Characteristics of the microbial community in rhizosphere of Camptotheca acuminata cultured with exotic invasive plant Eupatorium adenophorum

The traditional culture-dependent plate counting and culture-independent small-subunit-ribosomal RNA gene-targeted molecular techniques, Single-Strand Conformation Polymorphism (SSCP) and ter-minal Restriction Fragment Length Polymorphism (tRFLP) combined with 16S rDNA clone library were adopted to investigate the impacts of secretion from Camptotheca acuminata (abbreviated to Ca) roots on the quantities and structure of eukaryotic microbes and bacteria in the rhizosphere, and the possi-bility that Ca controls exotic invasive plant Eupatorium adenophorum (Ea). The counting results indi-cated that the number of bacteria increased in turn in rhizospheres of Ea, Ca-Ea mixed culture and Ca, while that of eukaryotic microbes decreased. PCR-SSCP profiles showed eukaryotic microbial bands (corresponding to biodiversity) in rhizosphere of Ea were more complex than those of Ca and CE. Meristolohmannia sp., Termitomyces sp. and Rhodophyllus sp. were the dominant populations in the rhizosphere of Ca. Bacterial terminal restriction fragments (TRFs) profiles showed no difference among three kinds of rhizospheres, and the sequences of the 16S rDNA clone library from Ca rhizospheres were distributed in 10 known phyla, in which phylum Proteobacteria were the absolute dominant group and accounted for 24.71% of the cloned sequences (δ-Proteobacteria accounted for up to 17.65%), and phyla Acidobacteria and Bacteroidetes accounted for 16.47% and 10.59% of the cloned sequences, respectively. In addition, high performance liquid chromatography detected a trace amount of camp-tothecin and hydroxycamptothecin in the rhizospheric soil of Ca and CE, but examined neither camp-tothecin nor hydroxycamptothecin in rhizospheric soil of Ea. Therefore, invasion and diffusion of Ea evidently depended on distinguishing the eukaryotic community structure, but not on that of the bac-terial pattern. Ca was able to alter the eukaryotic community structure of invasive Ea by secreting camptothecin and hydroxycamptothecin into rhizospheres, and may benefit the control of overspread of Ea. This study provided theoretical evidence for rhizospheric microbial aspects on substituting Ca for Ea.     

Camptothecin, over four decades of surprising findings

Camptothecin (CPT) is a modified monoterpene indole alkaloid produced by Camptotheca acuminata (Nyssaceae), Nothapodytes foetida, Pyrenacantha klaineana, Merrilliodendron megacarpum (Icacinaceae), Ophiorrhiza pumila (Rubiaceae), Ervatamia heyneana (Apocynaceae) and Mostuea brunonis (Gelsemiaceae), species belonging to unrelated orders of angiosperms. From the distribution of CPT and other secondary metabolites, it has been postulated that the genes encoding enzymes involved in their biosynthesis evolved early during evolution. These genes were presumably not lost during evolution but might have been "switched off" during a certain period of time and "switched on" again at some later point. The CPT derivatives, irinotecan and topotecan, are used throughout the world for the treatment of various cancers, and over a dozen more CPT analogues are currently at various stages of clinical development. The worldwide market size of irinotecan/topotecan in 2002 was estimated at about $750 million and at $1 billion by 2003. In spite of the rapid growth of the market, CPT is still harvested by extraction from bark and seeds of C. acuminata and N. foetida. All parts of C. acuminata contain some CPT, although the highest level is found in young leaves (approximately 4-5 mg g(-1) dry weight), approximately 50% higher than in seeds and 250% higher than in bark. The development of hairy root cultures of O. pumila and C. acuminata, and the cloning and characterization of genes encoding key enzymes of the pathway leading to CPT formation in plants has opened new possibilities to propose alternative and more sustainable production systems for this important alkaloid.     

喜树细胞悬浮培养中生理生化指标的测定

以喜树(Camptotheca acuminata Decsne.)幼嫩叶片为材料,诱导和筛选愈伤组织,进行细胞悬浮培养。初步研究了液体培养条件下光照对喜树细胞生长和生理生化特性的影响。结果显示：在光照条件下培养细胞生长周期30d,在黑暗条件下培养细胞生长周期为27d,细胞的增殖曲线呈“S”形;在两种不同光照条件下,其培养液pH值先下降后回升;培养细胞中可溶性蛋白质含量及过氧化物酶活性均出现两个峰值,但出现的时间不同。     

田间栽培喜树不同品系枝叶喜树碱产量动态分析

采用高效液相色谱法和生物量法,以四川种源的普通喜树为对照,对生长季内喜树新品系海喜1号不同冠层枝叶喜树碱产量进行了测定。结果表明：由于海喜1号枝叶喜树碱含量、生物量均高于普通喜树,其产量更加显著地高于普通喜树;虽然海喜1号幼嫩的上层枝叶喜树碱含量高于中层和下层,但由于生物量偏低,产量明显低于中下层枝叶;虽然基于喜树碱含量较高（即优质为目标时）,采收期应确定为6月份,但为了达到持续产量,在6月份可采收上层枝叶,7~10月以适度透光为方法连续采收中下部枝叶,11月份,即生长季末采收冠层下部全部1/3枝叶。     

不同种源喜树幼枝中喜树碱的含量

通过HPLC测定了喜树(Camptotheca acuminata)不同器官及不同种源幼枝中喜树碱的含量.结果表明,喜树碱含量在叶、种子和幼枝(幼叶和幼茎)中较高,木质部中较少,髓中最低.不同种源的幼枝中,来自成都种源喜树的喜树碱含量最高.通过比较,两年生喜树各器官中喜树碱含量普遍比一年生高,幼枝中顶枝的喜树碱含量比侧枝高.     

滤光膜对喜树幼苗叶片生长和喜树碱含量的影响

喜树 (Camptotheca acuminata)为中国特有树种 ,因其次生代谢产物喜树碱具有抗癌作用而闻名。通过用黄色、红色、蓝色 3种滤光膜对温室栽培的喜树幼苗进行遮光处理 ,研究了不同光照环境下喜树幼苗叶片生物量、叶绿素含量、光合作用和喜树碱含量的差异。结果表明在 30 d的遮光过程中 ,红膜和蓝膜遮光明显导致幼苗叶片生物量降低 ,黄膜遮光下幼苗叶片生物量在处理后 2 5 d才表现明显降低。不同滤光膜下幼苗叶片叶绿素含量先降低然后升高 ,遮光幼苗的叶绿素 a/ b明显低于日光幼苗。幼苗日最大净光合速率的顺序是 :日光 >黄膜 >红膜 >蓝膜。处理后第 2 0天 ,不同滤光膜下幼苗的光饱和光合速率 (Amax)、光饱和点 (Is)、光补偿点 (Ic)、最大表观量子效率 (AQYmax)都不同程度的低于日光幼苗。处理后第 10天至第 30天 ,遮光幼苗叶片喜树碱含量均显著高于日光下幼苗 ,以蓝膜下幼苗的喜树碱含量最高。蓝膜和黄膜下幼苗的喜树碱产量在后期处理中显著高于日光下幼苗 ,蓝膜下幼苗喜树碱产量在第 30天最高 ,是日光下幼苗的 2 .4 9倍。红膜下幼苗的喜树碱产量在第 10天后与日光下幼苗差异不显著。通过滤光膜遮光促进喜树碱在幼苗叶片中的积累 ,提高了叶片喜树碱产量 ,对喜树碱的生产实践有一定的意义     

喜树叶、枝水溶性糖提取的研究

用正交实验结果作方差分析表明：溶剂用量对试验结果有较显著的影响（a=0.10)，提取温度对试验结果也有较大的影响，而提取时间对试验结果的影响不显著。喜树叶中水溶性糖的提取最优方案是A3C2B3，即溶剂用量为60ml，提取温度80℃，提取时间60min；喜树叶水溶性糖的提取最优方案是A2C2B1，即溶剂用量为40ml，提取温度80℃，提取时间20min。通过对喜树叶与枝的成对比较分析，叶与枝的水溶性糖含量无显著差异。     

喜树毛状根的诱导及其喜树碱含量分析

研究了不同外植体类型(包括真叶、茎段、子叶及胚轴)、胚轴年龄和不同发根农杆菌菌株(包括A4、15834、R1601、C58C1)等因素对喜树毛状根诱导频率的影响,并用PCR对诱导出的毛状根进行了分子鉴定.结果表明:(1)最佳外植体为胚轴,5~10 d是胚轴最佳诱导年龄段,最佳诱导菌株为15834.(2)PCR鉴定结果表明,发根农杆菌的rolB基因已整合到喜树毛状根基因组中.(3)对不同菌株诱导的毛状根进行HPLC检测表明,C58C1菌株诱导的毛状根的喜树碱和羟基喜树碱含量最高,分别为1.219 mg/g和0.305 mg/g.研究结果为喜树碱的药源开发提供了一条新途径,并为进一步利用基因工程技术调控喜树碱的代谢合成奠定了基础.     

产喜树碱内生真菌的筛选及鉴定

从喜树Camptotheca acuminate树皮和果实中分离得到27株内生真菌,发酵后经HPLC检测,筛选出一株菌丝产喜树碱的菌,产量达774μg/L。对其ITS序列进行系统发育分析,结合其培养特征和显微特征,鉴定为拟茎点霉属(Phomopsis sp.)。这是首次报道分离自喜树的该属真菌发酵产喜树碱。     

丛枝菌根对喜树幼苗生长和氮、磷吸收的影响

喜树(Camptotheca acuminata)是我国特有的多年生亚热带落叶阔叶树种,因其次生代谢产物喜树碱具有良好的抗肿瘤活性而受到人们的广泛关注。该文通过温室盆栽接种试验,观察了2属6种丛枝菌根真菌即蜜色无梗囊霉(Acaulospora mellea)、光壁无梗囊霉(A. laevis)、木薯球囊霉(Glomus manihot)、地表球囊霉(G. versiforme)、幼套球囊霉(G. etunicatum)和透光球囊霉(G. diaphanum)对喜树幼苗生长和氮、磷养分吸收的影响。结果表明,丛枝菌根的形成对喜树幼苗的生长以及氮、磷营养的吸收均有影响。从生物量看,除幼套球囊霉和光壁无梗囊霉侵染形成的丛枝菌根喜树幼苗与无菌根幼苗(CK)差异不显著外,其余菌根幼苗的生物量均明显大于无菌根幼苗,透光球囊霉和蜜色无梗囊霉菌根幼苗尤为突出,分别达到无菌根幼苗的1.9和1.4倍。丛枝菌根的形成似乎不利于喜树幼苗的氮素营养吸收,并且主要体现在叶片的氮含量上。相反,丛枝菌根形成总体上促进喜树幼苗对磷素营养的吸收,并且主要体现在根的磷含量上。与无菌根幼苗比,所有菌根幼苗根的氮、磷分配比例增加,而叶片的氮、磷分配比例减少。