Effect of ethylene on enzymatic activities involved in the browning of Hevea brasiliensis callus

Browning, which is of varying intensity depending on species, develops in Hevea brasiliensis callus in vitro and can affect somatic embryogenesis. Endogenous ethylene appears to be involved since application of aminocyclopropane carboxylic acid (ACC) strongly enhances this browning. At the same time, peroxidases (EC 1.11.1.7), bonded polyphenol oxidase (EC 1.10.3.1) and NADH-quinone reductase (EC 1.6.99.5) are enhanced by ACC whereas superoxide dismutase (SOD: EC 1.15.1.1) and ascorbate peroxidase (EC 1.11.1.11) are decreased. In contrast, application of aminoacetic acid (AOA) causes spectacular decreases in bound and soluble polyphenol oxidase, peroxidase and NADH-quinone reductase activities whereas SOD and ascorbate peroxidase activities increase. Enzymatic activities in the control callus present a medium value, probably due to a considerable ethylene production in the culture medium. After 47 days culture. the initial catalase activity (EC 1.11.1.6) in the explants had disappeared completely. In contrast, after only 22 days, the silver nitrate and especially AOA treatments promoted considerable activities of catalase. The beneficial effect of the inhibitors of ethylene synthesis was reflected in the enhanced formation of embryogenic tissue, which varied from 1% (control or ACC) to 34% (AOA treatment).     

Seasonal Changes of Cambial Activity and Phloem Development in Hevea brasiliensis

There is definitely seasonal activity in cambial growth in the trunk of Hevea brasi- liensis planted on Hainan Island and the cambial growth ceases during leaf-fail and leaf-absent period simultaneously with lower temperature and dries. The sieve elements founction for one-half year to two years. The variation in conducting-phloem thicknesses in a year is not significant and the conducting-phloem retains in about the same thickness during leaf-fall and leaf-absent period as that during the other periods in a year.     

基因枪法获得GAI转基因橡胶植株的研究

将含有Camv35S启动子、卡那霉素抗性基因和GUS报告基因,目的基因为GAI基因的转化载体质粒pBI121,通过基因枪轰击巴西橡胶树(Hevea brasiliensis Muell-Arg.)花药愈伤组织,50 mg L~(-1)卡那霉素的继代培养基进行抗性筛选.获得了抗性再生植株,经过PCR、Southern检测结果表明:GAI基因已经成功转入橡胶树基因组中.     

AFLP在橡胶树优异种质研究中的应用

采用377DNA测序仪（P.E.Corp.)用AFLP技术对25种分别具有高产／低产、抗白粉病／感白粉病、抗寒／不抗寒、死皮／不死皮性状的橡胶树（Hevea brasiliensis Mull.Ary)无性系（其中Wickham种质15种,Amazon野生橡胶树种质10种）进行了指纹图谱分析,从64对引物组合中选出2对引物组合构建了所有被研究种质的指纹图谱。2对引物共扩增出518条带,多态性比率超过98．6％。遗传多样性分析表明,橡胶树种质间遗传距离为0．25－0．81,RRIM600种质内遗传距离为0．07－0．17。通过基因分型分析获得一条大小为320bp的抗白粉病种质特有的片段。根据以上的AFLP数据进行了聚类分析,结果表明所有被研究种质几乎是依次聚成一个大类。     

农杆菌介导法在橡胶树遗传转化中的应用与展望

综述农杆菌介导法在巴西橡胶树遗传转化中的应用进展,分析影响农杆菌转化的关键因素,如植物基因型与外植体、菌株与载体类型、菌液浓度与侵染时间、vir诱导物、筛选剂与抑菌剂、培养基的组成和附加成分等,并对提高巴西橡胶树转化效率的策略进行探讨。     

巴西橡胶树转基因研究现状与展望

由于存在遗传背景狭窄、高度杂合化、育种周期长等特点,巴西橡胶树育种进展非常缓慢。转基因技术为拓展其的遗传范围、加速育种进程提供了契机。在过去20年里,橡胶树转基因研究取得了很大进展：成功应用的转化技术包括基因枪法和农杆菌介导法,受体材料包括花药和内珠被愈伤,育种目标涉及到提高胶乳产量、抗死皮和作为生物反应器等。但同时也存在组培程序不够成熟、转化技术和外植体比较单一、转化效率低下等问题。最后,对以后转化体系的优化、转基因育种方向进行了分析和展望。     

AFLP Fingerprinting Analysis of Elite Hevea brasiliensis Germplasm

There are more than 6 000 clones of Hevea brasiliensis Mull. Ary germplasm in the germplasm garden of Chinese National Key Biotechnology Laboratory for Tropical Crops and some of them are elite germplasm demonstrated by production and previous studies. AFLP (amplified fragment length polymorphism) fingerprinting analysis was performed on 25 clones (15 of Wickham clones and 10 of Amazon wild clones which possess phenotypes with high-yielding/low-yielding, cold-tolerance/cold-sensitivity, oidium-resistance/oidium-sensitivity, tapping panel dryness (TPD)/healthy) respectively through a 377 DNA sequencer (P. E. Corp.) and PAGE electrophoresis results were analyzed by using GeneScanTMand GenotypeTM Analysis software (P.E.Corp.). The fragment profiles of different clones were obtained. Five hundred and eighteen fragments were generated by two primer combinations screened from 64 primer combinations and 511 fragments appeared to be polymorphic (98.6%). Genetic distance ranged from 0.25 to 0.81 between clones and ranged from 0.07 to 0.17 within RRIM600 clone. A specific 320 bp fragment of the oidium-resistant clones was found through genotype analysis. These results showed that AFLP fingerprints were highly reproducible and powerful and can be widely used in germplasm identification and genetic diversity analysis of Hevea brasiliensis. In addition, based on the AFLP data, cluster analysis was performed. Cluster results showed that all the clones studied were almost clustered into a group one by one.      

基因枪法获得GAI转基因橡胶树植株的研究

将含有Camv35S启动子、卡那霉素抗性基因和GUS报告基因,目的基因为GAI基因的转化载体质粒pBI121,通过基因枪轰击巴西橡胶树(Hevea brasiliensis Muell-Arg.)花药愈伤组织,50 mg L~(-1)卡那霉素的继代培养基进行抗性筛选.获得了抗性再生植株,经过PCR、Southern检测结果表明:GAI基因已经成功转入橡胶树基因组中.     

云南保存橡胶树魏克汉种质资源亲子系谱分析

对“农业部景洪橡胶树种质资源圃”（云南景洪）保存的290份魏克汉种质资源进行了亲子系谱分析。结果表明,无性系GT1、PR107、PB86、RRIM600、云研277-5、PB5/51、IAN873的后代为210份,占72.41%。从表型看,魏克汉种质资源群体变异丰富,实际上遗传基础很窄。本文以GT1、PR107、PB86、RRIM600、云研277-5、PB5/51、IAN873为主线建立了亲子系谱图,并对部分种质资源的性状进行了分析,获得一些性状的传递遗传规律,供育种工作者参考。     

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.     

燃油植物橡胶树籽的开发利用

橡胶籽是橡胶树种植产业中的一项副产品资源,估计我国每年可产橡胶籽油15万～18万t.论述了橡胶籽油用于生物柴油的可行性,如能因地制宜开发生物柴油,既可解决植胶农场柴油不足,也可充分利用资源,其生态效益、经济效益均很明显,值得引起有关方面的注意.     

橡胶树魏克汉种质群体结构分析

群体结构是引起关联分析假阳性的最主要因素。本研究基于25个EST-SSRs位点,分析了283份橡胶树魏克汉种质群体的哈迪-温伯格平衡和群体分层情况。结果表明,在被检查的25个位点中13个位点偏离哈迪-温伯格平衡;在共祖系数为50%、60%、70%和80%时,283份种质中具有明确亚群归属的分别只有155份、110份、61份和22份,分别占总种质的54.78%、38.87%、21.55%和7.78%。同时,25个多态位点共形成了300个位点组合,20个位点组合(6.67%)呈显著的连锁不平衡状态,其中5个位点组合(1.67%)达到极显著连锁不平衡。由上可知283份橡胶树魏克汉种质所构成的群体具有一定的群体分层,但分化程度较小,可用于关联分析。     

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Hevea brasiliensis差减cDNA文库中分离到微管相关蛋白（Microtubule-associated protein,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（Rapid Amplification ofcDNA Ends）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.     

巴西橡胶树一个AP2／EREBP转录因子的克隆及表达分析

本文采用RACE技术从巴西橡胶树中鉴定出一个AP2／EREBP转录因子。该转录因子全长cDNANl225bp,最长开放阅读框699bp,预测编码蛋白包含232个氨基酸;序列比对分析发现,该转录因子具有一段保守的AP2／EREBP结构域,与拟南芥Soloist（At4g13040）具有较高的相似性,将该基因命名HbSoloist。半定量胙PcR分析结果表明,HbSoloist在巴西橡胶树的胶乳、叶片、树皮和花中均有表达。与健康橡胶树相比,死皮树胶乳HbSoloist表达量明显下降。研究发HbSoloist表达受乙烯利、茉莉酸和低温处理调控,表明HbSoloist基因可能在巴西橡胶树乙烯、茉莉酸和低温反应中发挥作用。     

橡胶树AFLP银染体系的建立和优化

目的:扩增片段长度多态性(AFLP)为遗传图谱的构建及育种的辅助选择提供了有力的工具。建立一套适合橡胶树的AFLP技术优化体系。方法:以197个GT1×IAN873橡胶树杂交群体为材料,通过对影响AFLP的多种关键因素如模板DNA质量、酶切连接体系、酶切连接反应时间、预扩增体系、选择性体系的分析进行研究。结果与结论:找出一套适于热带植物基因组DNA提取及橡胶树AFLP技术。用改进的CTAB法,经多次抽提纯化,用细玻璃棒挑出DNA得到高质量的模板DNA;酶切连接时DNA模板为250ng,反应体系采用各3U的EcoRⅠ/MseⅡ/T4连接酶,反应时间为9h;预扩增模板为稀释1/2的酶切连接产物,用量为1μL;选择性扩增要用稀释至1/20的预扩增产物。     

巴西橡胶树HbAIH基因的克隆及表达分析

鲱精胺亚胺水解酶(Agmatine iminohydrolase,AIH)是植物通过精氨酸途径合成多胺过程中的重要酶之一。本研究首次从巴西橡胶树中克隆了AIH基因HbAIH。序列分析结果表明HbAIH全长1 462 bp,开放阅读框长为1 137 bp,编码378个氨基酸。预测 HbAIH 蛋白的分子量是42.28 kD,等电点为5.09,是一个亲水性蛋白。氨基酸相似性比对结果表明HbAIH与大戟科木薯MeAIH、蓖麻RcAIH和麻风树JcAIH等具有很高相似性,且含有植物AIH中高度保守的与酶活性位点形成和参与底物结合的11个氨基酸位点。qRT-PCR 分析表明,HbAIH表达无组织特异性,在雌花中表达量最高,树皮、茎尖、胶乳、叶片和雄花中表达量依次降低。HbAIH在不同发育时期叶片中的表达存在变化,稳定期表达量最低,淡绿期最高,说明HbAIH可能参与橡胶树叶片发育过程。此外,低温、干旱、高盐、过氧化氢、乙烯利、茉莉酸甲酯处理以及橡胶树死皮的发生均能引起HbAIH表达变化。这些结果说明HbAIH可能参与了橡胶树生长发育、产排胶调控及逆境应答过程。     

巴西橡胶树HbCMT的克隆与表达分析

通过PCR和RACE技术克隆了巴西橡胶树中一个染色质甲基化酶(Chromomethylase, CMT)基因（HbCMT）。HbCMT长2697 bp,含有2556 bp的阅读框,编码851个氨基酸。推测HbCMT分子量为95.67 KD,等电点为5.38,氨基酸序列与可可、葡萄、黄瓜、鹰嘴豆、番茄、拟南芥CMT家族成员的同源性分别为77％、74％、72%、67%、64%和52%。定量PCR分析表明HbCMT在巴西橡胶树的根、树皮、叶、胶乳中均有表达,其中在胶乳中表达量最低,此外HbCMT在橡胶树自根幼态无性系的胶乳中表达比老态无性系胶乳中的表达低。     

巴西橡胶树环锌指蛋白基因克隆及其分子特性

在先前的研究中通过抑制缩减杂交获得了一个在巴西橡胶树胶乳中特异表达的片段（HbSSH10）,该片段含有“RING finger”或“C3HC4”保守序列。根据HbSSH10的序列信息设计引物并通过3’-RACE和5＇-RACE的方法,获得了一个全长的cDNA（HbRZF）。该cDNA含有589个核苷酸,含有完整的阅读框架,编码156个氨基酸。从它推导出的氨基酸序列中含有“RING finger”或“C3HC4”保守区（氨基酸100～144）。该氨基酸序列与Poncirus trifoliata、Arabidopsis thaliana和Thellungiella halophila的环锌指蛋白的同源性分别为48％、52％和50％。Northern杂交分析表明HbRZF在胶乳中大量表达,在叶片中微量表达,而在根和花中几乎没有表达。茉莉酸处理可以诱导胶乳中HbRZF的表达,而乙烯对胶乳中HbRZF的表达基本上没有影响。     

巴西橡胶成龄无性系茎段的试管微繁技术研究

巴西橡胶成龄无性系茎段组织培养在生产中有很大的潜在应用价值,但一直是橡胶树组织培养的难点,至今国内外鲜有成功的报道.本研究以在中国热带地区大面积推广种植的巴西橡胶树优良品种热研7-33-97不同生长时期成龄树茎段为外植体,进行无性系试管微繁技术研究.研究结果表明不同生长时期的茎段在自然条件中受污染程度不同,茎段真菌污染率为稳定期＞淡绿期＞古铜期,细菌污染率为淡绿期＞稳定期＞古铜期.不同生长时期的成龄树茎段需要通过不同的灭菌方法才能显著提高外植体的利用率,外植体灭菌方法显著影响橡胶成龄树茎段的组织培养效率;与稳定期相比,古铜期和淡绿期茎段在组织培养过程中诱导丛生芽萌芽快、萌芽多,是较优的外植体材料;古铜期和淡绿期茎段在激素配比为4.0～5.0 mg/L6-BA+0.5 mg/L GA3的条件下能很好的诱导抽出丛生芽;丛生芽在激素配比为2.0 mg/L 6-BA+0.5 mg/LNAA,1.0 mg/t 6-BA+1.0 mg/L KT+0.5 mg/L NAA或0.5 mg/L 6-BA+1.5 mg/L KT+0.5 mg/L NAA的条件下能很好的诱导丛生芽抽出健壮的芽条;丛生芽抽出的健壮芽条在激素配比为0.5 mg/L IBA+0.5 mg/L IAA的条件下能较好的抽根成苗.外植体生长时期、激素种类和浓度配比都是影响巴西橡胶成龄无性系茎段的试管微繁的重要因素.     

种皮和环境温度对橡胶树种子萌发的影响

在人工气候箱控制的条件下,研究了种皮和环境温度对橡胶树(Hevea brasiliensis)种子萌发的影响.结果表明,种皮对橡胶树种子的吸水和萌发具有明显的机械限制作用;去除种皮后种子萌发速率加快,萌发指数增大,但萌发率从83.8%下降到47.8%,幼苗活力指数从332.3下降到187.9.15℃以下的温度明显推迟种子萌发,且萌发率下降.去除种皮后的种仁萌发率、萌发指数和幼苗活力指数均随着温度的升高而增大;有种皮种子的萌发率、萌发指数和幼苗活力指数分别在20℃(94.7%)、30℃(1.86)和25℃(374.4)时最大,而萌发速率系数随着温度的升高先增大后减小.在橡胶树栽培和胶园建设中,可利用种皮对种仁水分散失的限制作用以及对低温的不敏感性,有效延长橡胶树种子的贮藏时间,提高萌发率和籽苗质量.