水稻早衰突变体LS-es1的基因定位及候选基因分析

衰老是植物发育末期自主发生且不可逆的适应性反应, 叶片早衰相关分子机制研究对水稻(Oryza sativa)遗传改良以及抗衰老品种培育有重要意义。LS-es1是通过EMS诱变粳稻品种TP309获得的稳定遗传的早衰突变体。对LS-es1及其野生型的表型观察和生理生化分析表明, LS-es1叶片中积累了大量活性氧且细胞死亡更多, 同时LS-es1与产量相关的农艺性状均显著下降, 这也验证了LS-es1早衰的特征。对LS-es1及其野生型幼苗进行外源激素处理, 结果表明LS-es1对水杨酸(SA)、脱落酸(ABA)和茉莉酸甲酯(MeJA)更敏感。用图位克隆方法将LS-es1基因定位在水稻第7号染色体长臂46.2 kb区间内, 该区间共包括8个开放阅读框(ORF)。对该区间内的基因进行生物信息学分析, 结果发现Os07g0275300和Os07g0276000两个候选功能基因与早衰途径相关, 并且这2个基因的表达量在野生型和突变体中差异较大。研究结果为进一步克隆LS-es1基因并深入研究其生物学功能奠定了基础。     

Recent progress in understanding thiamin biosynthesis and its genetic regulation in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is able to synthesize thiamin pyrophosphate (TPP) de novo, which involves the independent formation of two ring structures, 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-β-hydroxyethylthiazole, in the early steps. In addition, this organism can efficiently utilize thiamin from the extracellular environment to produce TPP. Nineteen genes involved in the synthesis of TPP and the utilization of thiamin (THI genes) have been identified, and the function of several THI genes has been elucidated. All THI genes participating in the synthesis of the pyrimidine unit belong to multigene families. It is also intriguing that some thiamin biosynthetic proteins are composed of two distinct domains or form an enzyme complex. The expression of THI genes is coordinately induced in response to thiamin starvation. It is likely that the induction of THI genes is activated by a positive regulatory factor complex and that the protein–protein interaction among the factors is disturbed by TPP. Thiamin-hyperproducing yeast and fermented food containing a high content of thiamin are expected to be available in the future based on the progress in understanding thiamin biosynthesis and its genetic regulation in S. cerevisiae.     

Primary root and root hair development regulation by OsAUX4 and its participation in the phosphate starvation response

Among the five members of AUX1/LAX genes coding for auxin carriers in rice, only OsAUX1 and OsAUX3 have been reported. To understand the function of the other AUX1/LAX genes, two independent alleles of osaux4 mutants, osaux4-1 and osaux4-2, were constructed using the CRISPR/Cas9 editing system. Homozygous osaux4-1 or osaux4-2 exhibited shorter primary root (PR) and longer root hair (RH) compared to the wild-type Dongjin (WT/DJ), and lost response to indoleacetic acid (IAA) treatment. OsAUX4 is intensively expressed in roots and localized on the plasma membrane, suggesting that OsAUX4 might function in the regulation of root development. The decreased meristem cell division activity and the downregulated expression of cell cycle genes in root apices of osaux4 mutants supported the hypothesis that OsAUX4 positively regulates PR elongation. OsAUX4 is expressed in RH, and osaux4 mutants showing longer RH compared to WT/DJ implies that OsAUX4 negatively regulates RH development. Furthermore, osaux4 mutants are insensitive to Pi starvation (-Pi) and OsAUX4 effects on the -Pi response is associated with altered expression levels of Pi starvation-regulated genes, and auxin distribution/contents. This study revealed that OsAUX4 not only regulates PR and RH development but also plays a regulatory role in crosstalk between auxin and -Pi signaling.     

薰衣草叶片对低温胁迫的生理与分子响应机制

薰衣草(Lavandula angustifolia)作为名贵的芳香植物,其生长、繁育、品质和产量均受低温影响。前期研究已获得1个耐低温薰衣草品种。该研究将对其处理的温度从20℃降至0℃,揭示薰衣草响应冷胁迫的生理及分子调控机制,同时结合薰衣草的细胞质膜透性、可溶性糖和蛋白质含量及抗氧化酶活性等生理变化。采用转录组学和生物信息学方法挖掘分析相关耐寒基因,并探讨外施水杨酸缓解–10℃冻胁迫的可行性。研究发现7个编码脂肪酸去饱和酶和转移酶的基因(LaFADs)、3个参与合成可溶性糖的基因(LaBAM1和LaSS2)、19个编码胚胎晚期丰富蛋白的基因(LaLEAs)及7个编码过氧化物酶的基因(LaPODs),这些基因在低温胁迫下均上调表达,指导薰衣草合成并积累保护物质,维持膜稳定性以应对胁迫。此外, 150 mg·L^-1水杨酸预处理能有效缓解植株冻害,可作为低温保护剂。该研究丰富了薰衣草重要抗逆基因家族的遗传背景,为后续分子遗传学功能分析和定向品种改良奠定基础。     

Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis

Thiamin pyrophosphate (TPP) is an essential enzyme cofactor required for the viability of all organisms. Whether derived from exogenous sources or through de novo synthesis, thiamin must be pyrophosphorylated for cofactor activation. The enzyme thiamin pyrophosphokinase (TPK) catalyzes the conversion of free thiamin to TPP in plants and other eukaryotic organisms and is central to thiamin cofactor activation. While TPK activity has been observed in a number of plant species, the corresponding gene/protein has until now not been identified or characterized for its role in thiamin metabolism. Here we report the functional identification of two Arabidopsis TPK genes, AtTPK1 and AtTPK2 and the enzymatic characterization of the corresponding proteins. AtTPK1 and AtTPK2 are biochemically redundant cytosolic proteins that are similarly expressed throughout different plant tissues. The essential nature of TPKs in plant metabolism is reflected in the observation that while single gene knockouts of either AtTPK1 or AtTPK2 were viable, the double mutant possessed a seedling lethal phenotype. HPLC analysis revealed the double mutant is nearly devoid of TPP and instead accumulates the precursor of the TPK reaction, free thiamin. These results suggest that TPK activity provides the sole mechanism by which exogenous and de novo derived thiamin is converted to the enzyme cofactor TPP.     

甘蔗节间伸长过程赤霉素生物合成关键基因的表达及相关植物激素动态变化

以甘蔗(Saccharum officinarum)优良品种桂糖42号(GT42)为研究材料, 分别于未伸长期(9-10叶龄以前) (Ls1)、伸长初期(12-13叶龄) (Ls2)和伸长盛期(15-16叶龄) (Ls3)取甘蔗第2片真叶(自顶部起)对应的节间组织, 测定其赤霉素(GA)、生长素(IAA)、油菜素甾醇(BR)、细胞分裂素(CTK)、乙烯(ETH)和脱落酸(ABA)的含量, 并通过实时荧光定量PCR (qRT-PCR)分析赤霉素合成途径关键基因GA₂₀氧化酶基因(GA20-Oxidase1)、赤霉素受体基因(GID1)和DELLA蛋白编码基因(GAI)的差异表达。结果表明, 在甘蔗伸长期间, GA和IAA含量呈现上升趋势, CTK和ABA含量呈下降趋势, ETH含量先上升后下降, BR含量则变化不明显; GA20-Oxidase1和GID1的表达呈上升趋势, 而GAI的表达则呈下降趋势, 这与相关植物激素的变化基本一致。综上, 甘蔗节间伸长过程主要与GA和IAA相关, 其次为CTK和ABA, 而ETH受到IAA的调控影响节间伸长; 植物激素间通过相互作用调控GA20-Oxidase1、GID1和GAI的表达, 影响GA含量和GA的信号转导过程, 进而影响甘蔗节间的伸长。该研究揭示了甘蔗节间伸长过程中赤霉素生物合成途径和信号转导关键基因的差异表达及植物激素含量的动态变化规律。     

水稻黄绿叶调控基因YGL18的克隆与功能解析

叶色突变体往往伴随着叶绿素含量变化及叶绿体结构异常,是研究叶绿体发育与光合作用相关基因功能的重要材料。该研究通过甲基磺酸乙酯(EMS)诱变籼稻(Oryzasativasubsp.indica)品种华占(HZ)获得黄绿叶突变体,将其命名为ygl18 (yellow-green leaf 18)。与野生型相比,黄绿叶突变体ygl18自三叶期起叶片开始变黄且程度不断加深,同时伴随着光合速率与叶绿素含量下降,且结实率、千粒重及有效穗数均显著降低。透射电镜观察结果显示, ygl18的叶绿体结构紊乱,基质片层疏松,发育受到抑制,与叶片出现黄绿色表型一致。遗传分析表明, ygl18突变性状受1对隐性等位核基因控制,这对等位基因位于水稻第3号染色体长臂标记InDel2和InDel3之间115.2 kb范围内。进一步研究发现该突变体表型是编码铁氧还蛋白FdC2的基因LOC＿Os03g48040的5’UTR发生突变所致。通过CRISPR转基因实验验证了该基因对表型的控制作用。研究结果揭示了叶色调控网络的遗传基础,可为今后选育高光效水稻品种提供新线索。     

Transketolase: observations in alcohol-related brain damage research

Thiamin, or vitamin B1, is crucial for brain function. In its active form, thiamin pyrophosphate (TPP), it is a co-enzyme for several enzymes, including transketolase. Transketolase is an important enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP), a pathway responsible for generating reducing equivalents, which is essential for energy transduction and for generating ribose for nucleic acid synthesis. Transketolase also links the PPP to glycolysis, allowing a cell to adapt to a variety of energy needs, depending on its environment. Abnormal transketolase expression and/or activity have been implicated in a number of diseases where thiamin availability is low, including Wernicke-Korsakoff's Syndrome and alcoholism. Yet, the precise mechanism by which this enzyme is involved in the pathophysiology of these disorders remains controversial.     

稻镰状瓶霉促进水稻生长的机制

稻镰状瓶霉Falciphora oryzae是本实验室从野生稻根系分离获得的一株DSE,具有促生、防病等作用。本研究旨在对其促生机制进行初探。在室内平板共培养条件下,对水稻种子接种稻镰状瓶霉菌饼（4个/皿）,测定植株生长指标、营养元素含量及营养吸收相关基因表达量;温室盆栽条件下,将稻镰状瓶霉以菌肥形式（60g/桶）与水稻进行盆栽共培养,测定植株农艺性状指标。结果表明,接种稻镰状瓶霉后,平板上的水稻幼苗的株高、叶宽和茎秆直径显著增加,根长并未受影响。稻镰状瓶霉能够促进水稻根系对营养元素的吸收,提高地上部分和根系组织中N、P、K、S、Fe和Mg元素的含量,诱导根部与营养元素吸收相关的调控基因OsPTR9、OsAMT3;2、OsPT4、OsSULTR3;1、OsMRS2-8、OsHAK16、OsYSL15和OsIRO2的显著上调表达。盆栽试验表明,稻镰状瓶霉显著提高了水稻各农艺性状指标,包括叶宽、茎秆直径、植株鲜重、植株干重、叶绿素含量和光合强度。结果表明,稻镰状瓶霉的根部定殖能够诱导营养元素吸收相关基因的上调表达,从而促进根系对营养元素的吸收,进而促进水稻植株的生长。     

Crystal structure of thiamin pyrophosphokinase.

Thiamin pyrophosphate (TPP) is a coenzyme derived from vitamin B1 (thiamin). TPP synthesis in eukaryotes requires thiamin pyrophosphokinase (TPK), which catalyzes the transfer of a pyrophosphate group from ATP to thiamin. TPP is essential for central metabolic processes, including the formation of acetyl CoA from glucose and the Krebs cycle. Deficiencies in human thiamin metabolism result in beriberi and Wernicke encephalopathy. The crystal structure of mouse TPK was determined by multiwavelength anomalous diffraction at 2.4 A resolution, and the structure of TPK complexed with thiamin has been refined at 1.9 A resolution. The TPK polypeptide folds as an alpha/beta-domain and a beta-sandwich domain, which share a central ten-stranded mixed beta-sheet. TPK subunits associate as a dimer, and thiamin is bound in the dimer interface. Despite lacking apparent sequence homology with other proteins, the alpha/beta-domain resembles the Rossman fold and is similar to other kinase structures, including another pyrophosphokinase and a thiamin biosynthetic enzyme. Comparison of mouse and yeast TPK structures reveals differences that could be exploited in developing species-specific inhibitors of potential use as antimicrobial agents.     

水稻早衰突变体LS-es1的基因定位及候选基因分析

衰老是植物发育末期自主发生且不可逆的适应性反应, 叶片早衰相关分子机制研究对水稻(Oryza sativa)遗传改良以及抗衰老品种培育有重要意义。LS-es1是通过EMS诱变粳稻品种TP309获得的稳定遗传的早衰突变体。对LS-es1及其野生型的表型观察和生理生化分析表明, LS-es1叶片中积累了大量活性氧且细胞死亡更多, 同时LS-es1与产量相关的农艺性状均显著下降, 这也验证了LS-es1早衰的特征。对LS-es1及其野生型幼苗进行外源激素处理, 结果表明LS-es1对水杨酸(SA)、脱落酸(ABA)和茉莉酸甲酯(MeJA)更敏感。用图位克隆方法将LS-es1基因定位在水稻第7号染色体长臂46.2 kb区间内, 该区间共包括8个开放阅读框(ORF)。对该区间内的基因进行生物信息学分析, 结果发现Os07g0275300和Os07g0276000两个候选功能基因与早衰途径相关, 并且这2个基因的表达量在野生型和突变体中差异较大。研究结果为进一步克隆LS-es1基因并深入研究其生物学功能奠定了基础。     

Impact of the reduced folate carrier on the accumulation of active thiamin metabolites in murine leukemia cells

The thiamin transporter encoded by SLC19A2 and the reduced folate carrier (RFC1) share 40% homology at the protein level, but the thiamin transporter does not mediate transport of folates. By using murine leukemia cell lines that express no, normal, or high levels of RFC1, we demonstrate that RFC1 does not mediate thiamin influx. However, high level RFC1 expression substantially reduced accumulation of the active thiamin coenzyme, thiamin pyrophosphate (TPP). This decreased level of TPP, synthesized intracellularly from imported thiamin, resulted from RFC1-mediated efflux of TPP. This conclusion was supported by the following observations. (i) Efflux of intracellular TPP was increased in cells with high expression of RFC1. (ii) Methotrexate inhibits TPP influx. (iii) TPP competitively inhibits methotrexate influx. (iv) Loading cells, which overexpress RFC1 to high levels of methotrexate to inhibit competitively RFC1-mediated TPP efflux, augment TPP accumulation. (v) There was an inverse correlation between thiamin accumulation and RFC1 activity in cells grown at a physiological concentration of thiamin. The modulation of thiamin accumulation by RFC1 in murine leukemia cells suggests that this carrier may play a role in thiamin homeostasis and could serve as a modifying factor in thiamin nutritional deficiency as well as when the high affinity thiamin transporter is mutated.     

水稻籽粒维生素E QTL挖掘及候选基因分析

维生素E (V_E)是稻米营养品质的重要指标。水稻(Oryza sativa)是我国种植最广泛的粮食作物, 增加其籽粒的V_E含量是实现国民营养强化的一条便捷有效的途径。该研究以籼稻华占(HZ)为父本, 粳稻热研2号(Nekken2)为母本, 构建120个重组自交系(RILs)群体。采用高效液相色谱法(HPLC)对RILs群体的V_E各组分含量进行测定, 并基于构建的高密度分子遗传图谱进行QTL定位, 谱系分析后挖掘到122个V_E总量和分量相关QTLs, 分布在12条染色体上。其中qT3α/to2-1的LOD值高达10.32, qT3α2-1的LOD值高达9.91, 另有多个控制各异构体含量的主效QTLs, 且区间内包含OsGGR1、OsGGR2、OsTC及OsγTMT等V_E生物合成基因。通过qRT-PCR检测亲本中V_E合成基因的表达量, 发现在华占中候选基因的表达量均极显著高于热研2号, 推测这些基因的高表达是华占生育酚及生育三烯酚含量高于热研2号的原因。研究挖掘到的QTL数目较多, LOD值也较大, 为进一步筛选和培育高V_E含量的水稻新品种奠定了分子基础, 同时为揭示水稻V_E生物合成的分子调控机制提供了重要基因资源。