山黧豆160年研究历程及进展

在全球范围内曾多次发生因过量食用山黧豆导致神经中毒事件,使得国内外对于山黧豆的种植和利用存在一定的误解和偏见,山黧豆的优良农艺价值和潜在功能食品利用价值也受到一定限制。但山黧豆适应性强、分布范围广,是全球气候变化条件下农业可持续发展和地力维持的优选作物,在食品安全、生态文明建设和乡村振兴新形势下,进一步研究和挖掘利用这一古老的优良作物具有重要的战略意义。山黧豆相关研究报道可追溯到1861年,距今已有160年的历史。在同行学者的不懈努力下,山黧豆基础研究及种质资源利用等方面均取得了显著的阶段性成果。该文系统回顾了山黧豆研究160年以来的发展历程,依托历史文献进行了梳理和分析。首先,基于山黧豆神经活性物质β ODAP的分离和鉴定、神经山黧豆中毒机制的探索、β ODAP生物合成途径解析等重要研究节点将整个山黧豆研究进程划分为山黧豆中毒因素的探索、神经山黧豆中毒机理解析和神经山黧豆中毒及β ODAP生物学功能的再认识等三个阶段。其次,总结了山黧豆在毒理学研究、种质资源利用、品质改良基础研究等方面取得的重要进展。特别是以兰州大学为代表的中国学者在β ODAP的分析检测、生物合成途径、山黧豆生理生态学研究及种质资源利用等方面进行了深入探讨,确立了中国在国际山黧豆研究中的主流地位。最后,针对目前山黧豆分子生物学基础研究相对滞后、种质资源缺乏系统利用等问题进行了展望,提出了未来的重点发展方向,为山黧豆种质资源的进一步深入挖掘和应用提供参考。     

水分胁迫下山黧豆多胺代谢与 β-N-草酰-L-α ,β-二氨基丙酸积累相关性的研究

为了研究水分胁迫下山黧豆 (LathyrussativusL .)叶片中多胺代谢与 β_N_草酰_L_α,β_二氨基丙酸 (ODAP)积累的相关关系 ,利用聚乙二醇 (PEG)对山黧豆幼苗进行水分胁迫处理 ,同时加入腐胺 (Put) ,α_二氟甲基精氨酸(DFMA)和Put DFMA。实验结果表明 ,随PEG处理时间的延长 ,山黧豆幼苗叶片中Put、亚精胺 (Spd)和精胺 (Spm)含量逐渐增加 ,特别是Spm含量增加显著 ,同时ODAP逐渐积累 ;在PEG处理的同时 ,加入Put使得Put、Spd含量显著增加 ,但对Spm影响不大 ,同样对ODAP含量影响也较小 ;加入DFMA可显著抑制Put、Spd、Spm的积累 ,同时也抑制了ODAP的积累 ;加入Put DFMA ,Put可以部分地减缓DFMA对两种内源多胺 (Put和Spd)合成的抑制作用 ,但对Spm所受DFMA的抑制作用影响不大 ,这时ODAP的积累也受到抑制。由此可见 ,水分胁迫对山黧豆幼苗叶片中多胺特别是Spm含量的增加与ODAP的积累密切相关。     

家山黧豆及其毒素ODAP的研究

家山黧豆作为人类的食物及动物的饲料,最早可追溯到新石器时代。长期以来,因其营养丰富,具有耐旱、耐寒等优良生产性状,广受世界各地的青睐。特别是在大旱年份,在其它粮食作物绝收的情况下,仍有较好的收成,因此,被视为干旱及半干旱地区首选的优良作物。但由于其含有毒素β-ODAP,使山黧豆的种植受到限制。近年来,由于人类对肉质品的量与质的多元化的需求,家山黧豆这一潜在的、丰产的、高营养的豆科作物,已引起各国学者的广泛关注。从家山黧豆在植物学、遗传学、分子生物学、生态学、营养学、山黧豆中毒、毒理学、毒素ODAP、ODAP的分析方法、ODAP的生物合成途径、养殖业等几个方面的研究进展作了简要综述。     

山黧豆及其神经毒素(ODAP)的研究进展

山黧豆具有耐寒、耐旱等优良生产性状，且营养丰富，在恶劣的天气条件下仍能维持较高的产量，因此它是半干旱地区的潜在粮食作物。但长期食用山黧豆会引起中毒，这是由于山黧豆中含有的神经毒素（ＯＤＡＰ）所致，因此筛选无毒品系具有重要意义。本文对山黧豆神经毒素（ＯＤＡＰ）的生物合成毒素异构化等方面作简要综述。     

渗透胁迫对山黧豆幼苗H2O2及毒素积累的影响

用PEG经根部处理15d龄的山黧豆幼苗,取幼苗叶片为实验材料,测定过氧化氢酶（CAT）、过氧化物酶（POD）活性、过氧化氢（H2O2）和毒素（β-N-oxalyl-α,β-diaminopropionic acid,ODAP）的含量。结果表明,随着PEG处理时间的延长,POD和CAT活性降低,而H2O2和ODAP含量显著升高;在PEG处理液中加入二乙基二硫代氨基甲酸钠(diethyldithiocarbamate,DDC)和氨基三唑(aminotriazole,AT)后分别抑制和促进H2O2的产生,DDC可降低叶片中ODAP的含量,AT则使ODAP积累。由此我们推测,水分胁迫条件下活性氧代谢与ODAP的积累有关。     

山黧豆毒素的薄层分离

通过对山黧豆中常见的20几种游离氨基酸薄层层析发现,其毒素ODAP的Rf值与好几种相关氨基酸的接近,而用阳离子交换柱对氨基酸抽提样进行简单的分离纯化,第一个洗脱出来的即为ODAP,这样在排除其它氨基酸干扰的情况下再进行ODAP的薄层分析将得到准确的结果。     

渗透胁迫对山黧豆幼苗H_2O_2及毒素积累的影响

用PEG经根部处理 15d龄的山黧豆幼苗 ,取幼苗叶片为实验材料 ,测定过氧化氢酶 (CAT)、过氧化物酶 (POD)活性、过氧化氢 (H2 O2 )和毒素 ( β N oxalyl α ,β diaminopropionicacid ,ODAP)的含量。结果表明 ,随着PEG处理时间的延长 ,POD和CAT活性降低 ,而H2 O2 和ODAP含量显著升高 ;在PEG处理液中加入二乙基二硫代氨基甲酸钠 (diethyldithiocarbamate ,DDC)和氨基三唑 (aminotriazole ,AT)后分别抑制和促进H2 O2 的产生 ,DDC可降低叶片中ODAP的含量 ,AT则使ODAP积累。由此我们推测 ,水分胁迫条件下活性氧代谢与ODAP的积累有关     

水分胁迫下山黧豆中ABA及ODAP的积累研究

用PEG、PEG+ABA、ABA分别处理15d龄的山黧豆幼苗,取其叶片为实验材料,测定内源ABA、ODAP、MDA和H₂O₂含量以及几种抗氧化酶活性,结果表明,与对照相比处理材料叶片中ABA和ODAP含量显著增加;外源ABA的加入降低了PEG胁迫引起的MDA和H₂O₂含量的增加,延缓了PEG胁迫引起的CAT活性的衰减,提高了GR活性.用外源ABA长时间处理山黧豆,发现叶片中ABA含量显著增加,随后出现ODAP的积累;ABA处理初期(0～3d)对叶片中活性氧代谢影响不大,随着ABA处理时间的延长(7～15d),可引起叶片中SOD、POD、CAT、GR活性的降低,MDA、H₂O₂含量的增加,表明ABA确实可促进ODAP的积累.     

山黧豆LsSULTR基因的克隆与表达分析

为研究山黧豆(Lathyrus sativus)硫酸盐转运蛋白(sulfate transporter,SULTR)与内源活性物质β N 草酰 L α,β 二氨基丙酸(β N oxalyl L α,β diaminopropionic acid,β ODAP)含量之间的关系,该研究采用序列比对的方法,从山黧豆转录组数据(SRP145030)中查找SULTR基因序列,并进行生物信息学分析;采用PCR方法克隆基因全长序列并进行组织特异性表达分析,以筛选与β ODAP含量相关的SULTR基因。结果表明：(1)经序列比对共查找到13条SULTR基因序列,其编码蛋白分别隶属于SULTR Ⅰ-Ⅳ。其中LsSULTR3;3和LsSULTR3;5具有SULTR蛋白家族保守的STAS结构域 (PF01740) 和Sulfate_transp结构域(PF00916),且二者活性受到转录因子MYB和激素响应等多个顺式作用元件的共同调节,并受蛋白水平互作及磷酸化等翻译后修饰调控。(2)成功获得LsSULTR3;3和LsSULTR3;5基因全长cDNA分别为1 962 bp和1 923 bp,分别编码653和640个氨基酸。(3)半定量RT PCR分析显示,LsSULTR3;3在山黧豆主根、成熟叶、茎、花、盛荚初期(S2)种子和鼓粒满期(S6)种子中表达,并在茎中的表达量较高;LsSULTR3;5在山黧豆主根、侧根、花、S2时期种子中均有表达,且花中的表达量最为显著。该研究结果为进一步研究山黧豆中硫酸盐的吸收、转运及同化、β ODAP的生物合成途径等奠定了基础。     

山黧豆属作物在土耳其的栽培与利用状况北大核心CSCD

众所周知,山黧豆起源于巴尔干半岛并传播到世界各地。在土耳其,山黧豆属作物有73个分类群(约1/3为地方特有种),在全国各地均有分布种植,但主要集中在黑海中部、东南部和安纳托利亚东部地区。该文介绍了山黧豆属作物在土耳其全国的分布和在局部地区的食用、观赏等资源利用和保存情况。通常,不同国家人民会因为文化差异形成不同的饮食习惯,但山黧豆大多被用于制作汤、面包或其他咸点。山黧豆的栽培技术则因地制宜,土耳其在边缘土地上进行旱作种植,中国和印度部分地区多采用轮作种植方法等。作为一种极“小众”的作物,山黧豆研究工作的从业人员极少,而且缺乏大型联合研究项目。因此,需要联合化学、表型和分子分析等方法,以保护世界各地包括土耳其在内的地方种质资源,尤其是培育适合土耳其、中国等世界各国的食饲兼用型低β-ODAP品种。总之,选育低β-ODAP或不含β-ODAP的山黧豆品种将有助于丰富人类在食用、饲用和观赏等方面的物种资源利用。     

Analysis of the molecular cascade responsible for mesodermal limb chondrogenesis: Sox genes and BMP signaling

Here, we have studied how Sox genes and BMP signaling are functionally coupled during limb chondrogenesis. Using the experimental model of TGFbeta1-induced interdigital digits, we dissect the sequence of morphological and molecular events during in vivo chondrogenesis. Our results show that Sox8 and Sox9 are the most precocious markers of limb cartilage, and their induction is independent and precedes the activation of BMP signaling. Sox10 appears also to cooperate with Sox9 and Sox8 in the establishment of the digit cartilages. In addition, we show that experimental induction of Sox gene expression in the interdigital mesoderm is accompanied by loss of the apoptotic response to exogenous BMPs. L-Sox5 and Sox6 are respectively induced coincident and after the expression of Bmpr1b in the prechondrogenic aggregate, and their activation correlates with the induction of Type II Collagen and Aggrecan genes in the differentiating cartilages. The expression of Bmpr1b precedes the appearance of morphological changes in the prechondrogenic aggregate and establishes a landmark from which the maintenance of the expression of all Sox genes and the progress of cartilage differentiation becomes dependent on BMPs. Moreover, we show that Ventroptin precedes Noggin in the modulation of BMP activity in the developing cartilages. In summary, our findings suggest that Sox8, Sox9, and Sox10 have a cooperative function conferring chondrogenic competence to limb mesoderm in response to BMP signals. In turn, BMPs in concert with Sox9, Sox6, and L-Sox5 would be responsible for the execution and maintenance of the cartilage differentiation program.     

Alterations of rx1 and pax6 expression levels at neural plate stages differentially affect the production of retinal cell types and maintenance of retinal stem cell qualities

rx1 and pax6 are necessary for the establishment of the vertebrate eye field and for the maintenance of the retinal stem cells that give rise to multiple retinal cell types. They also are differentially expressed in cellular layers in the retina when cell fates are being specified, and their expression levels differentially affect the production of amacrine cell subtypes. To determine whether rx1 and pax6 expression after the eye field is established simply maintains stem cell-like qualities or affects cell type differentiation, we used hormone-inducible constructs to increase or decrease levels/activity of each protein at two different neural plate stages. Our results indicate that rx1 regulates the size of the retinal stem cell pool because it broadly affected all cell types, whereas pax6 regulates more restricted retinal progenitor cells because it selectively affected different cell types in a time-dependent manner. Analysis of rx1 and pax6 effects on proliferation, and expression of stem cell or differentiation markers demonstrates that rx1 maintains cells in a stem cell state by promoting proliferation and delaying expression of neural identity and differentiation markers. Although pax6 also promotes proliferation, it differentially regulates neural identity and differentiation genes. Thus, these two genes work in parallel to regulate different, but overlapping aspects of retinal cell fate determination.     

At least two regions of the oncoprotein Tre2 are involved in its lack of GAP activity

The product of the human Tre2 oncogene is structurally related to the Ypt/Rab GTPase-activating proteins (Ypt/Rab GAPs). Particularly, the oncoprotein shares with the yeast proteins Msb3p and Msb4p, and with the human protein RN-tre the highly conserved TBC domain, forming the catalytically active domain of Ypt/Rab GAPs. Yet, the Tre2 oncogene seems to encode a nonfunctional Rab GAP. As regions flanking the TBC domain may be crucial for catalytic activity, regions located N- and C-terminally with respect to this domain were explored. For this, chimeric proteins created by sequence exchanges between the Tre2 oncoprotein and RN-tre were tested for their ability to replace functionally the Msb3p and Msb4p proteins in double-mutant yeast cells. These complementation experiments revealed, in addition to the TBC domain, a second Tre2 region involved in the oncoprotein's lack of GAP activity: a 93-aa region flanking the TBC domain on the C-terminal side.     

Six1 and Six4 are essential for Gdnf expression in the metanephric mesenchyme and ureteric bud formation, while Six1 deficiency alone causes mesonephric-tubule defects

Interaction between the ureteric-bud epithelium and the metanephric mesenchyme is important for kidney development. Six1 and Six4 are the mammalian homologs of Drosophila sine oculis, and they are coexpressed in the nephrogenic mesenchyme. Six1-deficient mice show varying kidney defects, while Six4-deficient mice have no apparent abnormalities. Here, we report Six1/Six4-deficient mice that we generated in order to elucidate the functions of Six4 in Six1-deficient kidney development. The Six1/Six4-deficient mice exhibited more severe kidney phenotypes than the Six1-deficient mice; kidney and ureter agenesis was observed in all the neonates examined. The Six1/Six4-deficient metanephric mesenchyme cells were directed toward kidney lineage but failed to express Pax2, Pax8, or Gdnf, whereas the expression of these genes was partially reduced or unchanged in the case of Six1 deficiency. Thus, Six4 cooperates with Six1 in the metanephric mesenchyme to regulate the level of Gdnf expression; this could explain the absence of the ureteric bud in the Six1/Six4-deficient mice. In contrast, Six1 deficiency alone caused defects in mesonephric-tubule formation, and these defects were not exacerbated in the Six1/Six4-deficient mesonephros. These results highlight the fact that Six1 and Six4 have collaborative functions in the metanephros but not in the mesonephros.     

Fruit load modulates flowering-related gene expression in buds of alternate-bearing ‘Moncada’ mandarin

Background and Aims

Gene determination of flowering is the result of complex interactions involving both promoters and inhibitors. In this study, the expression of flowering-related genes at the meristem level in alternate-bearing citrus trees is analysed, together with the interplay between buds and leaves in the determination of flowering.

Methods

First defruiting experiments were performed to manipulate blossoming intensity in ‘Moncada’ mandarin, Citrus clementina. Further defoliation was performed to elucidate the role leaves play in the flowering process. In both cases, the activity of flowering-related genes was investigated at the flower induction (November) and differentiation (February) stages.

Key Results

Study of the expression pattern of flowering-genes in buds from on (fully loaded) and off (without fruits) trees revealed that homologues of FLOWERING LOCUS T (CiFT), TWIN SISTER OF FT (TSF), APETALA1 (CsAP1) and LEAFY (CsLFY) were negatively affected by fruit load. CiFT and TSF activities showed a marked increase in buds from off trees through the study period (ten-fold in November). By contrast, expression of the homologues of the flowering inhibitors of TERMINAL FLOWER 1 (CsTFL), TERMINAL FLOWER 2 (TFL2) and FLOWERING LOCUS C (FLC) was generally lower in off trees. Regarding floral identity genes, the increase in CsAP1 expression in off trees was much greater in buds than in leaves, and significant variations in CsLFY expression (approx. 20 %) were found only in February. Defoliation experiments further revealed that the absence of leaves completely abolished blossoming and severely affected the expression of most of the flowering-related genes, particularly decreasing the activity of floral promoters and of CsAP1 at the induction stage.

Conclusions

These results suggest that the presence of fruit affects flowering by greatly altering gene-expression not only at the leaf but also at the meristem level. Although leaves are required for flowering to occur, their absence strongly affects the activity of floral promoters and identity genes.