橡胶树死皮病胶乳C-乳清差异表达蛋白质的筛选与鉴定

橡胶树死皮病(Tapping Panel Dryness,TPD)在世界各橡胶种植园普遍发生,给橡胶种植业带来严重的危害。为了更好地了解和阐明死皮病发生、发展的分子机制,本研究应用双向凝胶电泳技术（2-DE）比较橡胶树死皮株与健康株胶乳C-乳清蛋白质组表达的差异。采用固相pH梯度双向凝胶电泳分离橡胶树死皮株与健康株C-乳清的总蛋白质,凝胶经考染显色后,用PDQuest7.40图像分析软件进行比较分析,识别差异表达的蛋白质。这些点经过胶内酶切后进行基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)分析获取肽质指纹图谱(PMF),Mascot软件搜索SWISS-PROT和NCBInr数据库鉴定蛋白质。结果：①橡胶树死皮株与健康株C-乳清凝胶的平均蛋白质点数分别为1075±35和1134±27,其平均匹配的点数分别为982±38和1008±22,组内图像匹配率达91.89﹪和88.72﹪。②橡胶树死皮株与健康株C-乳清组间的平均匹配蛋白点数为970±25。利用MALDI-TOF-MS质谱技术对40个差异明显的蛋白点进行分析鉴定,通过查询数据库鉴定了27个蛋白质。本研究建立了分辨率高且重复性较好的橡胶树死皮株与 健康株胶乳C-乳清的双向凝胶电泳图谱,并应用质谱技术鉴定了其中表达差异的蛋白质点,这些差异表达的蛋白质可能参与了死皮发生和发展的过程。     

巴西橡胶树HbICE1基因酵母双杂交诱饵载体的构建及互作蛋白的筛选

寒害是我国植胶区的主要自然灾害之一,不仅影响橡胶产量还威胁到橡胶树的生存。拟南芥中,ICE1是低温胁迫信号通路中的重要转录激活因子,但橡胶树冷胁迫信号途径中包括Hb ICE1在内的大部分关键基因尚未被克隆与鉴定,与Hb ICE1蛋白发生互作的蛋白也不清楚,成为严重阻碍橡胶树抗寒分子机理研究的瓶颈。为了筛选和鉴定与巴西橡胶树Hb ICE1蛋白发生互作的蛋白,阐明橡胶树抵御寒害胁迫的分子机理,本文首先通过PCR扩增出Hb ICE1基因的编码序列(约1400 bp),然后将目标序列插入p GBKT-7载体,构建酵母双杂交诱饵载体;将该载体转入酵母Y2H菌株感受态细胞中,并在缺陷型培养基上检测其自激活活性,发现Hb ICE1基因的自转录激活在含有30 mmol/L和更高浓度3-AT(3-氨基-1,2,4-三氮唑)的培养基上能得到有效抑制;进一步通过与橡胶树c DNA文库进行酵母双杂交,筛选出一些可能与Hb ICE1发生互作的蛋白,包括DNA结合蛋白、核糖体蛋白和功能未知蛋白。本研究结果为橡胶树抗寒机理研究提供了重要理论依据。     

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。     

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。     

橡胶树花药和小孢子发生与发育过程的观察

应用石蜡切片法．观察橡胶树的实生树和RRIM600、GT-1品系的花药壁以及小孢子的发生和发育过程,得到如下结果：1．实生树的花药壁通常由四层细胞组成,发育形式为双子叶型。药室内壁细胞在发育后期进行径向条纹加厚．至花药开裂时仍保留着原生质体。中层由一层或不规则的两层细胞组成,在小孢子单核期消失。绒毡层细胞具单棱或双核,属分泌型,至花粉发育到三细胞时消失。小孢子母细胞减数分裂为同时型。成熟花粉粒具三十细胞。精细胞椭圆形,在光镜下不能区分出细胞质鞘和核仁。所观察的实生树雄花,多数发育正常,很少有空秕的花粉。2．RRIMB00品系的花药和小孢子发生与发育和实生树相似,但至后期只有少数花粉发育正常,多数成为大小不等的败育花粉;此外也有一些败育的雄花。3．GT-1的花药在小孢子母细胞减数分裂时,绒毡层细胞的体积开始异常增大并液泡化．小孢子在四分体内解体或分离后成为空秕花粉。     

硒镧复合作用对巴西蘑菇子实体形态和数量特征的影响

采用混合培养料试验和电子显微技术,观察了不同硒镧配施水平对巴西蘑菇子实体盖皮和菌褶表面形态及孢子和囊状体数量特征的影响.结果表明,低浓度硒镧配施(Na2SeO3 21.905mg/kg LaCl317.667mg/kg)处理时,孢子数极显著增加,随着硒镧配施浓度升高,孢子数极显著增加,达到峰值后极显著减少;低浓度(Na2SeO 21.905mg/kg LaCl17.667mg/kg)和高浓度(Na2SeO 109.524mg/kg LaCl88.340mg/kg)处理后囊状体数极显著减少,中浓度(Na2SeO365.714mg/kg LaCl353.002mg/kg)处理囊状体数显著增加.研究还发现硒镧配施对巴西蘑菇孢子长、宽、囊状体最大直径和最小直径以及菌盖皮菌丝最大宽度具有促进或抑制作用.扫描电镜观察结果表明,与对照相比,中浓度硒镧配施(Na2SeO365.714mg/kg LaCl3 53.002mg/kg)时,孢子由正常椭圆形变成全部萎缩,菌丝粗壮;囊状体在较低硒镧配施浓度(Na2SeO343.810mg/kg LaCl3 35.335mg/kg)处理时表面纹饰模糊、消失,个别囊状体破裂,表面带有藤蔓状的组织.     

Cutaneous and systemic cellular responses induced by the feeding of the argasid tick Ornithodoros parkeri

Carolyn M. Johnston and Stephen J. Brown 1985. Cutaneous and systemic cellular responses induced by the feeding of the argasid tick Ornithodoros parkeri. International Journal for Parasitology15: 621–628. Initial feeding by Ornithodoros parkeri ticks induced a significant blood basophilia in guinea pigs, with a minimal cutaneous basophil response. Hosts challenged 14 days later, however, exhibited significantly depressed blood basophil levels associated with a marked accumulation of these cells at tick feeding sites in the tissue. Blood eosinophil levels in primary and secondary hosts were comparable, but eosinophil levels at tick feeding sites in challenged animals were significantly greater than levels in primary hosts. Furthermore, challenge tick feeding resulted in the activation of primary tick feeding sites on the opposite flank that became erythematous 90 min after challenge and indurated within 24 h. Histologically, these activated primary feeding sites 90 min after challenge on the opposite flank were marked by a dominant eosinophil response (314 ± 128 cells, 59% of the infiltrate) with a marked basophil component (145 ± 67 cells, 28% of the infiltrate) that resembled the active challenge feeding sites 24 h after infestation (24 ± 52 cells, 76% of the infiltrate); 90 min after challenge active feeding sites had a weak basophil response (2 ± 1 cells) similar to uninfested controls. These results suggest the chronic nature of tick bites with an apparent continual recruitment of basophils that is probably a result of slow antigen release over time by appropriately sensitized antigen presenting cells. Primary tick feeding sites in guinea pigs previously exposed to Xenopsylla cheopis fleas, on the opposite flank, contained a marked eosinophilia (63 ± 25 cells) compared to primary tick feeding sites in naive guinea pigs (2 ± 0 cells); suggesting the possibility of cross reactivity between flea and tick antigens     

橡胶树魏克汉种质资源生长与形质性状变异分析

于2009-2016年每年12月中旬,对2008年定植保存在"农业部景洪橡胶树种质资源圃"内来自4个国家的364份橡胶树魏克汉种质资源1740株林木的年度径围生长量进行了跟踪观测,2016年12月底对其枝下高、主干通直度、主干分杈、侧枝分枝轮、侧枝粗细、侧枝分枝角、侧枝伸展等指标进行了观测,统计分析了年度径围生长规律及变异情况;以364份种质资源的调查数据对8个指标的遗传多样性指数进行了分析,并分别以基因型和国家为单位进行了多指标综合聚类,旨在为揭示橡胶树魏克汉种质资源遗传多样性及合理保护和利用提供理论依据。研究结果表明,可以根据林木的茎粗生长量将保存的364份魏克汉种质资源划分成3个类群,依次为缓慢型(径围50 cm)、中间型(径围在50~60 cm)、速生型(径围60 cm),其中,8年生时径围总生长量平均为52.88 cm,变幅为41.50~64.00 cm;缓慢型均值为47.49 cm,变幅为40.50~49.75 cm;中间型均值为54.02 cm,变幅为50.00~59.75 cm;速生型均值为61.07 cm,变幅为60.00~64.00 cm;速生型与缓慢型之间均值相差13.58 cm。从变异程度来看,总体、缓慢型、中间型、速生型等年度间变异系数变幅依次为10.87%~25.81%、10.20%~26.89%、9.32%~25.62%、8.64%~20.78%。随着林龄的增加,3类群体连年生长量与年平均生长量均呈现出先增大后降低的趋势,且两者在8年间只出现了1次相交,相交时间在3~4年生之间,速生型相交时间较晚一些。年度间径围生长量相关性分析表明,缓慢型和中间型群体相关性均达到极显著正相关(除中间型1年生与8年生间相关性不显著外),速生型群体3年生后各年度间相关性达到极显著正相关。8个指标的遗传多样性指数从大到小排序依次为:枝下高(1.835)径围生长量(1.713)侧枝分枝轮(1.222)侧枝粗细(1.192)侧枝分枝角(1.079)主干通直度(1.032)主干分杈数(0.991)侧枝伸展(0.579),多数指标遗传多样性指数偏小。聚类结果表明,来自同一国家的种质大部分聚在一组或几组;4个国家种质关系最为密切的是印度尼西亚和马来西亚,其次是印度尼西亚和中国,距离较远的是斯里兰卡。综上可知,国内保存的橡胶树魏克汉种质资源8个性状变异程度不大,遗传多样性丰度低。建议在进行早期选择时,以较小的林龄(如2年生)进行淘汰选择,以较大的林龄(如4年生)进行速生资源选择,其中7份速生型种质资源可直接用来培育胶木兼优无性系或作为亲本材料进行杂交育种。     

Role of the armadillo Dasypus novemcinctus in the epidemiology of paracoccidioidomycosis

A study conducted in a rural area of Ibiá, State of Minas Gerais, Brazil, where Paracoccidioides brasiliensis was recently isolated from soil, sought to determine if the armadillo Dasypus novemcinctus developed paracoccidioidomycosis. Out of 21 armadillos captured in the area, one had a lung granuloma containing fungal cells attributable to those of P. brasiliensis. The present report presents the first histopathological evidence for the presence of this pathogen in the lungs of an armadillo. This finding permitted us to establish a linkage between the two species and to suggest that fungal infections of humans and of armadillos occur in the same agricultural and forest microenvironment that they share. This revised version was published online in August 2006 with corrections to the Cover Date.     

The Colletotrichum gloeosporioides perilipin homologue CAP 20 regulates functional appressorial formation and fungal virulence

Previous studies of the CAP20 gene in Colletotrichum gloeosporioides show that the CAP20 gene may affect virulence in avocados and tomatoes. In this study, we characterized the function of CAP20 from C. gloeosporioides, the causal agent of Colletotrichum leaf fall disease of Hevea brasilience. CAP20 encodes a perilipin homologue protein. Further investigations showed that the Cap20‐GFP fusion protein localized in lipid droplets in hypha and conidia. A C. gloeosporioides mutant, lacking CAP20, had thinner spores and smaller appressoria, and its turgor pressure generation was dramatically reduced and pore size was enlarged. Furthermore, we tested the pathogenicity of conidia from the wild type, gene‐deleted mutant and complemented transformant C.gloeosporioides on the leaves of rubber trees in sterile water and 0.19 M PEG2000. Conidia from the wild type and complemented transformant C. gloeosporioides in 0.19 M PEG2000 caused necrotic lesions and did not produce any lesion with the CAP20 null mutant. But all of them had developed normal disease lesions when they were inoculated in water. These results suggest that CAP20 is a perilipin homologue protein and is involved in functional appressoria development in C. gloeosporioides. CAP20 gene only affects fungal virulence to some extent by reducing the penetration of the immature appressoria into host cuticle in C. gloeosporioides.