巴西橡胶树橡胶生物合成关键基因REF,SRPP,HRT1和HRT2的表达分析

橡胶延长因子REF、小橡胶粒子蛋白SRPP、橡胶转移酶HRT1和HRT2是巴西橡胶树胶乳中的主要橡胶粒子蛋白,它们在橡胶生物合成中发挥重要作用,与橡胶树胶乳产量密切相关。为进一步探明REF、SRPP、HRT1和HRT2的基因表达与橡胶树胶乳产量之间的关系,以成龄未开割橡胶树无性系热研7-33-97胶乳为材料,通过实时荧光定量PCR的方法,对割胶伤害、外源乙烯利和茉莉酸刺激的处理条件下,橡胶树胶乳中的REF、SRPP、HRT1和HRT2的基因表达进行了分析。结果表明,随着割胶刀次的增加,REF基因在第4刀的表达量最高,而SRPP、HRT1和HRT2基因则在第6刀表达量达到最高;而在乙烯利和茉莉酸刺激处理下,REF、SRPP、HRT1和HRT2基因均在刺激8 h后表达量达到最高。因此,割胶(机械伤害)、外源乙烯利和茉莉酸刺激促进橡胶树产胶可能与它们提高橡胶生物合成相关橡胶粒子蛋白REF、SRPP、HRT1和HRT2的基因表达具有密切关系。     

巴西橡胶树HbSIP2互作蛋白的筛选和鉴定

可溶性无机焦磷酸酶在天然橡胶生物合成中具有重要的调控作用,HbSIP2是胶乳中关键的可溶性无机焦磷酸酶基因。为了深入了解HbSIP2调控橡胶生物合成的机理,本研究对HbSIP2互作蛋白进行了筛选和鉴定。结果表明:诱饵载体p GBKT7-Hb SIP2无自激活活性,且对酵母无毒性作用,可以用于酵母文库筛选。将诱饵载体与橡胶树胶乳cDNA文库进行杂交,初步筛选获得20个与HbSIP2互作的蛋白。进一步通过双分子荧光互补实验证实,Hb SIP2能够与橡胶延伸因子发生蛋白互作。本研究结果为HbSIP2调控橡胶生物合成的机理研究提供了重要的理论依据。     

新型抗体结合蛋白CBD—SPG的设计与表达

设计并表达可用于纯化IgG的新型高栽量抗体结合蛋白CBD—SPG。利用基因重组技术将纤维素结合结构域（Cellulose Binding Domain,CBD）基因插入到表达载体pET28a—SPG中,获得重组质粒pET28a—CBD—SPG,并转化大肠杆菌曰位J（DE3）。IPTG诱导CBD—SPG融合蛋白表达,并用SDS—PAGE和Westernblot对表达产物进行鉴定。重组表达质粒pET28a—CBD-SPG经双酶切及测序验证无误;表达产物经SDS．PAGE和WesternBlot分析表明融合蛋白的表观分子量约为40kD;CBD—SPG具有良好的结合纤维素和抗体的能力,晶体纤维素Avicelphl01对CBD—SPG的载量可达11．61mg／g（w／w）。成功构建并运用原核系统表达CBD-SPG;CBD—SPG在保持良好抗体结合能力的同时,更具有了结合纤维素的能力,有望成为一种新型的亲和材料。     

小麦低分子量麦谷蛋白亚基组成研究

利用改良的两步一向SDS—PAGE(two—step one—dimensional SDS—PAGE)分析了几种小麦低分子量麦谷蛋白亚基(LMW-GS)组成。70％热乙醇提取总谷蛋白,11％分离胶进行第一步SDS—PAGE分离．电泳1h后切取顶端1cm胶条并置于巯基乙醇溶液进行还原,还原后的胶条于11%～16．5％的梯度胶进行第二步SDS—PAGE分离。结果显示。两步一向SDS—PAGE可以彻底除去清蛋白、球蛋白和醇溶蛋白对LMW—GS分离的背景干扰。提高LMW—GS的分辨率。对几种小麦低分子量麦谷蛋白亚基分析表明：LMW-GS组合比HMW—GS更为丰富,每种小麦含有2～5种B亚基,2～4种C亚基．B、C亚基的总数量为4～8种。     

巴西橡胶树胶乳中DUF1262结构域蛋白结构与基因表达分析

为了揭示天然橡胶生物合成酶互作蛋白结构及其在天然橡胶生物合成过程中的功能。本研究以橡胶树胶乳橡胶粒子总蛋白为研究对象,采用免疫共沉淀实验技术以天然橡胶合成关键酶顺式-异戊二烯基转移酶（CPT）抗体从胶乳中捕获了1个含DUF1262结构域的未知功能蛋白。生物信息学分析表明橡胶树基因组中包含50个编码含DUF1262结构域蛋白的基因序列;蛋白质相互作用网络分析表明DUF1262结构域蛋白可能参与调节信号转导或转录调控等过程;荧光定量PCR结果表明编码该蛋白基因的转录本在根、叶、花、枝和胶乳等组织中广泛分布,但在胶乳中表达较低,在树皮表达较高;水杨酸、脱落酸、过氧化氢及干旱处理可增强该基因在叶片中的转录水平。本研究证明DUF1262参与橡胶树逆境反应等生理过程,为揭示胶乳生物合成调控机制提供新线索。     

结核分枝杆菌菌体蛋白双向电泳技术的优化

目的：提取结核分枝杆菌菌体蛋白并建立一种利用双向电泳分离结核分枝杆菌蛋白质组的方法。方法：分离提取结核分枝杆菌菌体蛋白。样品采用不同pH梯度的鹏胶条进行第一向等电聚焦,12％SDS—PAGE凝胶进行二向电泳。银染后双向电泳图谱用Molecular Image Fx激光图像扫描仪扫描,PDQuest6．0软件完成配比分析。结果：优化了结核分枝杆菌菌体蛋白的提取方法,用裂解液8mol／L尿素结合2mol／L硫脲,140mmol／LDTT,0．5％biolyte,4％CHAPs,400mg／m1lOG处理,成功提取了蛋白,并通过结核分枝杆菌双向电泳技术体系的优化,建立了结核分枝杆菌菌体蛋白的分解图谱。pH4—7及pH7—10两胶面上共1387个点,占所检测到的蛋白总数的86％,绝大部分（1194个）蛋白位于pH4—7范围内。结论：为进一步开展结核分枝杆菌的比较蛋白质组学研究提供了方法学参考。     

团头鲂卵壳蛋白的分离与纯化

报道了一种从团头鲂卵壳中提取卵壳蛋白的有效方法。该提取淮经SDS－PAGE电泳鉴定后,证明有三条主要的蛋白带,其分子量分别为64KDa,56KDa和52KDa,借助于制备型SDS－PAGE电泳和电洗脱分离纯化技术,获得了三种高纯度的印壳蛋白。     

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。     

一种快速、高效的橡胶树胶乳总RNA 提取方法

胶乳是橡胶树(Hevea brasiliensis)乳管中特殊的细胞质, 主要由橡胶粒子、黄色体、F-W粒子和普通细胞质成分构成, 其中橡胶粒子占20%-40%, 蛋白含量高达1%-2%。由于高比例橡胶粒子和蛋白质的干扰, 目前使用的胶乳RNA提取方 法都具有步骤繁琐、胶乳需求量大、操作技巧性强不易掌握等缺点。为快速、高效地获取高质量的胶乳RNA, 我们在现有方法的基础上摸索出一套步骤简单、容易操作、快速、高效提取橡胶树胶乳总RNA的简易方法, 获得了较好的实验效果。紫外分光光度计、RT-PCR和RACE分析结果表明, 使用该方法提取的胶乳RNA质量完全能够满足相应的分子操作, 但所需时间仅为目前常用方法的50%, RNA获得率提高了2-3倍, 操作难度大大降低。     

一种快速、高效的橡胶树胶乳总RNA提取方法

胶乳是橡胶树（Hevea brasiliensis）乳管中特殊的细胞质,主要由橡胶粒子、黄色体、F-W粒子和普通细胞质成分构成,其中橡胶粒子占20％-40％,蛋白含量高达1％-2％。由于高比例橡胶粒子和蛋白质的干扰,目前使用的胶乳RNA提取方法都具有步骤繁琐、胶乳需求量大、操作技巧性强不易掌握等缺点。为快速、高效地获取高质量的胶乳RNA,我们在现有方法的基础上摸索出一套步骤简单、容易操作、快速、高效提取橡胶树胶乳总RNA的简易方法,获得了较好的实验效果。紫外分光光度计、RT-PCR和RACE分析结果表明,使用该方法提取的胶乳RNA质量完全能够满足相应的分子操作,但所需时间仅为目前常用方法的50％,RNA获得率提高了2-3倍,操作难度大大降低。     

The micromorphology and protein characterization of rubber particles in Ficus carica,Ficus benghalensis and Hevea brasiliensis

Rubber biosynthesis takes place on the surface of rubber particles. These particles are surrounded by a monolayer membrane in which the rubber transferase is anchored. In order to gain better insight into whether rubber particles from different plant species share common structural characteristics, the micromorphology of rubber particles from Ficus carica, Ficus benghalensis, and Hevea brasiliensis was examined by electron microscopy. Rubber particles of all three species were spherical in shape, and the size of rubber particles of H. brasiliensis was much smaller than those of F. carica and F. benghalensis. In addition, investigations were undertaken to compare the cross-reactivity of the antibody raised against either the H. brasiliensis small rubber particle protein (SRPP) which is suggested to be involved in rubber biosynthesis, or the cis-prenyltransferase (CPT) which has an activity similar to rubber transferase. Both western analysis and TEM-immunogold labelling studies showed that rubber particles of F. carica and F. benghalensis do not contain the SRPP. None of the rubber particles in F. carica, F. benghalensis and H. brasiliensis contained the CPT, suggesting that the CPT itself could not catalyse the formation of high molecular weight rubber. These results indicate that rubber particles in the three different plant species investigated share some degree of similarity in architecture, and that the SRPP and CPT themselves are not the core proteins necessary for rubber biosynthesis.     

Micromorphological characterization and label-free quantitation of small rubber particle protein in natural rubber latex

Commercial natural rubber is traditionally supplied by Hevea brasiliensis, but now there is a big energy problem because of the limited resource and increasing demand. Intensive study of key rubber-related substances is urgently needed for further research of in vitro biosynthesis of natural rubber. Natural rubber is biosynthesized on the surface of rubber particles. A membrane protein called small rubber particle protein (SRPP) is a key protein associated closely with rubber biosynthesis; however, SRPP in different plants has been only qualitatively studied, and there are no quantitative reports so far. In this work, H. brasiliensis was chosen as a model plant. The microscopic distribution of SRPP on the rubber particles during the washing process was investigated by transmission electron microscopy–immunogold labeling. A label-free surface plasmon resonance (SPR) immunosensor was developed to quantify SRPP in H. brasiliensis for the first time. The immunosensor was then used to rapidly detect and analyze SRPP in dandelions and prickly lettuce latex samples. The label-free SPR immunosensor can be a desirable tool for rapid quantitation of the membrane protein SRPP, with excellent assay efficiency, high sensitivity, and high specificity. The method lays the foundation for further study of the functional relationship between SRPP and natural rubber content.     

Identification of natural rubber and characterization of rubber biosynthetic activity in fig tree

Natural rubber was extracted from the fig tree (Ficus carica) cultivated in Korea as part of a survey of rubber producing plants. Fourier transform infrared and (13)C nuclear magnetic resonance analysis of samples prepared by successive extraction with acetone and benzene confirmed that the benzene-soluble residues are natural rubber, cis-1,4-polyisoprene. The rubber content in the latex of fig tree was about 4%, whereas the rubber content in the bark, leaf, and fruit was 0.3%, 0.1%, and 0.1%, respectively. Gel-permeation chromatography revealed that the molecular size of the natural rubber from fig tree is about 190 kD. Similar to rubber tree (Hevea brasiliensis) and guayule (Parthenium argentatum Gray), rubber biosynthesis in fig tree is tightly associated with rubber particles. The rubber transferase in rubber particles exhibited a higher affinity for farnesyl pyrophosphate than for isopentenyl pyrophosphate, with apparent K(m) values of 2.8 and 228 microM, respectively. Examination of latex serum from fig tree by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed major proteins of 25 and 48 kD in size, and several proteins with molecular mass below 20 and above 100 kD. Partial N-terminal amino acid sequencing and immunochemical analyses revealed that the 25- and 48-kD proteins were novel and not related to any other suggested rubber transferases. The effect of EDTA and Mg(2+) ion on in vitro rubber biosynthesis in fig tree and rubber tree suggested that divalent metal ion present in the latex serum is an important factor in determining the different rubber biosynthetic activities in fig tree and rubber tree.     

In-depth proteome analysis of the rubber particle of Hevea brasiliensis (para rubber tree)

The rubber particle is a special organelle in which natural rubber is synthesised and stored in the laticifers of Hevea brasiliensis. To better understand the biological functions of rubber particles and to identify the candidate rubber biosynthesis-related proteins, a comprehensive proteome analysis was performed on H. brasiliensis rubber particles using shotgun tandem mass spectrometry profiling approaches—resulting in a thorough report on the rubber particle proteins. A total of 186 rubber particle proteins were identified, with a range in relative molecular mass of 3.9–194.2 kDa and in isoelectric point values of 4.0–11.2. The rubber particle proteins were analysed for gene ontology and could be categorised into eight major groups according to their functions: including rubber biosynthesis, stress- or defence-related responses, protein processing and folding, signal transduction and cellular transport. In addition to well-known rubber biosynthesis-related proteins such as rubber elongation factor (REF), small rubber particle protein (SRPP) and cis-prenyl transferase (CPT), many proteins were firstly identified to be on the rubber particles, including cyclophilin, phospholipase D, cytochrome P450, small GTP-binding protein, clathrin, eukaryotic translation initiation factor, annexin, ABC transporter, translationally controlled tumour protein, ubiquitin-conjugating enzymes, and several homologues of REF, SRPP and CPT. A procedure of multiple reaction monitoring was established for further protein validation. This comprehensive proteome data of rubber particles would facilitate investigation into molecular mechanisms of biogenesis, self-homeostasis and rubber biosynthesis of the rubber particle, and might serve as valuable biomarkers in molecular breeding studies of H. brasiliensis and other alternative rubber-producing species.     

Initiator-independent and initiator-dependent rubber biosynthesis in Ficus elastica

The rubber-producing tree, Ficus elastica (the Indian rubber tree), requires the same substrates for rubber production as other rubber-producing plants, such as Hevea brasiliensis (the Brazilian or Para rubber tree), the major source of commercial natural rubber in the world, and Parthenium argentatum (guayule), a widely studied alternative for natural rubber production currently under commercial development. Rubber biosynthesis can be studied, in vitro, using purified, enzymatically active rubber particles, an initiator such as FPP, IPP as the source of monomer, and a metal cofactor such as Mg2+. However, unlike H. brasiliensis and P. argentatum, we show that enzymatically active rubber particles purified from F. elastica are able to synthesize rubber, in vitro, in the absence of added initiator. In this paper, we characterize, for the first time, the kinetic differences between initiator-dependent rubber biosynthesis, and initiator-independent rubber biosynthesis, and the effect of cofactor concentration on both of these processes.     

Rubber elongation factor from Hevea brasiliensis. Identification, characterization, and role in rubber biosynthesis

The presence of a protein, rubber elongation factor (REF), which is tightly bound to serum-free rubber particles purified from Hevea brasiliensis latex, is necessary for prenyltransferases from a number of sources to add multiple cis-isoprene units to rubber molecules. These prenyltransferases show normal farnesyl pyrophosphate synthase activity (two trans additions of isopentenyl pyrophosphate to dimethylallyl pyrophosphate) in the absence of REF bound to rubber particles. REF bound to rubber molecules can be highly purified from all other proteins in whole latex by treatment of rubber particles with low concentrations of detergent. Treatment of rubber particles with trypsin which hydrolyzes bound REF, removal of REF with high concentrations of various detergents, or treatment of whole latex with polyclonal antibodies specific for REF all prevent prenyltransferase from adding [14C]isopentenyl pyrophosphate to rubber molecules. However, we have not been successful using detergent-solubilized REF in the reconstitution of in vitro rubber biosynthesis with either REF-depleted rubber particles or allylic pyrophosphate primers. REF has a molecular mass of 14,600 Da and is associated specifically with rubber particles in whole latex. It makes up between 10-60% of the total protein in whole latex but is absent in C-serum, the supernatant fluid obtained when rubber particles are removed by centrifugation. The amount of REF in whole latex is proportional to the rubber content. Based on a number average molecular mass of 500,000 Da for rubber and the content of rubber and REF in whole latex or serum-free rubber particles, the stoichiometry of REF molecules to rubber molecules is 1:1 in both cases. There is sufficient REF to form a monomolecular protein layer coating large rubber particles (700-1,000 nm). In the electron microscope, serum-free rubber particle preparations contain particles with diameters from 800 to as small as 10 nm. In the presence of 1% sodium dodecyl sulfate no particles smaller than 100 nm are observed. We suggest that the smaller particles may be mainly composed of REF molecules.