De Novo Transcriptome Analysis in Hevea brasiliensis to Unveil Genes Involved in Low Temperature Stress Response

Low temperature is one of the adversities threatening the growth and development and reduces the yield of rubber trees.However,molecular mechanisms toward rubber trees in response to low temperature are largely unclear.In this study,7,159 and 7,600 differentially expressed genes(DEGs)were identified in‘Reyan 73397’rubber trees.Through GO analysis,the catalytic activity was the representative of the GO term in the only DEGs at the two studied temperatures(room temperature and 4°C,respectively),while KEGG analysis showed that carbon metabolism was the most important grouping under the comparison of these two temperatures.In addition,expression of 9 members of transcription factor MYB family genes were further verified by qRT-PCR,and MYB family genes may play important roles in the regulation of rubber trees under low temperature stress.This study provided a theoretical foundation for(1)revealing the molecular mechanisms of rubber trees in response to low temperature and(2)breeding of tolerant varieties of rubber trees.

Evaluation of unconstrained and constrained mathematical functions to model girth growth of rubber trees (Hevea brasiliensis) using young agemeasurements

No attempt has been made to date to model growth in girth of rubber tree （Hevea brasiliansis）. We evaluated the few widely used growth functions to identify the most parsimonious and biologically reasonable model for describing the girth growth of young rubber trees based on an incomplete set of young age measurements. Monthly data for girth of immature trees （age 2 to 12 yearsi from two locations were sub- jected to modelling. Re-parameterized, unconstrained and constrained growth functions,of Richards （RM）, Gompertz （GM） and the monomo- lecular ＇model ^（MM） were fitted to data. Duration of growth was the firsf constraint introduced. In the stagel We attempted a population aver- age （PA） model to capture the trend in growth. The best PA model was fitted as a subject specific （SS） model. We used appropriate error vari- ance-covariance structure to account for correlation due to repeated measurements over time. Unconstrainecl functions underestimated the asymptotic maximum that did not reflective carrying capacity of the locations. Underestimafions were attributed to the partial set＇ of meas- urements made during the early growth phase of the trees. MM proved superior to RM and GM. In the randomcoefficient models, both Gf and Go appeared to be influenced by tree level effects. Inclusion of diagonal definite positive matrix removed the correlation between random effects. The results were similar at both locations. In the overall assessment MM appeared as the candidate model for studying the girth-age relationships in Hevea trees. Based on the fitted model we conclude that, in Hevea trees, growth rate is maintained at maximum value at to, then decreases until the final state at dG/dt 〉 0, resulting in yield curve with no period of accelerating growth. One physiological explanation is that photosynthetic activity in Hevea trees decreases as girth increases and constructive metabolism is larger than destructive metabolism.

Effect of natural and synthetic antioxidants on oxidative stability of FAMEs obtained from hevea brasiliensis

Biodiesel(fatty acid alkyl esters),an alternate to fossil fuel,has the tendency of autoxidation and hence requires antioxidants for long term storage.The influence of synthetic and natural antioxidants on the oxidative stability was analysed for fresh FAMEs(fatty acid methyl esters)obtained from hevea brasiliensis at 140 C.Higher activity was observed for synthetic antioxidants following the order of GA】BHT】DTBP】Q】GT-M】PH-M】GT-C,whereas the oxidative stability of stored FAMEs samples measured at 110 C reveals a nearly inverse trend.Storage stability was tested for the FAMEs obtained from hevea brasiliensis stored at 30 C,after addition of synthetic and natural antioxidants—butylated hydroxytoluene(BHT),2,6-di-tert-butylphenol(DTBP),quercetin(Q),gallic acid(GA),methanol extracts from green tea(GTM),pomegranate hull(PH-M),and chloroform extract of green tea(GT-C).Antioxidant activities above 1500 ppm was in the order of DTBP】BHT】GA】GT-C】GT-M】Q】PH-M.Synthetic antioxidants have been found more efficient to improve the storage stability of FAMEs obtained from hevea brasiliensis.DTBP in particular has the highest protection factor.

Molecular Cloning and Analysis of LEA14L2 Gene from Rubber Tree( Hevea brasiliensis Muell. Arg. )

Using an isotig encoding a putative polypeptide with high similarity to Arabidopsis LEA14 as a query, a 613 bp long cDNA was in silico cloned from the transeriptome data of rubber tree. The sequence nominated as HbLEA14L2 contains an ORF of 456 bp with 3 bp 5＇ UTR and 154 bp 3＇ UTR. Subsequently, a 464 bp eDNA and an 834 bp genome sequence containing this ORF was amplified and sequenced. Sequence analysis suggested that HbLEA14L2 has one intron and encodes 151 amino acids with a theoretical molecular weight of 16.55 kDa, isoleetric point of 4.93 and GRAVY value of -0.022, indicating a cytoplasmle localization pattern; HbLEA14L2 protein contains a conserved LEA_2 domain and belongs to LEA_2 subfamily, sharing 91%, 76%, 75%, 72% and 63% similarity with the homologous proteins in castor bean, leafy spurge, poplar, cotton, and Arabidopsis, respectively. Swiss-Model indicated that the tertiary structure of HbLEA14L2 is consisted of one α-helix and seven β-sheets, which was proposed to serve as a regulatory protein to prevent cellular desiccation.

Intra-and inter-clonal tree growth variations of Hevea brasiliensis

We evaluated the effects of planting densities （500, 1,000, 1,500 and 2,000 trees.ha-1） on tree growth performance （diameter at base, diameter at breast height, and clear bole height） of two clones （RRIM 2020 and PRIM 2025） of nine years old plantations of rubber tree （Hevea brasiliensis Muell. Arg） in Malaysia. For the four planting densities of the two clones, basal area and diameter at breast height declined with increasing planting density. Clear bole heights were greatest at 1,500 trees.haland lowest at 500 trees.ha-1 for the clone RRIM 2020, and at 2,000 trees.ha-1and 500 trees.halfor clone RRIM 2025. We conclude that the ideal planting density is 2,000 trees.ha＂l for obtaining high volume of wood production and 500 trees-ha-1 for high wood quality.

Molecular Cloning and Sequence Analysis of an Aquaporin Gene Related to Tapping Panel Dryness in Rubber Tree( Hevea brasiliensis Muell. Arg. )

As a syndrome with tapping incision blocked partly or entirely during latex exploiting in rubber tree （Hevea brasiliensis ）, ＂Tapping panel dryness （TPD）＂ causes great yield losses, thereby becoming the most important factor limiting rubber production. On the basis of an EST down-regulated in TPD-affected rubber trees, a 903 bp cDNA denoted HbPIP2;2 was isolated from the bark tissue with a combination of in silico cloning and RT-PCR. The cDNA contains an 867 hp ORF, 13 bp 5＇UTR and 23 bp 3＇ UTR. Sequence analysis indicated that HbPIP2;2 encodes 288 amino acids with a theoretical molecular weight （Mw） of 30. 71 kDa and isolectric point （Pi） of 8.20. Bioinformatics analysis suggested that the deduced protein is predicted to have six transmembrane helices located to the plasma membrane and harbor one conserved MIP domain that can be grouped into plasma membrane intrinsic proteins （PIPs） of aquaporin （AQP） family. Homolo- gy search revealed that the protein shares a similarity of more than 90% with the homologues in Ricinus communis, Popttlus trichocarpa, Juglans regia and Theobro- ma cacao, suooorting a hie.hly conserved evolution. This study provided basis for further uncovering the regulatory role of AOPs in TPD occurrence.

Advances in Tissue Culture of Rubber( Hevea brasiliensis L. )

Natural rubber, which is produced as a secondary plant metabolite, has been well recognized as an important raw industrial material due to its unique properties that can not be substituted by any alternative synthetic materials. Although more than 2000 plant species have been shown to possess the ability to synthe- size natural rubber, Hevea brasiliensis L. is yet the only one qualified as a commercial source for natural rubber production, which not only has important economic value, but ＂also leads to significant ecological and social benefits to maintain ecological forest microclimate and ease current ecological issues caused by dwindling natural forests. With the growing demand for natural rubber, providing a large number of high-quality rubber planting materials has become a major issue to be solved. In this paper, advances on tissue culture of H. brasiliensis were summarized, which laid the foundation for breeding high-quality rubber planting materials.

Molecular Cloning and Sequence Analysis of a Tonoplast Aquaporin Gene TIP1 Associated with Tapping Panel Dryness in Hevea brasiliensis

[Objective]To address the role of aquaporins（AQPs）in the occurrence of Tapping Panel Dryness（TPD）,a phenomenon with tapping incision blocked partly or entirely during latex exploiting in the rubber tree（Hevea brasiliensis）that causes great losses on rubber production,a tonoplast aquaporin gene associated with TPD occurence was cloned and analyzed.[Method]Based on an EST down-regulated in TPD-affected rubber trees,a 774 bp cDNA designated HbTIP1 was isolated from the bark tissue of Hevea brasiliensis with a combination of in silico cloning and RT-PCR.And the gene structure and sequence characteristics were analyzed using bioinformatics tools.[Result]The cDNA includes a 759 bp ORF,8 bp 5’UTR and 7 bp 3’UTR.Sequence analysis indicated that HbTIP1putatively encodes 252 amino acids with a theoretical molecular weight（Mw）of 25.88 kDa and isolectric point（pI）of 4.96.Bioinformatics analysis suggested that the deduced protein is predicted to have six transmembrane helices located to the vacuolar membrane and to harbor one conserved MIP domain that can be grouped into the tonoplast intrinsic proteins（TIPs）of aquaporin family.Homology search revealed that the protein shares a similarity of more than 90%with the homologues in Theobroma cacao,Prunus persica,Citrus sinensis and Ricinus communis,supporting a highly conserved evolution.[Conclusion]This study provided basis for further revealing the regulatory role of AQPs in the TPD occurrence.

Exploration and Practice of Rubber Based Agroforestry Complex Systems in China

Agroforestry ecosystems are constructed by simulating natural ecosystems, applying the principles of symbiosis in nature, and organizing multiple plant populations to coexist, while conducting targeted cultivation and structural control scientifically. Rubber agroforestry complex ecosystems aim for sustainable development in terms of industry, ecology, resource utilization, and the livelihoods of producers. Rubber agroforestry complex ecosystems create a complex production structure system that integrates biology, society, and the economy through species combinations. Rubber trees and associated biological components coordinate with each other, mutually promote growth, and yield a variety of products for producers. Cultivation techniques and patterns of rubber agroforestry are essential components of these ecosystems. This study analyzes the production practices of rubber agroforestry complex cultivation, with a focus on the development and characteristics (complexity, systematicity, intensity, and hierarchy) of rubber agroforestry systems using a literature analysis and a survey approach. It explores the types and scales of complex planting, specifications and forms, and major effects of complex cultivation. This study identifies successful rubber agroforestry cultivation patterns and practical techniques, as well as the potential benefits of developing rubber agroforestry cultivation. It also points out the shortcomings in the development of complex planting, including an emphasis on production practices but insufficient theoretical research, a focus on production but inadequate attention to the market, and an emphasis on yield while overlooking the improvement of standards, brands, and added value. There are various complex patterns for young rubber plantations, but relatively fewer for mature plantations. Based on this analysis, this study suggests that future efforts should focus on in-depth research on interspecies and environmental interactions in rubber agroforestry ecosystems, clearly define key roles, accelerate the innovation of development patterns, and strengthen the foundation for development. It recommends promoting and demonstrating successful rubber agroforestry complex patterns and providing technical training, developing product branding for rubber agroforestry patterns, enhancing product value, expanding the application functions of rubber-forest mixed crop products, and establishing a stable and sustainable industry chain. This study provide practical experience and theoretical insights in rubber agroforestry complex systems from China the potential to enrich the knowledge of rubber agroforestry composite systems, provide practical experience to improve the operating income of smallholders, and even promote the sustainable development of rubber plantations.

Divergent DNA Methylation Patterns Associated with Abiotic Stress in Hevea brasiliensis

Cytosine methylation is a fundamental epigenetic mechanism for gene-expression regulation and development in plants. Here, we report for the first time the identification of DNA methylation patterns and their putative relationship with abiotic stress in the tree crop Hevea brasiliensis （source of 99% of natural rubber in the world）. Regulatory sequences of four major genes involved in the mevalonate pathway （rubber biosynthesis pathway） and one general defense-related gene of three high-yielding popular rubber clones grown at two different agroclimatic conditions were analyzed for the presence of methylation. We found several significant variations in the methylation pattern at core DNA binding motifs within all the five genes. Several consistent clone-specific and location-specific methylation patterns were identified. The differences in methylation pattern observed at certain pivotal cis-regulatory sites indicate the direct impact of stress on the genome and support the hypothesis of site-specific stress-induced DNA methylation. It is assumed that some of the methylation patterns observed may be involved in the stress-responsive mechanism in plants by which they adapt to extreme conditions. The study also provide clues towards the existence of highly divergent phenotypic characters among Hevea clones despite their very similar genetic make-up. Altogether, the observations from this study prove beyond doubt that there exist epigenetic variations in Hevea and environmental factors play a significant role in the induction of site-specific epigenetic mutations in its genome.

In vitro Transient Expression System of Latex C-serum was used for Analysis of Hevein Promoter in Response to Abscisic Acid in Hevea brasiliensis

Hevein has been found to be an essential element in coagulation of rubber particles in latex of rubber trees. In a previous study, we cloned a 1 241-bp fragment of a 5＇ upstream region of the hevein gene by genome walking. This fragment was analyzed by a 5＇ end nested deletion method in the present study, fused with a uidA （gus） gene to produce a series of tested constructs, which were transferred into C-serum of latex and the Gus activities were detected. Results showed that the fragment from -749 to -292 was sufficient for expression of gus gene in latex, and the fragment from -292 to -168 was crucial in response to abscisic acid inducement. In a transient transgenic test of rubber leaf with particle bombardment, construct Hev749 conferred gus-specific expression in veins, in which the latex tubes mainly distributed. This implies that the fragment from -749 to -292 was laticiferous-specific.

The rapid determination of volatile fatty acid number in para rubber latex using fourier transform-near infrared spectroscopy based on quantification and discrimination model

Volatile Fatty Acid number(VFA no.)is one of the parameters indicating the state of quality of Para rubber latex at that particular time.Most factories analyze this parameter using standard analytical method as in ISO 506:1992(E).Nevertheless,this procedure is complicated,chemical and time consuming,as well as skilled analyst required.Therefore,near infrared(NIR)spectroscopy which is rapid,accurate and nonchemicals method was applied to determine the VFA no.infield latex and concentrated latex based on quantification and discriminant model.The best calibration equation was obtained from standard normal variate(SNV)spectra in the region of 6109.7–5770.3,4613.1–4242.9 cm^(-1) with R?0:832,SECV?0:036 and no bias.From the performance check,statistically it was found that SECV and bias were low enough for practical acceptance and the predicted VFA no.was not di®erent significantly from actual VFA no.at 95% confidence intervals.In addition,discriminant model was developed to separate good quality latex from the deteriorated latex using VFA no.at 0.06 as standard as in ISO 2004:2010(E).The discriminant model can be used to screen the latex with overall accuracy of 91.86%in validation set.

Weed Control Strips Influences on the Rubber Tree Growth

Hevea brasiliensis (rubber tree) is the most widely cultivated tree species for producing natural rubber latex whose productivity can be reduced by weeds. Field experiment was carried out during two years to evaluate the growth of rubber tree plants submitted to different weed control strips (0-weed-free, 50, 100, 150, 200, 250, 300, 350, and 400-(weedy cm) and to determine the critical width of weed control strip during the establishment of rubber tree plantation. Weed community was composed mainly by Urochloa decumbens (90%). The width of the weed control strip influenced on the initial plant growth of rubber trees, reducing the plant height, stem diameter, leaf area, and aboveground dry mass accumulation. In consequence, crop plantation was affected by the width of weed control. The critical width of the weed control strip for establishment of a rubber tree plantation was within 100 cm.

Relationship between the number of tapping-induced secondary laticifer lines and rubber yield among Hevea germplasm

The lack of suitable early selection parameters means that traditional rubber breeding for yield is time consuming and inefficient. Laticifer is a tissue specific fo natural rubber biosynthesis and storage in rubber tree. The number of the secondary laticifers in the trunk bark tissues is positively correlated with rubber yield in the matured rubber trees that are regularly tapped. In the present study the rubber yield from 280 of 4–5 year-old virgin trees from7 cross combinations was compared with the number o newly differentiated secondary laticifers caused by tapping. Results showed that the number of tapping induced lines of secondary laticifers varied in differen germplasm and was positively related to the rubber yield indicating this could be used as a suitable parameter fo early evaluation of yield potential of rubber trees.

植物激素对橡胶树热激转录因子家族成员表达的影响

热激转录因子(heat shock transcription factor,Hsf)是一类重要的转录因子,调控ROS清除酶和热激蛋白(heat shock protein,HSP)的活性,从而提高植物的抗逆性。为了研究Hsf调控橡胶树响应非生物胁迫(低温,干旱等),我们分析了橡胶树HbHsfs基因家族的亚类及亚类成员特征;同时,通过Q-PCR技术分析了Hsf家族在中响应茉莉酸甲酯(methyl jasmonate,Me JA)、脱落酸(abscisic acid,ABA)和乙烯(ethylene,ET)的表达模式。橡胶树Hsf家族30个成员中分别响应MeJA、ABA和ET上调表达的有20、9和22个,其中HbHsfA3b、HbHsfA4b、HbHsfA6a和HbHsfA8a均受其诱导表达,但受激素诱导表达的变化规律与响应的时间和强度相关。橡胶树HbHsfs基因受多种植物激素诱导表达,暗示着基因功能的多样性和复杂性。本研究探索了HbHsfs参与MeJA、ABA、ET等激素介导的信号转导途径,为揭示HbHsfs调控橡胶树对低温、干旱等非生物胁迫的耐受性提供理论依据。

巴西橡胶树HbHMGR1在开割前后胶乳中的互作蛋白筛选

3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)是调控"碳源"进入类异戊二烯代谢的关键限速酶。天然橡胶是一种类异戊二烯化合物,主要来自于巴西橡胶树(以下简称橡胶树)。割胶是获取天然橡胶的主要方式。在前期研究中,发现了开割树胶乳的橡胶合成效率远高于未开割树。本研究分别构建了未开割树和开割树胶乳的酵母双杂交c DNA文库,利用前期制备的pGBKT7-HbHMGR1诱饵载体分别对两个文库进行互作蛋白筛选,并对得到的阳性克隆进行回转验证。文库构建结果显示两个酵母文库的库容量均在1.2×10^(7)CFU以上,平均插入片段均大于1.3 kb,质量较高,可用于后续筛库实验。文库筛选结果显示从未开割树胶乳文库中筛选到3个候选互作蛋白,其中包含HbHMGR1自身(scaffold0419_22144),推测割胶前HbHMGR1可能以二聚体形式存在;在开割树胶乳文库中筛选到6个候选互作蛋白,其中包含一个丰度极高的丝氨酸/苏氨酸激酶受体相关蛋白(STRAP,scaffold0891_7224),表明该蛋白很可能通过与HbHMGR1的结合来调控其活性。本研究为深入认识HbHMGR1在调控"碳源"在橡胶树割胶前后之间的分配模式提供了参考依据。

橡胶间种百香果的不同栽培模式探讨

通过对橡胶(Hevea brasiliensis)林下间作百香果(Passiflora edulia Sims)的立体栽培模式进行比较研究,希望能够提高经济收入,给橡胶林下间作作物的种植模式提供启发和参考。

HbTCTP及其截短体酵母双杂交诱饵载体的构建及鉴定

为进一步鉴定橡胶树翻译控制肿瘤蛋白(HbTCTP)与橡胶延伸因子1 (HbREF1)互作的分子区域,构建Hb TCTP及其截短体的诱饵载体。本研究利用RT-PCR技术从橡胶树胶乳中扩增出包含HbTCTP不同结构域的片段,克隆到p GBKT7表达载体,通过酶切、菌落PCR和测序鉴定后,转化酵母Y2HGold感受态细胞,检测诱饵蛋白是否具有自激活作用和毒性。结果显示,成功构建6个包含HbTCTP不同结构域的诱饵载体,且诱饵载体对酵母宿主细胞无毒性和无自主激活报告基因的功能。本研究结果为进一步筛选HbTCTP与HbREF1互作的分子区域及阐明HbTCTP的功能提供科学依据。

后基因组时代橡胶树分子生物学

天然橡胶是一种重要的工业原材料。橡胶树(Hevea brasiliensis Muell. Arg.)是天然橡胶的主要来源。由于缺乏基因组信息以及基因功能验证平台,长期以来,橡胶树分子生物学研究进展缓慢,严重限制了生产技术的改进和产业的转型升级。2013年,橡胶树基因组草图的发布标志着橡胶树生理与分子生物学的研究进入后基因组时代。本研究从基因组、转录组与蛋白质组、重要功能基因和基因家族鉴定、高密度连锁图谱构建及群体遗传学等方面对近年来橡胶树分子生物学领域的重要进展进行综述,并对今后的研究重点作出展望。

巴西橡胶树小孢子单细胞体外扩增体系的优化

高质量单细胞的获得且进行高效率的扩增是单细胞测序的前提,而植物细胞因为存在细胞壁使得单细胞扩增变得困难。本研究以巴西橡胶树‘热研7-33-97’幼嫩雄花为试验材料,使用5种不同的酶液在不同的时间和渗透压稳定剂下探索获得高质量且易扩增的巴西橡胶树小孢子单细胞,同时对单细胞扩增过程进行相应的探索优化。结果表明,使用8%纤维素酶+6%果胶酶+3%蜗牛酶混合酶,0.6 mol/L甘露醇作为渗透压稳定剂,37℃酶解45 min后可以得到品质优良的巴西橡胶树小孢子单细胞;单细胞扩增过程确定细胞裂解酶用量为0.5μL,细胞裂解时间为80 min,预扩增循环数为10个循环,可以得到准确且覆盖度高的单细胞扩增产物,经Dot-blot杂交验证其确实来自于巴西橡胶树基因组。本研究为后续巴西橡胶树单细胞全基因组测序,构建巴西橡胶树高分辨SNP遗传图谱提供基础性技术支持。