Selection of Transcripts Affecting Initial Growth Rate of Rice Backcrossed Inbred Lines Using RNA Sequencing Data

Seedling growth is an important factor for direct seeding of rice. However, the genetic and transcriptomic factors involved in this process are largely unknown. In this study, transcripts affecting shoot weight were identified in rice (Oryza sativa L.) using RNA sequencing (RNA-Seq) data from 20 backcrossed inbred lines (BILs) and their parental cultivars. The selection frequency of the genes for the regression model was determined using repeated analysis of random subsets of the transcriptome. The qLTG3-1gene, controlling low-temperature germinability, and short grain 1 gene (SG1), known to decrease organ elongation, showed high frequency. The quantitative trait loci (QTLs) analysis performed for BILs revealed that qLTG3-1 was included in the QTLs for shoot weight but SG1 was not. No nucleotide polymorphisms were found in the coding region of SG1 in either of the parental cultivars. Quantitative real-time PCR showed that SG1 expression was negatively correlated with shoot weight for all 104 BILs analyzed in this study. Expression QTL (eQTLs) analysis showed an eQTL for SG1 expression located in the same region as the QTL for shoot weight. However, no eQTLs were detected on the same chromosome as SG1, suggesting that nucleotide polymorphisms around the gene do not affect its expression in analyzed growth stage. Overall, these results indicate that RNA-Seq is a useful tool for identifying transcripts that can be related to seedling growth rate

Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae.

An acyclic diterpene alcohol, (E, E, E)-geranylgeraniol (GGOH), is one of the important compounds used as perfume and pharmacological agents. A deficiency of squalene (SQ) synthase activity allows yeasts to accumulate an acyclic sesquiterpene alcohol, (E, E)-farnesol, in their cells. Since sterols are essential for the growth of yeasts, a deficiency of SQ synthase activity makes the addition of supplemental sterols to the culture media necessary. To develop a GGOH production method not requiring any supplemental sterols, we overexpressed HMG1 encoding hydroxymethylglutaryl-CoA reductase and the genes of two prenyl diphosphate synthases, ERG20 and BTS1, in Saccharomyces cerevisiae. A prototrophic diploid coexpressing HMG1 and the ERG20-BTS1 fusion accumulated GGOH with neither disruption of the SQ synthase gene nor the addition of any supplemental sterols. The GGOH content on the diploid cultivation in a 5-l jar fermenter reached 138.8 mg/l under optimal conditions

Characterization of sugar metabolism in the stem of Tachisuzuka, a whole-crop silage rice cultivar with high sugar content in the stem

Tachisuzuka, a rice cultivar for whole-crop silage, is characterized by a small panicle and high sugar content in the stem. Our previous study suggests that the high sugar content in the stem of Tachisuzuka is due to a factor other than the small panicle. To characterize sugar metabolism in the stem of Tachisuzuka, here we compared carbohydrate content, enzyme activity, and the expression of genes involved in sugar metabolism in the stem between Tachisuzuka and its parental variety, Kusanohoshi. Thinning the panicles of Kusanohoshi increased the starch content in the leaf sheath and internode but did not increase the sucrose content in the leaf sheath to the same level as that of Tachisuzuka. This suggests that Tachisuzuka has high potential to accumulate sucrose in its leaf sheath. Comparison of enzyme activity showed that the hexokinase activities in the leaf sheath tended to be higher in Tachisuzuka than Kusanohoshi or panicle-thinned Kusanohoshi, suggesting that glucokinase or fructokinase affects sugar accumulation in the stem of Tachisuzuka. Comparative transcriptome analysis revealed the differences in expression levels of carbohydrate-related genes between Tachisuzuka and Kusanohoshi. In particular, the expression levels of ISA2, which encodes starch-debranching enzyme, and TMT2, which encodes tonoplast monosaccharide transporter – both of which maybe involved in sugar accumulation in grass stems – were higher in Tachisuzuka than Kusanohoshi. Thus, these enzymes and transporters may contribute to the high sugar content in the stem of Tachisuzuka

Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae▿

(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter−1) rather than GGOH (0.2 mg liter−1) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter−1 GGOH and 6.5 mg liter−1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter−1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering

Characterization of sugar metabolism in the stem of Tachisuzuka, a whole-crop silage rice cultivar with high sugar content in the stem

<p>Tachisuzuka, a rice cultivar for whole-crop silage, is characterized by a small panicle and high sugar content in the stem. Our previous study suggests that the high sugar content in the stem of Tachisuzuka is due to a factor other than the small panicle. To characterize sugar metabolism in the stem of Tachisuzuka, here we compared carbohydrate content, enzyme activity, and the expression of genes involved in sugar metabolism in the stem between Tachisuzuka and its parental variety, Kusanohoshi. Thinning the panicles of Kusanohoshi increased the starch content in the leaf sheath and internode but did not increase the sucrose content in the leaf sheath to the same level as that of Tachisuzuka. This suggests that Tachisuzuka has high potential to accumulate sucrose in its leaf sheath. Comparison of enzyme activity showed that the hexokinase activities in the leaf sheath tended to be higher in Tachisuzuka than Kusanohoshi or panicle-thinned Kusanohoshi, suggesting that glucokinase or fructokinase affects sugar accumulation in the stem of Tachisuzuka. Comparative transcriptome analysis revealed the differences in expression levels of carbohydrate-related genes between Tachisuzuka and Kusanohoshi. In particular, the expression levels of <i>ISA2</i>, which encodes starch-debranching enzyme, and <i>TMT2</i>, which encodes tonoplast monosaccharide transporter – both of which maybe involved in sugar accumulation in grass stems – were higher in Tachisuzuka than Kusanohoshi. Thus, these enzymes and transporters may contribute to the high sugar content in the stem of Tachisuzuka.</p