineering an improved Rubisco. Meanwhile, we found that sorbitol in the culture of induced cells promoted the production of active assembled enzyme and shortened the time to reach maximal expression.

Two-cistron system overexpression of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunit B and B-derivatives from spinach in Escherichia coli.

A gene coding for the subunit B (GapB) of chloroplast glyceraldehyde-3-phosphate dehydrogenase from spinach and its two derivatives (GapBc) lacking the GapB-specific C-terminal extension have been cloned by RT-PCR. These three genes have been overexpressed with full activity in Escherichia coli when a two-cistron expression system controlled by an inducible promoter P(trc) is used. With a suitable base composition of the first cistron, the expression level of GapB and the derivatives GapBc are expressed up to 15-20% of the total cell protein and around 20 mg of recombinant GapBcs with full activity are purified from 1 liter of cultured bacteria. The specific activity of the two derivatives GapBc (40-60 u/mg) is similar to that of GapA (50-70 u/mg) and lower than that of reported GapBc derivative (E. Baalmann, R. Scheibe, R. Cerff, and W. Martin, 1996, Plant Mol. Biol. 32, 505-513).

Overexpression in Escherichia coli and characterization of the chloroplast fructose-1,6-bisphosphatase from wheat.

An important Calvin cycle enzyme, chloroplast fructose-1, 6-bisphosphatase (FBPase) from wheat, has been cloned and expressed up to 15% of the total cell protein using a pPLc expression vector in Escherichia coli by replacing the codons in the 5'-terminal encoding sequence with optimal and A/T-rich ones. The overexpressed wheat FBPase is soluble, fully active, and heat stable. It can be purified by chromatography in turn on DEAE-Sepharose and Sephacryl S-200, and around 15 mg of purified enzymes (>95%) is obtained from 1 liter of cultured bacteria. Its special activity is 8.8 u/mg, K(cat) is 22.9/S, K(m) is 121 microM, and V(max) is 128 micromol/min. mg. The recombinant FBPase can be activated by DTT, Na(+), or low concentrations of Li(+), Ca(2+), Zn(2+), GuHCl, and urea, while it can be inhibited by K(+) or NH(+)(4).

Overexpression in Escherichia coli and characterization of the chloroplast triosephosphate isomerase from spinach.

An important Calvin cycle enzyme, chloroplast triosephosphate isomerase (cpTPI) from spinach, has been cloned and expressed in up to 15% of the total cell protein using the P(L) expression vector in Escherichia coli. An even higher level expression, up to 36% of the total protein, was achieved by replacing the nucleotide sequence between the ribosomal binding site and the initial codon, ATG, with an AT-rich sequence. Computer modeling revealed that the moderate change in the standard free energy (5'-DeltaG degrees ) of mRNA secondary structure in the translation initial region might be the major factor which led to the later high-level expression. The overexpressed spinach cpTPI was soluble and fully active and was able to be purified beyond 95% purity by DEAE-Sepharose and Sephadex G-75, and around 55 mg of purified enzymes was obtained from 1 liter of cultured bacteria. With d-glyceraldehyde 3-phosphate as substrate, K(m (D-3-P)) is 0. 68 mM, V(max (G-3-P)) is 3.16 x 10(4) micromol/min. mg, and K(cat (G-3-P)) is 4.51 x 10(3)/s; with dihydroxyacetone phosphate as substrate, the corresponding values are 7.27 mM, 1.04 x 10(3) micromol/min. mg, and 1.16 x 10(2)/s, respectively.

A new method for the synthesis of a structural gene.

A novel method of synthesizing a structural gene or gene fragment, consisting of the first synthesis of a single-stranded DNA (ssDNA), has been developed. As a preliminary test of this method, four synthetic genes or gene fragments have been synthesized. The first one with 396 base pairs (b.p.) codes for the mature rbcS from wheat, the next two with 370 and 342 b.p. respectively, for two half molecules of a gene for trichosanthin and the last one with 315 b.p. for the N-terminal 1-102 residues of human prourokinase. In all these syntheses, a plus-stranded DNA of the target gene was generally assembled by a stepwise or one step T4 DNA ligase reaction of six oligonucleotides (A, *pB, *pC, *pD, *pE and *pF) of 30-71 nucleotides long in the presence of two terminal complementary oligonucleotides (Ab' and eF') and three short inter-fragment complementary oligonucleotides (bc, cd and de). After purification, the synthetic ssDNA was inserted into a cloning vector, pWR13. The resulting product was directly used to transform a host cell. The structure of the cloned synthetic gene was confirmed by DNA sequence analysis.