ウメにおける自家および他家受粉雌ずいのトランスクリプトーム解析

Solanaceae, Rosaceae, and Plantaginaceae exhibit the S-RNase-based gametophytic self-incompatibility (GSI) system. This type of GSI is controlled by a single polymorphic locus (S locus) containing the pistil S determinant gene, S-ribonuclease (S-RNase), and the pollen S determinant, the S locus F-box gene (SFB/SLF). In addition to these determinant genes, non-S factors, called modifier genes, are required for the GSI reaction. Here, we conducted large-scale transcriptome analysis of unpollinated, self-pollinated, and cross-pollinated pistils of Japanese apricot (Prunus mume Sieb. et Zucc. cv. Nanko) to capture all of the molecular events induced by the GSI reaction in Prunus, using next-generation sequencing technologies. We obtained 40,061 unigenes from 77,521,310 reads from pollinated and unpollinated pistils and pollen grains. Among these unigenes, 29,985 and 27,898 unigene sequences showed at least one hit against the NCBI nr and TAIR10 protein databases, respectively, in BLASTX searches using an E-value cutoff of 1e-6. Digital expression analysis showed that 8,907 and 10,190 unigenes were expressed at significantly different levels between unpollinated (UP) and cross-pollinated (CP) pistils and between UP and self-pollinated (SP) pistils, respectively. The expression of 4,348 unigenes in both CP and SP pollination was commonly and significantly different from that in UP, while the expression of 4,559 and 5,842 unigenes in CP and SP, respectively, was specifically and significantly different from UP. The expression of 2,227 unigenes was up-regulated both in CP and SP compared with UP. Genes supposedly involved in S-RNase-based GSI were included among the unigenes up-regulated by pollination, while no unigenes homologous to the solanaceous pistil modifiers HT-B or 120K were included among the unigenes up-regulated by pollination or in the whole unpollinated/pollinated pistil transcriptome. We discuss the distinct molecular mechanism of S-RNase-based GSI in Prunus.ナス科, バラ科, オオバコ科の多くの種は S-RNase 依存性配偶体型自家不和合性を示す. このタイプの不和合性は雌ずい側因子 (S-RNase)と花粉側因子 (SFB/SLF)が座乗する S 遺伝子座によって支配されている. また, 自家不和合性共通因子と呼ばれる S 因子以外の因子も配偶体型自家不和合性反応に必要であることが明らかになっている. そこで本研究では, ウメの配偶体型自家不和合性機構解明を目的として, 次世代シークエンス技術を用いた大規模トランスクリプトーム解析を行った. ウメの受粉雌ずい, 未受粉雌ずい, 花粉から合計 77,521,310 個の配列が得られ, それらから 40,061 個の unigene が得られた. これら 40,061 個の unigene を query に, 米国の国立生物工学情報センター (NCBI)の非冗長タンパク質データベース (nr)ならびにシロイヌナズナのタンパク質データベース (TAIR10)に対して, BLASTX 検索したところ, 29,985 個 (全体の 74.8%) の unigene が NCBI の少なくとも1 つ以上のデータに, また 27,898 個 (全体の 69.9%)の unigene が TAIR10 の少なくとも1 つ以上のデータにヒット (E-value cutoff 値 1e-6)した. デジタル発現解析により, 8,907の unigene が未受粉と他家受粉で有意に発現が異なることが, また 10,190 個の unigene が未受粉と自家受粉で有意に発現が異なることが示された. これらのうち, 4,348 個の unigene は両受粉で共通であり, 4,559 個の unigene は他家受粉で特異的に, また 5,842 個の unigene は自家受粉で特異的に発現が変化していた. さらに, 両受粉で発現が変化していた unigene のうち, 2,227 個は両受粉で発現が増加していた. しかし, ナス科で花柱側共通因子であることが示されている HT-B や 120K の相同遺伝子はトランスクリプトーム全体の中に存在しなかった. Prunus 属の配偶体型自家不和合性に特異な認識機構について考察した

マイクロアレイを用いた長期低温遭遇時のウメ休眠芽のトランスクリプトーム解析

Bud dormancy is a critical developmental process for perennial plant survival, and also an important physiological phase that affects the next season’s growth of temperate fruit trees. Bud dormancy is regulated by multiple genetic factors, and affected by various environmental factors, tree age and vigor. To understand the molecular mechanism of bud dormancy in Japanese apricot (Prunus mume Sieb. et Zucc.), we constructed a custom oligo DNA microarray covering the Japanese apricot dormant bud ESTs referring to the peach (P. persica) genome sequence. Because endodormancy release is a chilling temperature-dependent physiological event, genes showing chilling-mediated differential expression patterns are candidates to control endodormancy release. Using the microarray constructed in this study, we monitored gene expression changes of dormant vegetative buds of Japanese apricot during prolonged artificial chilling exposure. In addition, we analyzed seasonal gene expression changes. Among the 58539 different unigene probes, 2345 and 1059 genes were identified as being more than twofold up-regulated and down-regulated, respectively, following chilling exposure for 60 days (P < 0.05). Cluster analysis suggested that the expression of the genes showing expression changes by artificial chilling exposure were coordinately regulated by seasonal changes. The down-regulated genes included P. mume DORMANCY-ASSOCIATED MADS-box genes, which supported previous quantitative RT-PCR and EST analyses showing that these genes are repressed by prolonged chilling exposure. The genes encoding lipoxygenase were markedly up-regulated by prolonged chilling. Our parametric analysis of gene-set enrichment suggested that genes related to jasmonic acid (JA) and oxylipin biosynthesis and metabolic processes were significantly up-regulated by prolonged chilling, whereas genes related to circadian rhythm were significantly down-regulated. The results obtained from microarray analyses were verified by quantitative RT-PCR analysis of selected genes. Taken together, we have concluded that the microarray platform constructed in this study is applicable for deeper understanding of the molecular network related to agronomically important bud physiology, including dormancy release.永年性作物において越冬芽の休眠は, 環境に適応するために進化した成長制御機構のひとつであり, かつ翌年の成長を左右する重要な農業形質のひとつである. 芽の休眠には多くの遺伝的要因が関わるばかりでなく, 多岐にわたる環境要因や樹勢, 樹齢の影響もうけており, 多くの遺伝子の発現制御が休眠に関与していると考えられる. そこで本研究では, 休眠関与候補遺伝子の単離を目的に, カスタムマイクロアレイを用いたウメ休眠芽のトランスクリプトーム解析をおこなった. まず, 我々が先行研究で獲得したウメ芽 EST 配列情報をもとに, ゲノム全体をできるだけ偏りなく網羅するように選びだした 58539 ユニジーンに相当するプローブを搭載したマイクロアレイを構築した. 次いで, 休眠覚醒を誘導する長期の低温処理を施したウメ休眠芽の遺伝子発現変動を調査した. その結果, 有意に 2 倍以上発現が上方制御あるいは下方制御されるプローブが 2345 個あるいは 1059 個同定された. これらの変動遺伝子のなかには季節的な発現同調性がみられるものがあった. 下方制御される遺伝子のなかには, 先行研究で休眠への関与が示唆されている DORMANCY ASSOCIATED MADS-box が含まれており, 上方制御される遺伝子にはリポキシゲナーゼが含まれていた. Parametricanalysis of gene set enrichment 解析の結果, 長期の低温遭遇によってジャスモン酸やオキシリピン生合成・代謝の GO タームが有意に上昇し, 概日リズムの GO タームが有意に減少した. これらの GO タームに含まれる遺伝子について定量 RT-PCR を行った結果, マイクロアレイで検出された発現変動パターンとほぼ同様であった. 以上の結果より, 本研究で構築したマイクロアレイはウメ休眠芽の網羅的な遺伝子発現解析に有効であることが示された. 本実験では, ウメの休眠覚醒に関与する遺伝子ネットワークあるいは新規候補遺伝子の探索に活用した

Sulfur stable isotope data

Sulfur stable isotope ratios of water and vertebral sections of masu salmon. Each vertebral centrum was subdivided equally into 10 sections numbered from the center (01) to the margin (10). Fish and water samples were collected in the Churui River, Hokkaido Islands, japan. All fish samples analyzed consisted of bone collagen

Data from: Incremental analysis of vertebral centra can reconstruct the stable isotope chronology of teleost fishes

1. Isotope analysis has high potential for understanding fish ecology and food-web structure in aquatic ecosystems. The utility of isotope analysis will be greatly improved if we can reconstruct the chronology of several isotopes at multiple growth stages of individual fish. However, no practical methods exist for reconstructing the chronology of light-element isotopes (e.g. δ13C, δ15N, δ34S, and Δ14C) in teleost fishes. Here, we present and test a new analytical approach for reconstructing the isotopic ratios of light isotopes at multiple life-stages in teleost fishes. 2. We sampled an anadromous salmon species, masu salmon Oncorhynchus masou (n = 3), along with water from its natal stream and from the ocean. We subdivided the vertebral centra of the salmon equally into 10 sections and extracted bone collagen from each sample. We then measured the stable sulfur isotope ratios of each vertebral section and compared them with δ34S values of the river water and sea water. We also measured the 87Sr/86Sr ratios of otoliths as a reference indicator of salmon migration between fresh water and the ocean. 3. In all samples, the bone section closest to the centre of the centrum had the lowest δ34S values, which were similar to those of fresh water. The δ34S values gradually increased from the centre to marginal sections, finally reaching constant values similar to those of seawater. The 87Sr/86Sr ratios of sagittal otolith sections had significant inter-individual differences and were consistent with the patterns of variation of the δ34S values of the vertebral sections. 4. Our results show that the vertebral centra of teleost fishes record isotopic information from juvenile to adult life-stages. We suggest that our method can provide reproducible isotopic chronology, even in teleost fishes smaller than 50 cm. This method can be used in isoscape studies and in studies of the ecology of marine teleost fishes

Strontium stable isotope data

Results of elemental and 87Sr/86Sr analyses of fish otolith sections from masu salmon (identified as OM-01, OM-02 and OM-03) collected from the Churui River, water from the Churui River, and sea water collected approximately 10 km south of the mouth of the Churui River

The parthenocarpic gene Pat-k is generated by a natural mutation of SlAGL6 affecting fruit development in tomato (Solanum lycopersicum L.)

Abstract Background Parthenocarpy is a desired trait in tomato because it can overcome problems with fruit setting under unfavorable environmental conditions. A parthenocarpic tomato cultivar, ‘MPK-1’, with a parthenocarpic gene, Pat-k, exhibits stable parthenocarpy that produces few seeds. Because ‘MPK-1’ produces few seeds, seedlings are propagated inefficiently via cuttings. It was reported that Pat-k is located on chromosome 1. However, the gene had not been isolated and the relationship between the parthenocarpy and low seed set in ‘MPK-1’ remained unclear. In this study, we isolated Pat-k to clarify the relationship between parthenocarpy and low seed set in ‘MPK-1’. Results Using quantitative trait locus (QTL) analysis for parthenocarpy and seed production, we detected a major QTL for each trait on nearly the same region of the Pat-k locus on chromosome 1. To isolate Pat-k, we performed fine mapping using an F4 population following the cross between a non-parthenocarpic cultivar, ‘Micro-Tom’ and ‘MPK-1’. The results showed that Pat-k was located in the 529 kb interval between two markers, where 60 genes exist. By using data from a whole genome re-sequencing and genome sequence analysis of ‘MPK-1’, we could identify that the SlAGAMOUS-LIKE 6 (SlAGL6) gene of ‘MPK-1’ was mutated by a retrotransposon insertion. The transcript level of SlAGL6 was significantly lower in ovaries of ‘MPK-1’ than a non-parthenocarpic cultivar. From these results, we could conclude that Pat-k is SlAGL6, and its down-regulation in ‘MPK-1’ causes parthenocarpy and low seed set. In addition, we observed abnormal micropyles only in plants homozygous for the ‘MPK-1’ allele at the Pat-k/SlAGL6 locus. This result suggests that Pat-k/SlAGL6 is also related to ovule formation and that the low seed set in ‘MPK-1’ is likely caused by abnormal ovule formation through down-regulation of Pat-k/SlAGL6. Conclusions Pat-k is identical to SlAGL6, and its down-regulation causes parthenocarpy and low seed set in ‘MPK-1’. Moreover, down-regulation of Pat-k/SlAGL6 could cause abnormal ovule formation, leading to a reduction in the number of seeds

The CDK-PLK1 axis targets the DNA damage checkpoint sensor protein RAD9 to promote cell proliferation and tolerance to genotoxic stress

二刀流のがん増殖戦略. 京都大学プレスリリース. 2017-12-20.Genotoxic stress causes proliferating cells to activate the DNA damage checkpoint, to assist DNA damage recovery by slowing cell cycle progression. Thus, to drive proliferation, cells must tolerate DNA damage and suppress the checkpoint response. However, the mechanism underlying this negative regulation of checkpoint activation is still elusive. We show that human Cyclin-Dependent-Kinases (CDKs) target the RAD9 subunit of the 9-1-1 checkpoint clamp on Thr292, to modulate DNA damage checkpoint activation. Thr292 phosphorylation on RAD9 creates a binding site for Polo-Like-Kinase1 (PLK1), which phosphorylates RAD9 on Thr313. These CDK-PLK1-dependent phosphorylations of RAD9 suppress checkpoint activation, therefore maintaining high DNA synthesis rates during DNA replication stress. Our results suggest that CDK locally initiates a PLK1-dependent signaling response that antagonizes the ability of the DNA damage checkpoint to detect DNA damage. These findings provide a mechanism for the suppression of DNA damage checkpoint signaling, to promote cell proliferation under genotoxic stress conditions

Additional file 1 of The parthenocarpic gene Pat-k is generated by a natural mutation of SlAGL6 affecting fruit development in tomato (Solanum lycopersicum L.)

Table S1. Raw data status of whole genome re-sequencing of ‘MPK-1’. Table S2. Primer sequences used for fine mapping. Table S3. Primer sequences used for primer walking of the insertion in the Solyc01g093960 gene. Table S4. Primer sequences used for quantitative RT-PCR analysis of the Solyc01g093960 gene. Table S5. The information of SNPs used in QTL analysis in this study. Table S6. The information of SNPs for fine mapping of Pat-k in this study. Figure S1. Amplification products produced by the primers of 093960_fwd and 093960_rev. (XLSX 136 kb