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.

橡胶树与白粉病菌Oidium heveae亲和互作组织细胞学研究

白粉病是橡胶树生产中最重要的病害之一,是由粉孢属病菌Oidium heveae Steinm.引起。目前,对该病原菌在寄主中侵染行为及其与寄主互作的组织细胞学尚缺乏系统研究。本文利用显微技术,结合多种染色方法,观察了橡胶树白粉菌侵染橡胶树叶组织的细胞学变化及寄主的抗性反应。O.heveae在寄主上发育要经历5个关键发育时间点,即分生孢子萌发高峰(4hpi)、附着胞形成高峰(8hpi)、侵入结构(初生吸器)形成高峰(15hpi)、次生菌丝形成高峰(24hpi)、分生孢子梗形成高峰(5dpi);分生孢子萌发侵入寄主前,其能量来自自身贮存的能量物质。在互作过程中,病原菌初生吸器形成之后,橡胶树叶组织开始出现明显的氧暴发、胼胝质和乳突等抗性反应,活性氧在橡胶树与病原菌互作中起到了重要的作用,当橡胶树叶片中活性氧积累较低时,有利于O.heveae的发育及入侵,活性氧积累较高时,则引发寄主氧暴发等以阻止病原菌进一步扩展。O.heveae在寄主上发育的5个关键发育时间点分别属于病原菌侵染前期、潜育期和侵染后期,橡胶树叶组织早期亲和互作与中后期非亲和互作保持了专性寄生菌与寄主间发育平衡的进化关系。

不同树龄橡胶(Hevea brasiliensis)林细根生物量的垂直分布和年内动态

用根钻法对幼树期（5a）、初产期（9a）和旺产期（16a）3个树龄橡胶树细根（〈2 mm）生物量变化、垂直分布及动态变化进行研究。结果表明：5、9、16a橡胶林0～60cm土层内细根总生物量分别为2056.18、1557.42、1174.90 kg·hm^-2。细根的生物量与橡胶树不同生长或生产期有密切的关系,但幼龄橡胶树细根生物量还容易受环境的影响而改变。橡胶树细根总生物量80%左右分布在0～40cm土层,细根生物量随着土层深度的增加而减少,回归模型分析以指数方程y=a.ebx（a、b均为常数）拟合效果较好。通过不同月份细根生物量比较,发现橡胶树细根生物量年动态变化呈“双峰型”,5a、9a橡胶树峰值分别出现在4月份和8月份;16a橡胶树则出现在6月份和10月份,对环境因子的响应具有一定的滞后性。不同树龄橡胶树表层细根生物量年内变化不一致,其它土层细根生物量年内变化也呈“双峰型”,土层越深,变化越趋于缓慢。

巴西橡胶树(Hevea brasiliensis)微管相关蛋白全长cDNA克隆及表达分析

从巴西橡胶树Heveabrasiliensis差减cDNA文库中分离到微管相关蛋白(Microtubule-associatedprotein,MAPs)基因片段,根据该基因片段序列信息,设计特异引物,采用cDNA末端快速扩增技术RACE(RapidAmplificationofcDNAEnds)进行差异片段的5’和3’端的扩增,获得了长度为788bp的全长cDNA,该基因在GenBank中的登录号为AY461412。序列分析表明该基因包含完整的开放阅读框,编码144个氨基酸,与微管相关蛋白基因家族具有很高的同源性,推测该基因是微管相关蛋白基因。半定量RT-PCR检测证实它在胶乳中的表达强于叶中,胁迫处理(伤害及乙烯处理)使其表达上调。

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.

橡胶白粉菌(Oidium heveae Steinmann)基因组DNA的提取方法

比较了2种菌源、3种方法提取橡胶树白粉病病原真菌橡胶白粉菌(Oidium heveae BA.Steinmann)基因组DNA的效果。结果表明:无论采用单斑分离法收集还是直接收集橡胶白粉菌菌体,无论采用石英砂振荡破壁法、氯化苄法还是尿素法提取,均能获得满足常规分子生物学操作要求的基因组DNA;在基因组DNA的提取产率、完整性方面,各种方式之间略有差异,但无显著差别;2种来源菌体的基因组DNA的ITS序列分析结果一致,说明在无法进行单斑分离的情况下,可以利用直接收集的菌体提取基因组DNA。

橡胶树(Hevea brasiliensis Muell.Arg.)幼茎木材的超微结构观察

橡胶树是中国重要的热带经济作物,橡胶种植的副产物橡胶木是我国木材供应的重要来源。我们以不同发育阶段的橡胶树幼茎木材为材料,借助扫描电子显微镜技术,对木质部细胞的超微结构进行了观察。结果表明,在橡胶树幼茎木材中,导管和木纤维细胞壁随着木质部发育成熟会发生明显的次生加厚,加厚方式主要为螺纹加厚;木质部各类型细胞均存在大量纹孔,纹孔排列方式主要有散生、网状、梯状和单串状等类型;在木质部发育过程中,木射线和部分薄壁细胞中会逐渐积累大量淀粉粒;木质部细胞内壁及其填充物表面存在不同类型的附着物。研究结果将为橡胶木材材性及其形成机制的研究提供一定理论参考。

Preliminary Study on Seedling Growth Rhythm and Grey Correlation Analysis of Rubber (Hevea brasiliensis) Seedlings in Danzhou District, Hainan

The rubber tree physiological and ecological process quantitatively described by using mathematical method is an important means to the analysis of rubber tree growth process and mechanism. The study on growth simulation model of rubber tree will lay the foundation for the application of rubber tree cultivation intelligent decision system. A Richards equation was formulated to describe the height and stem diameter growth dynamics of the annual rubber seedlings. An area correlation analysis was done according to the closeness of the observed parameters to the dynamic curve on the gray system composed of the seedling growth increment and the meteorological factors including aerial temperature, precipitation and solar radiation hours that influence upon the seedling growth. The results showed that rubber seedling response fitted the Richards equation quite well. The growth increment displayed a distinct alternation of 'slow—fast—slow— fast—slow' rhythm. The growth course of the seedlings might be partitioned into three periods of time by the sequential clustering analysis, namely pre-growing, fast-growing, late-growing stage. The tray correlation analysis revealed that air temperature had the most significant influence while precipitation had the least impact on height growth of the rubber seedlings. In conclusion, the air temperature had the most significant influence while solar radiation hours had the least impact on stem diameter growth of the rubber seedlings.

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.

An Analysis of the Patents concerning Hevea brasiliensis

This paper mainly analyzes the application data of patents concerning Hevea brasiliensis in the world and China from the perspectives of time,research field,and the geographical distribution of applicants. It also interprets the patents for invention in China from time,research field,and types of applicants. Based on the study,it shows that although the patent applications fluctuate,the general trend is rising. And the patents are mostly concentrated in plant protection,and agricultural biotechnology. The Chinese applicants of patents for invention largely work in the research institutes,and most of these patents focus on the field of plant protection,agricultural biotechnology,and agro-processing.However,the foreign applicants mainly work in companies,and most of these patents only focus on plant protection. These findings will provide a reference for formulating development policies about natural rubber industry in China.

Studies on Rubber (<i>Hevea brasiliensis</i>) Trees Exist Plant Type after Planting and Available Tapping Tree of Rubber Plantation in China

Existing plant types of rubber tree after planting and available tapping tree were investigated, and there were about 28 rubber plantations with different tapping years of 8 varieties “CATAS7-33-97”, “CATAS8-79”, “CATAS7-20-59”, “PR107”, “RRIM600”, “GT1”, “INA873”, “93-114”in South China. The results showed that there were six kinds of existing plant types of rubber tree after planting of rubber plantations, which were available tapping trees, wind damaged trees, cold damaged trees, tapping panel dryness trees, absent trees and weak trees, respectively. These data investigated also showed rubber trees under available tapping, stoppage due to tapping panel dryness, absence, wind damage, cold damage and weakness were counted and calculated and made up for 72.21%, 14.75%, 5.61%, 3.86%, 2.68% and 1.89%. Tapping panel dryness trees, wind damage and absent trees are major factors for the loss of tapping rubber trees in the rubber plantations. Of these investigated varieties, available tapping trees per 100 trees of rubber plantation of “PR107”at the 1st, 12th, 14th, 16th, 20th, 24th tapping year were 96, 67, 70, 75, 66, 46 trees in Hainan planting zone, respectively. Available tapping trees per 100 trees of rubber plantation of “RRIM600”at the 9th, 15th, 20th, 22nd tapping year were 88, 62, 55, 36 trees in Yunnan planting zone, respectively. Available tapping trees per 100 trees of rubber plantation of “93-114” at the 10th, 19th, tapping year were 94, 62 trees in Guangdong planting zone. These results showed that available tapping trees of rubber plantation decreased with increasing tapping age under different planting zones in China.

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.

Seasonal Changes Impact on Growth of Rubber (<i>Hevea brasiliensis</i>) Seedlings under Different Cultivation

The vegetative growth of rubber tree is an important basis for rubber tree cultivation and economic management. Stem diameter and height growth patterns of rubber seedlings and the seasonal variability are still poorly understood. Studying the annual growth patterns and seasonal variation of rubber seedling will help understanding biological characteristics of rubber tree and provide a reference for field cultivation and management. Height growth and stem diameter of field-sown rubber seedlings cultivation and polybag-raised cultivation all displayed a distinct annual pattern of “slow-fast-slow”. Height growth and stem diameter growth response fitted the logistic equation preferably, supporting the observed pattern. Fast-growth of plant height for polybag-raised rubber seedlings cultivation and field-sown rubber seedlings cultivation occurred from June to November and May to November, respectively. This period saw the greatest accumulations of plant height growth for polybag-raised rubber seedlings cultivation and field-sown rubber seedlings cultivation accounting for 83.99% and 86.65% of total growth, respectively. Fast-growth of stem diameter for polybag-raised rubber seedlings cultivation and field-sown rubber seedlings cultivation occurred from June to November and May to November, respectively. This period saw the greatest accumulations of stem growth for polybag-raised rubber seedlings cultivation and field-sown rubber seedlings cultivation accounting for 86.71% and 84.60% of total growth, respectively. Polybag-raised rubber seedlings cultivation and field-sown rubber seedlings cultivation for the whole year had nine extension units and each month had one extension unit from May to November. Extension unit of for the whole year did not stop period and plant height growth rate has the seasonal difference. However, leaf phenophase of field-sown rubber seedlings cultivation was earlier than that of polybag-raised rubber seedlings cultivation and each extension unit of field-sown rubber seedlings cultivation was greater than that of poly-bag-raised rubber seedlings cultivation. The precipitation had the greatest impact on seedlings growth under different cultivation. There was no period annually in which height growth and stem diameter growth did not occur and the fastest growth occurred during the rainy season. Plant height and stem diameter of rubber seedlings under different cultivation showed isogony phenomenon. Extension unit and leaf phenophase of polybag-raised rubber seedlings and field-sown rubber seedlings showed consistency and synchronization characteristic. Different cultivation of rubber seedling should take corresponding measures tending management and operation.

Herbicidal Effects of Periconia heveae Culture Filtrate on Mikania micrantha

[Objective] The paper was to explore the herbicidal effects of Periconia heveae culture filtrate on Mikania micrantha. [Method] Through indoor bioassay method, the inhibition effects of culture filtrates of seven species of plant disease fungi on seed germination and seedling growth of M. micrantha were studied, and the security of P. heveae culture filtrate on pepper （Capsicum annuum） and tomato （Solanum lycopersicum） was also evaluated. [Result] The culture filtrates of P. heveae had strong inhibition effects on seed germination and the growth of root, shoot and seedling of M. micrantha, and the inhibition rates were 72.7%, 93.0%, 32.4%, and 54.6% （fresh weight）, respectively. Security tests showed that culture filtrates of P. heveae were safe to seed germination and root and shoot growth of tomato, and the growth rate had no significant difference with control, but they inhibited seed germination of pepper. [Conclusion] As potential biological herbicide, the culture filtrate and metabolites of P. heveae is worthy of further development. .

Evaluation of Brazilian wild Hevea germplasm for cold tolerance：genetic variability in the early mature growth

Hevea brasiliensis is a commercially cultivated species for its natural rubber（NR） latex in South East Asian countries. To meet the ever-increasing demand, NR cultivation has been extended to non-traditional regions due to the limited scope of further expansion in traditional rubbergrowing areas in India. These areas are often confronted with various abiotic stresses, especially high and low temperatures, which cause reduction in plant growth, thereby increasing its uneconomical immaturity period. Eighteen wild Hevea accessions along with two modern clones RRII203 and PB 235 and two check clones RRIM 600 and Haiken 1 were evaluated in the early mature growth phase.The site was at Nagrakata, West Bengal, the sub-Himalayan cold-prone region of India. In Hevea, crop production is governed by two major factors, growth-vigor and production capacity. Growth-vigor is of special importance because the production of rubber is a process linked with the early growth of the plant, which results in early tappability and early economic gains. The genotypes exhibited highly significant clonal differences（P = 0.05） for all the growth traits. Tappability percentage in the seventh year, ranged from 0.33 %（AC 3074, AC 3075, AC 3293） to 89.67 %（RO 2727）. The most vigorously growing accession（RO2727） reached tappabile girth early in the seventh year when the girth of plant ranged from 22.38 cm（AC 3293） to53.12 cm（RO 2727）. The general mean was 43.32 cm, and the similar growth trend was exhibited by these accessions in the tenth year also. Annual girth increment（cm a-1） over3 years ranged from 1.81 cm（AC 3075） to 6.80 cm（RO2727）. The mean winter girth increment（cm a-1） over4 years ranged from 0.13 cm（AC 3075） to 0.96 cm（RO2727） as compared to the check clone RRIM 600（1.11 cm）and Haiken 1（1.10 cm）. Wide differences between the phenotypic coefficient of variation（50.29） and genotypic coefficient of variation（24.82） were observed for winter girth increment. Girth in the tenth year recorded the highest heritability（87 %）. Girth was significantly correlated with the other growth traits. The top 30 % of the potential accessions showing high growth vigour and early tappability under cold stress were identified. These ecotypes/selections have high potential value for the development of coldtolerant clones for these regions and also in broadening the genetic base of the present-day cultivated rubber.

Methanolysis of Mixed Crop Oils (<i>Hevea brasiliensis</i>and <i>Jatropha curcas</i>L.) into Biodiesel: Kinetics Study

In this study, the methanolysis of mixed crop oils (Hevea brasiliensis and Jatropha curcas L.) was investigated. Due to the oils contain high impurities, a particular pre-treatment of which was carried out. For alkali-catalyzed methanolysis, few parameters (such as: molar ratio, catalyst percentage, and reaction time) were studied. The results showed that the methanolysis optimum conditions were 5.5 of molar ratio, 0.9% w/w of catalyst, and 60 minutes of reaction time with the highest yield of 98.9%. The kinetics study indicated that first order reaction mechanism with reaction rate constant and activation energy were 1.5 × 10-2 minute-1 and 23.93 KJ?mol-1 respectively. As fuel, the physicochemical properties of biodiesel met the standard values required by ASTM D6751.