【作者】 刘永红；

【导师】 梁宗锁；

【作者基本信息】 西北农林科技大学 ， 植物学， 2010， 博士

【摘要】 半夏为传统中药,含有多种化学成分,生物碱为其药理作用的主要成分之一。半夏单一生物碱中,葫芦巴碱可用于半夏的质量控制,鸟苷为半夏的指标性成分,而肌苷为半夏的鉴别性成分。目前,许多研究集中在半夏生物碱的分离与检测,而关于半夏生物碱的次生代谢调控未见报道。本研究通过组织培养的方法建立了两种体系—愈伤组织颗粒悬浮培养体系和小块茎悬浮培养体系,作为半夏生物碱代谢调控研究的模式材料。系统分离了半夏内生菌,测定了生物诱导子和非生物诱导子对半夏小块茎悬浮培养体系中生物碱含量的影响,以及代谢前体物质对半夏嘌呤生物碱代谢途径中对关键酶sAMP合成酶和IMP脱氢酶活性的影响,主要研究结果如下:(1)以半夏栽培块茎为外植体,在不同激素组合处理下,诱导出三种不同生长速度、颜色、质地、生物碱含量的愈伤组织细胞系;总生物碱含量检测结果表明,MS + 2.0 mg/L 2,4-D + 1.0 mg/L Kin培养基上的细胞系生物碱含量为0.0293%;MS + 0.2 mg/L 2,4-D + 2.0 mg/L 6-BA培养基上的细胞系生物碱含量为0.0234%;MS + 0.5 mg/L 2,4-D + 1.0 mg/L Kin培养基上的细胞系生物碱含量为0.0175%;三种愈伤组织细胞系的生物碱含量都高于栽培块茎的生物碱含量(0.0072%)。通过对愈伤组织细胞系显微结构的观察,推断2,4-D的应用可提高半夏愈伤组织细胞系的生物碱含量。(2)以筛选出的高生物碱含量细胞系为材料,建立半夏细胞悬浮培养体系。试验结果表明在MS + 1.0 mg/L IBA + 3.0 mg/L 6-BA液体培养基中可建立半夏愈伤组织颗粒悬浮培养体系;通过方法学试验,建立了半夏栽培块茎及培养细胞中鸟苷、肌苷和葫芦巴碱含量测定的HPLC方法;对悬浮培养体系中颗粒状细胞团生物碱含量的HPLC检测结果表明,其葫芦巴碱含量为412μg/g,比栽培块茎的提高出了6.8倍,肌苷含量为69μg/g,比栽培块茎的降低了8%,颗粒状细胞团中没有检测到鸟苷的存在。试验结果显示半夏愈伤组织颗粒悬浮培养体系可作为一种模式材料用于半夏葫芦巴碱的生化研究。(3)半夏组培小块茎是组培苗再生过程中的中间体,它和栽培块茎不仅形态上相似,而且具有相似的组织结构。以栽培块茎为外植体,在MS + 0.5 mg/L NAA + 1.0 mg/L 6-BA激素组合的培养基上,诱导出处于器官状态未分化的小块茎;以组培苗的叶片叶柄为外植体,在MS + 0.2 mg/L NAA + 1.0 mg/L 6-BA激素组合的培养基上,诱导出未分化的小块茎。小块茎可继代培养于1/2MS无激素固体培养基或添加0.6或1.2mg/LABA的1/2MS固体培养基上。在添加0.6mg/LABA的1/2MS液体培养基中建立了小块茎的悬浮培养体系。两种组培小块茎的总生物碱含量都高于栽培块茎。以栽培块茎为外植体诱导的组培小块茎中,鸟苷、肌苷和葫芦巴碱含量分别为857μg/g,27μg/g,155μg/g;以叶片叶柄为外植体诱导的组培小块茎中,鸟苷、肌苷和葫芦巴碱含量分别为83μg/g,21μg/g,117μg/g。试验结果显示半夏组培小块茎可作为一种模式材料用于半夏生物碱的代谢调控。(4)用平板分离法从不同产地的半夏根、茎、叶中共分离到内生菌87株,其中内生真菌48株、细菌34株、放线菌5株,内生真菌通过点植法、插片法和印片法根据其形态特征分类鉴定为4科、15属,从而可以看出半夏内生菌多样性丰富。同时,经HPLC法检测,共筛选到12株产半夏生物碱的内生菌,这一结果为工业生产生物碱提供了候选菌株。(5)筛选出两株对生物碱合成具有诱导作用的半夏内生真菌,其分别属于Penicillium sp.和Ozonium sp.。相比对照,Penicillium sp.菌丝体提取物使小块茎的鸟苷含量增加了13-115%,肌苷的含量增加了2-21%,葫芦巴碱含量增加了1-153%。添加Ozonium sp.菌丝体提取物时,相比对照,鸟苷的含量增加了7-35%,肌苷的含量增加了4-41%,葫芦巴碱的合成受到抑制。在培养的前期加入诱导子的诱导效果优于在培养的后期添加。对于不同生物碱的合成,两种诱导子的适宜浓度不同。两株真菌的发酵液中检测到了葫芦巴碱,其分别为:Penicillium sp.和Rhizoctonia sp.。(6)分离鉴定了两株半夏内生细菌,其分别属于Pseudomonas sp.和Enterobacter sp.。两株细菌诱导子都能促进半夏小块茎的生长,Pseudomonas sp.可促进半夏小块茎的萌发,Enterobacter sp.抑制了半夏小块茎的萌发。相比对照,细菌诱导子的添加使鸟苷含量增加了9-166%,肌苷含量增加了2-33%,葫芦巴碱含量增加了114-1140%。Pseudomonas sp.细胞提取物比其活体细胞更能促进小块茎中鸟苷和肌苷的生产,肌苷的含量在两种诱导方式下差别不大。Enterobacter sp.活体细胞和小块茎共培养比其细胞提取物更能促进小块茎中鸟苷、肌苷和葫芦巴碱的生产。这两株半夏内生细菌都可产半夏植株的一些生物碱。(7)考查了非生物诱导子氯化钙、肌醇、水杨酸、壳聚糖和前体化合物烟酸、天冬氨酸、甘氨酸和肌苷酸对半夏小块茎悬浮培养体系中生物碱合成的影响。试验结果显示,除了氯化钙和肌醇诱导子的添加抑制了鸟苷的合成,其它诱导子都可促进小块茎中鸟苷、肌苷和葫芦巴碱的合成。考察了鸟苷和肌苷代谢前体物质甘氨酸和肌苷酸添加对嘌呤生物碱代谢途径中关键酶sAMP合成酶和IMP脱氢酶比活力的影响。试验结果表明,添加了IMP的半夏组织中酶比活力比没有添加任何试剂的酶比活力要稍高,添加了甘氨酸的培养基中培养的半夏组织中酶比活力较大,一般都达到十倍以上。更多还原

【Abstract】 Pinellia ternata is a traditional Chinese medicine. It has multiple chemicals, and alkaloids are regarded as its main pharmacological component. Among the single alkaloids isolated, trigonelline can be used to investigate the quality control of P. ternata. Guanosine is the index component of P. ternata. Inosine is regarded as the authenticated component of P. ternata. Most researches have focused on isolation and detection alkaloids of P. ternata, whereas there has no report on study the secondary metabolism of Pinellia alkaloids. In this study, two systems were set up to study secondary metabolism in P. ternata, eg. callus aggregate suspension culture and protocorm-like body （PLB） suspension culture. The endophytes of P. ternata were isolated. Both biotic and abiotic elicitors were used to elicit the biosynthesize of Pinellia alkaloids in PLB suspension cultures. The effect of metabolize substrates on the activity of key enzymes involved in purine alkaloids metabolism pathway were also studied. The main results are as follows:（1） Three callus types, which varied in growth rate, color, texture and alkaloid content, were induced from tuber explants with various hormone combinations. Total alkaloid contents were determined. Callus cultured on MS + 2.0 mg/L 2,4-D + 1.0 mg/L Kin had an alkaloid content of 0.0293%. Callus cultured on MS + 0.2 mg/L 2,4-D + 2.0 mg/L 6-BA had an alkaloid content of 0.0234%. Callus cultured on MS + 0.5 mg/L 2,4-D + 1.0 mg/L Kin had an alkaloid content of 0.0175%. Alkaloid content of all three calli were higher that that of field-grown tuber, which was 0.0072%. Compared the microstructures of calli, it may be concluded that 2,4-D could improve the alkaloid content in Pinellia calli.（2） Callus cultured on MS + 2.0 mg/L 2,4-D + 1.0 mg/L Kin had the highest alkaloid content. This kind of callus was used to set up cell suspension culture of P. ternata. It indicated that callus aggregate suspension culture could be obtained in liquid MS containing 1.0 mg/L IBA and 3.0 mg/L 6-BA. With methodological experiment, the detection condition of guanosine, inosine and trigonelline was set up. Guanosine, inosine and trigonelline in both tubers and cultured cells could be detected by HPLC. Alkaloid contents of callus aggregate in suspension culture were determined. Trigonelline content was 412μg/g, which was 6.8 times higher than that in field-grown tuber. Inosine content was 69μg/g, which was 8% lower than that in field-grown tuber. No guanosine was detected in callus aggregate.（3） PLBs of P. ternata are a kind of intermediate before the formation of tissue cultured seedlings. PLBs are similar to field-grown tubers morphologically and histologically. It was observed that MS medium containing 0.5 mg/L NAA and 1.0 mg/L BA induced the highest frequency of undifferentiated PLBs from tuber explants; whereas, a combination of 0.2 mg/L NAA and 1.0 mg/L BA was best suited for inducing undifferentiated PLBs from leaf and petiole explants. When these PLBs were subcultured on solid MS medium containing 0.6 or 1.2 mg/L abscisic acid （ABA）, ABA promoted proliferation of PLBs, but inhibited their germination. To elicit alkaloid biosynthesis, suspension cultures of PLBs were established in half-strength MS （1/2 MS） liquid medium supplemented with 0.6 mg/L ABA. Total alkaloid contents of two kinds of PLBs were higher than that of field-grown tubers. Guanosine content of PLBs derived from field-grown tuber was 857μg/g. Inosine content of that was 27μg/g, Trigonelline content of that was 155μg/g. Guanosine content of PLBs derived from leaf and petiole was 83μg/g, Inosine content of that was 21μg/g. Trigonelline content of that was 117μg/g. Results indicated that PLBs could be used as a model material for study alkaloids metabolism in P. ternata.（4） Eighty seven entophytes were isolated by spot-planting method from P. ternata collected from three differernt habitations. Endophytic fungi were 48. Endophytic bacteria were 34. Endophytic fungi were morphologically identified belonging to 15 genera, and 4 families by three methods. The results suggested that entophytes were abundance in different organs of P. ternata. With HPLC, it was found that 12 endophytes can produce the alkaloids of P. ternata. The strains may be used to produce alkaloid in fermentation industry.（5） Two endophytic fungi, Penicillium sp.and Ozonium sp., could elicit the biosynthesize of alkaloids in PLB suspension cultures of P. ternata. Extracts from mycelium of Penicillium sp. increased guanosine content in PLBs by 13-115%, inosine content by 2-21%, trigonelline content by 1-153%, compared to control. In contrast, extracts from mycelium of Ozonium sp. increased guanosine content in PLBs by 7-35%, inosine content by 4-41%, wheras trigonelline synthesis was restrained, compared to control. The elicitation effects were better when elicitors were added to PLB suspension culture at the beginning. Trigonelline was detected in the fermentation broth of two strains of endophytic fungi, Penicillium sp.and Rhizoctonia sp..（6） Two strains of endophytic bacteria, Pseudomonas sp. and Enterobacter sp. were isolated and identified. Both bacterial strains increased the growth rate of P. ternata PLBs. Pseudomonas sp. promoted the differentiation of the PLBs, whereas Enterobacter sp. inhibited PLB differentiation. The bacterial strains increased guanosine production in PLBs by 9 to 166%, inosine production by 2 to 33%, and trigonelline production by 114 to 1140% compared to the control. For Pseudomonas sp., guanosine and trigonelline production was greater when bacterial extracts were added to the PLB suspension cultures rather than living cells （co-culture treatment）. Inosine production was similar in both the bacterial extract and co-culture treatments. For the Enterobacter sp., guanosine, inosine, and trigonelline production tended to be greatest when living cells were added to the PLB suspension cultures rather than bacterial extracts. We also observed that Pseudomonas sp. and Enterobacter sp. produced some of the same alkaloids as their host plants.（7） The effect of abiotic elicitors on the biosynthesize of alkaloids in PLB suspension cultures of P. ternata was investigated. These elicitors were CaCl₂, inositol, salicylate, chitosan, nicotinic acid, glycin, aspartic acid, IMP. Results indicated that CaCl2 and inositol restrained the accumulation of guanosine in PLBs. As to other elicitors, they could stimulate the accumulation of guanosine, inosine and trigonelline in PLBs. Two substrates, glycin and IMP, were investigate their effects on the specific activity of two key enzymes, IMP dehydrogenase and sAMP synzyme. It indicated that IMP could increase the specific activity of both the two enzymes slightly, compared to control. Glycin could increase the specific activity of the two enzymes notablely compared to control, usually a increase of more than 10 times.更多还原