橡胶草90年来主要研究成果及最新研究进展

天然橡胶是重要的国防战略物质,巴西橡胶树( Hevea brasiliensis (Willd. ex A. Juss.) Muell. Arg)是天然橡胶的唯一来源,天然橡胶商业化形式极为单一,潜在的供给不足问题亟待解决。因此,寻找可替代巴西橡胶树的产胶植物一直受到全世界高度重视。蒲公英属橡胶草( Taraxacum kok-saghyz Rodin)根部含有与橡胶树橡胶类似的天然橡胶分子,该植物主要分布在温带和寒带地区,具有易于机械化收获、生长周期短、遗传转化相对容易等特点,是最具开发潜力的产胶植物。本文对橡胶草90年(1931 - 2018)来的研究历史和主要成果进行了概括,对近10年取得的最新成果进行了深度分析,并预测橡胶草在未来天然橡胶产业中的作用,期望为开展橡胶草商业化生产和橡胶生物合成相关基础研究提供一定的参考。     

产胶植物橡胶转移酶的研究进展

天然橡胶合成中,橡胶转移酶催化异戊二烯焦磷酸的多聚化过程,这一过程对天然橡胶的品质及产量至关重要。橡胶转移酶及其性质、橡胶生物合成分子机理及橡胶的分子量大小决定机制是亟待解决的重要科学问题。本文介绍了以巴西橡胶树为主的产胶植物橡胶转移酶的性质和生物学功能,对橡胶转移酶的分离与鉴定及其活性调节等研究进展进行了综述。     

橡胶草分子生物学研究进展及展望

重点总结了国外对橡胶草分子生物学领域的最新研究成果,并对该领域今后的研究方向做了简要展望。     

橡胶草的研究进展

天然橡胶是一种不可替代的重要战略工业原料, 用途广泛。巴西橡胶树作为天然橡胶的主要来源, 受种植面积限制, 难以满足全球日益增长的对天然橡胶的需求。而南美叶疫病也是巴西橡胶树(Hevea brasiliensis)安全的潜在威胁。蒲公英属产胶植物(橡胶草)最早发现于20世纪30年代, 可产生高质量的天然橡胶, 具有生长周期短、地理适应范围广、适合机械化生产等特点, 被认为是一种理想的产胶备选作物。该文从橡胶草种质资源、遗传改良、栽培技术及产胶生物学机制等方面综述了国内外橡胶草的研究进展和存在的问题, 并为我国开展橡胶草相关研究提出建议。     

橡胶树橡胶生物合成调控相关基因表达丰度比较

蛋白质是构成生命系统的基本元件之一,是大部分生物学功能的执行者.蛋白质丰度与其生物学功能息息相关,其丰度受基因表达过程中各环节严格精密的调控.其中,蛋白质丰度与其相应mRNA丰度存在较强的相关性,蛋白质丰度差异的40％可由mRNA丰度来解释.茉莉酸信号途径调节巴西橡胶树中的天然橡胶生物合成,但相关基因彼此间的表达丰度差...     

植物焦磷酸酶(PPase)的研究进展

植物焦磷酸酶(PPase)可分为存在于细胞质中可溶性的无机焦磷酸酶和与膜结合的不可溶性焦磷酸酶.后者不仅能水解焦磷酸,同时还具有质子泵的功能.橡胶树乳管中与黄色体膜结合的不可溶性焦磷酸酶,是调控橡胶生物合成的一个必不可少的酶.对植物焦磷酸酶的结构及其功能和分子生物学研究的进展进行了综合论述,并着重阐述了焦磷酸酶在橡胶树橡胶生物合成中的作用.     

橡胶树橡胶生物合成调控相关基因的表达相关性分析

该文通过q PCR获得了正常割胶条件下,9个茉莉酸信号途径关键环节基因Hb COI1、Hb JAZ1、Hb JAZ2、Hb JAZ3、Hb MYC1、Hb MYC2、Hb MYC3、Hb MYC4、Hb MYC5和6个橡胶生物合成相关基因Hb HRT2、Hb SRPP、HbREF、Hb HMGR1、Hb HRT1、Hb GAPDH在5个橡胶树魏克汉种质和5个1981’IRRDB种质胶乳中的表达数据;通过皮尔逊相关系数分析了这15个基因彼此间的表达相关性,分别获得105对基因的双变量相关系数(r12)和偏相关系数(r12.3),所有105对基因的双变量相关系数(︱r12︱)和偏相关系数(︱r12.3︱)的平均值分别为0.486±0.220和0.304±0.211,达到0.05的差异显著水平。其中,r12与r12.3方向相同的63对(60%),方向相反的42对(40%);︱r12︱<︱r12. 3︱的23对(21. 905%),︱r12︱>︱r12.3︱的82对(78.095%);双变量相关显著性P<0.05的76对(72.38%),P<0.01的59对(56.19...     

橡胶树1981'IRRDB野生种质主要性状鉴定——1986-1994年大田鉴定区

为了加速橡胶树野生种质资源的开发利用,在进行产量等性状预测鉴定的基础上,1986-1994年共筛选162份1981′IRRDB种质布置于大田鉴定区。2004-2005年对这批种质进行了产胶量、树皮乳管数目鉴定以及茎围、抗风性调查,结果表明种质间存在较大差异,但从中不能直接筛选出可供商业化栽培的品种材料,只能选择其中存在有益基因源的种质纳入现有橡胶树的育种进程。早期预测鉴定结果与大田鉴定结果间的相关分析表明二者间相关程度较低,表明目前以Wickham栽培种质为基础试验材料形成的产量等方面的预测方法,难以适应对这些野生种质资源的鉴定评价,因此除继续利用常规的33月龄树皮解剖、试割等方法,还需要继续探索其他早期预测方法。     

巴西橡胶树43 kD橡胶粒子膜蛋白基因的cDNA克隆及表达

对43 kD的橡胶粒子膜蛋白进行了分离纯化和其N端氨基酸序列分析,根据N端氨基酸序列,设计一简并引物,通过3'RACE(Rapid Amplification ofcDNA Ends)的方法,获得了43 kD的橡胶粒子膜蛋白的cDNA.该cDNA含有1 385个核苷酸,含有完整的阅读框架,编码381个氨基酸.在终止密码子下游,包含有一个239bp的3'非编码区.该cDNA由5个首尾相连的重复单元组成,每个单元编码76个氨基酸组成的泛素(ubiquitin)单体.编码43 kD橡胶粒子蛋白的基因具有多个拷贝,在胶乳、叶片和树皮都表达.     

橡胶树茉莉酸信号途径相关基因表达与橡胶产量的相关性

割胶促进橡胶树合成天然橡胶与激活乳管细胞的茉莉酸信号途径密切相关,但茉莉酸信号途径关键环节的基因表达水平与干胶产量的相关性尚不清楚。为了找到与产量相关的分子标记,该研究采用qPCR技术,分析了割胶条件下茉莉酸信号途径关键环节的9个相关基因在5个橡胶树魏克汉种质和5个1981’IRRDB种质乳管细胞中的表达。结果表明:大多魏克汉种质的株次干胶产量显著高于1981’IRRDB种质。在9个基因中,除了HbMYC4和HbMYC5,其余7个基因在大多橡胶树魏克汉种质中的表达量均显著高于1981’IRRDB种质,尤其是HbMYC3基因表达差异性好,与干胶产量相关性高,有望作为橡胶树产量育种的一个分子标记。这对育种周期长的橡胶树产量育种具有实际应用价值。     

我国市售婴儿配方乳粉油脂配料使用情况分析

目的：了解我国市售婴儿配方乳粉的油脂配料使用情况及脂肪酸提供情况,为提升婴儿配方乳粉的营养水平及制定产品相关标准提供参考。方法：多渠道收集婴儿配方乳粉标签信息,统计分析油脂配料的种类、组合、最高添加量构成比及标识含量,比较全脂乳产品与脱脂乳产品、牛乳基产品与羊乳基产品、高必需脂肪酸产品与全部产品间的差异。均数和率的比较分别采用t检验和卡方检验。结果：共纳入269个婴儿配方乳粉。配料表分析显示,85%的产品使用了4种及以上的油脂配料,葵花籽油和椰子油在全部产品中的添加率最高,分别为88%、76%。牛、羊乳基配方粉的油脂配料使用情况存在差异,牛乳基配方粉中脂肪、亚油酸及α-亚麻酸的标识含量略高于羊乳基配方粉（P<0.05）。脱脂乳配方粉中,棕榈油添加率为32%,显著高于全脂乳产品 （P<0.05）。44例使用了棕榈油的产品中仅有4例强化了1,3-二油酸2-棕榈酸甘油三酯。结论：牛、羊乳基配方粉中的必需脂肪酸标识含量基本一致。现市售婴儿配方乳粉以多种油脂组合使用的方式,以尽可能模拟母乳脂肪酸模式,但有些油脂类原料使用的科学性还有待进一步研究。     

橡胶草TkAPC10基因的鉴定及其表达模式分析

APC/C是一类泛素连接酶E3复合体,在调控细胞周期过程中发挥重要作用。为了揭示橡胶草APC/C蛋白复合体的功能,鉴定了橡胶草TkAPC10基因,并对其表达模式进行了分析,初步确定了其功能。TkAPC10基因的ORF为579 bp,编码192个氨基酸,其基因组DNA序列为1 092 bp,包含6个外显子和5个内含子。基因组分析发现,TkAPC10以单拷贝的形式存在,其启动子序列除了含有TATA-box和CAAT-box增强子元件外,还有ABA、JA、光以及逆境响应相关的顺式作用元件。系统进化关系分析发现,不同物种的APC10蛋白具有很高的同源性,TKAPC10与莴苣LsAPC10的相似性最高达到99%,而与其他菊科植物的APC10蛋白相似性也达到95%以上。进一步采用qRT-PCR技术对TKAPC10的表达模式进行分析,结果表明,该基因在细胞分裂旺盛的组织（花、叶和根）中的表达量显著高于细胞分裂活动相对缓慢的组织（花梗）。外源ABA处理后,TKAPC10基因转录水平显著下降;而MeJA和ET处理后,该基因显著上调表达。经PEG6000以及甘露醇处理后,TKAPC10表达水平显著下降;而...     

橡胶草E2泛素结合酶基因TkUBC2基因的克隆及其表达分析

为解析橡胶草E2泛素结合酶在胁迫响应和信号转导中的功能,本文从橡胶草品系1151中克隆得到一个E2泛素结合酶基因,命名为TkUBC2,该基因编码区cDNA为459 bp,编码152个氨基酸。序列比对分析发现,不同物种间的UBC2高度同源,TkUBC2与莴苣、向日葵的UBC2相似性高达99%以上。采用荧光定量qRT-PCR技术分析该基因在橡胶草中的表达模式,结果表明,TkUBC2在不同组织中均有表达;而且PEG6000模拟干旱胁迫、甘露醇介导的渗透压胁迫以及植物激素茉莉酸甲酯(MeJA)、脱落酸(ABA)和乙烯利(ET)可诱导TkUBC2下调表达;而NaCl盐胁迫和UV辐射处理则上调TkUBC2表达。结果表明TkUBC2参与橡胶草抗逆反应、激素信号转导以及DNA损伤修复过程,以上结果为进一步解析该基因的功能奠定良好的基础。     

Natural rubber biosynthesis in plants,the rubber transferase complex,and metabolic engineering progress and prospects

Natural rubber (NR) is a nonfungible and valuable biopolymer, used to manufacture ~50 000 rubber products, including tires and medical gloves. Current production of NR is derived entirely from the para rubber tree (Hevea brasiliensis). The increasing demand for NR, coupled with limitations and vulnerability of H. brasiliensis production systems, has induced increasing interest among scientists and companies in potential alternative NR crops. Genetic/metabolic pathway engineering approaches, to generate NR‐enriched genotypes of alternative NR plants, are of great importance. However, although our knowledge of rubber biochemistry has significantly advanced, our current understanding of NR biosynthesis, the biosynthetic machinery and the molecular mechanisms involved remains incomplete. Two spatially separated metabolic pathways provide precursors for NR biosynthesis in plants and their genes and enzymes/complexes are quite well understood. In contrast, understanding of the proteins and genes involved in the final step(s)—the synthesis of the high molecular weight rubber polymer itself—is only now beginning to emerge. In this review, we provide a critical evaluation of recent research developments in NR biosynthesis, in vitro reconstitution, and the genetic and metabolic pathway engineering advances intended to improve NR content in plants, including H. brasiliensis, two other prospective alternative rubber crops, namely the rubber dandelion and guayule, and model species, such as lettuce. We describe a new model of the rubber transferase complex, which integrates these developments. In addition, we highlight the current challenges in NR biosynthesis research and future perspectives on metabolic pathway engineering of NR to speed alternative rubber crop commercial development.     

Synchronous flowering of the rubber tree (Hevea brasiliensis) induced by high solar radiation intensity

How tropical trees flower synchronously near the equator in the absence of significant day length variation or other meteorological cues has long been a puzzle. The rubber tree (Hevea brasiliensis) is used as a model to investigate this phenomenon. The annual cycle of solar radiation intensity is shown to correspond closely with the flowering of the rubber tree planted near the equator and in the subtropics. Unlike in temperate regions, where incoming solar radiation (insolation) is dependent on both day length and radiation intensity, insolation at the equator is due entirely to the latter. Insolation at the upper atmosphere peaks twice a year during the spring and autumn equinoxes, but the actual solar radiation that reaches the ground is attenuated to varying extents in different localities. The rubber tree shows one or two flowering seasons a year (with major and minor seasons in the latter) in accordance with the solar radiation intensity received. High solar radiation intensity, and in particular bright sunshine (as distinct from prolonged diffuse radiation), induces synchronous anthesis and blooming in Hevea around the time of the equinoxes. The same mechanism may be operational in other tropical tree species.     

Cloning and Sequencing of the meso-Diaminopimelate-d-dehydrogenase (ddh) Gene of Corynebacterium glutamicum

The ddh gene of Corynebacterium glutamicum encoding raesodiaminopimelate meso-diaminopimelate (meso-DAP)-d-dehydrogenase (DDH) involved in the lysine biosynthesis was cloned in a DAP auxotroph (dapD4) of Escherichia coli by complementation of the DAP auxotroph. Deletion analysis revealed that a ~1.7-kb XhoI-KpnI fragment contained the ddh structure gene. The specific activity of DDH was increased fourteen-fold when C. glutamicum was transformed with a recombinant plasmid harbouring the cloned ddh gene. Furthermore, the ddh gene has been sequenced [S. Ishino et al., Nucleic Acids Res., 15, 3917 (1987)] and some properties of the ddh gene are discussed.     

Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein,TkSRPP3, result in qualitative and quantitative changes in rubber metabolism

Several proteins have been identified and implicated in natural rubber biosynthesis, one of which, the small rubber particle protein (SRPP), was originally identified in Hevea brasiliensis as an abundant protein associated with cytosolic vesicles known as rubber particles. While previous in vitro studies suggest that SRPP plays a role in rubber biosynthesis, in vivo evidence is lacking to support this hypothesis. To address this issue, a transgene approach was taken in Taraxacum kok-saghyz (Russian dandelion or Tk) to determine if altered SRPP levels would influence rubber biosynthesis. Three dandelion SRPPs were found to be highly abundant on dandelion rubber particles. The most abundant particle associated SRPP, TkSRPP3, showed temporal and spatial patterns of expression consistent with patterns of natural rubber accumulation in dandelion. To confirm its role in rubber biosynthesis, TkSRPP3 expression was altered in Russian dandelion using over-expression and RNAi methods. While TkSRPP3 over-expressing lines had slightly higher levels of rubber in their roots, relative to the control, TkSRPP3 RNAi lines showed significant decreases in root rubber content and produced dramatically lower molecular weight rubber than the control line. Not only do results here provide in vivo evidence of TkSRPP proteins affecting the amount of rubber in dandelion root, but they also suggest a function in regulating the molecular weight of the cis-1, 4-polyisoprene polymer.     

Root apical diameter and root elongation rate of rubber seedlings (Hevea brasiliensis) show parallel responses to photoassimilate availability

The hypothesis that root apical diameter may be used to evaluate root growth potential was tested. Temporal variations in the apical diameter of individual roots of rubber seedlings ( Hevea brasiliensis ) were studied together with their elongation patterns, using root observation boxes under controlled conditions. This study confirmed the overall positive correlation between apical diameter and growth rale. Moreover, the two parameters, varied in the same way during the life of a given root. For roots with short growth duration, there was a parallel quick decrease in both apical diameter and elongation rate, whereas roots that grew for longer periods showed synchronous fluctuations for both parameters. Since the mean values for the secondary roots within a root system exhibited the same trends, variations in apical diameter and elongation rates should depend on factors influencing the whole root system. When related to shoot rhythmic growth, both apical diameter and elongation rates were depressed during the periods of leaf growth. These effects were enhanced and/or prolonged by shading, hence reinforcing the hypothesis that this development depends on assimilate availability. Such results can be interpreted in terms of a source-sink relationship within the whole plant by considering the apical diameter, representing the size of the meristem related to the number of rneristematic cells, as an indicator of each root's growth potential.     

The sucrose transporter HbSUT3 plays an active role in sucrose loading to laticifer and rubber productivity in exploited trees of Hevea brasiliensis (para rubber tree)

Efficient sucrose loading in rubber‐producing cells (laticifer cells) is essential for retaining rubber productivity in Hevea brasiliensis, but the molecular mechanisms underlying the regulation of this process remain unknown. Here, we functionally characterized a putative Hevea SUT member, HbSUT3, mainly in samples from regularly exploited trees. When expressed in yeast, HbSUT3 encodes a functional sucrose transporter that exhibits high sucrose affinity with a K_m value of 1.24 mm at pH 4.0, and possesses features typical of sucrose/H ⁺ symporters. In planta, when compared to the expression of other Hevea SUT genes, HbSUT3 was found to be the predominant member expressed in the rubber‐containing cytoplasm (latex) of laticifers. The comparison of HbSUT3 expression among twelve Hevea tissues demonstrates a relatively tissue‐specific pattern, i.e. expression primarily in the latex and in female flowers. HbSUT3 expression is induced by the latex stimulator Ethrel (an ethylene generator), and relates to its yield‐stimulating effect. Tapping (the act of rubber harvesting) markedly increased the expression of HbSUT3, whereas wounding alone had little effect. Moreover, the expression of HbSUT3 was found to be positively correlated with latex yield. Taken together, our results provide evidence favouring the involvement of HbSUT3 in sucrose loading into laticifers and in rubber productivity.