【作者】 杨先国；

【导师】 刘塔斯；

【作者基本信息】 湖南中医药大学 ， 方剂学， 2012， 博士

【摘要】 本文以经过“神舟七号”太空飞船搭载选育出的太空丹参(SP1-1)为研究材料,未搭载的丹参为对照(CK),从植物学特性、抗病性、DNA分子标记、生理生化、光合效应与产量、内在质量以及药效学等方面进行较为系统的对比研究,探讨空间环境对丹参的诱变效应,为筛选与培育产量高、质量优以及抗病性强的优质丹参种质资源奠定基础。实验结果如下：1.太空丹参SP1-1的植物学特性研究对航天搭载丹参中培育出的太空丹参(SP1-1)的根、茎、叶、花等植物学特性进行研究表明,与CK比较,SP1-1的主要特性为植株较矮,茎节间距短,叶片肥厚、表面皱缩,深绿色,叶形较大,根表面红棕色,二者在植物学特性上差异较为明显；根部常见病害调查表明,SP1-1感染根腐病与根结线虫病的发病率低于CK；因此,太空丹参SP1-1为植物学性状优良、抗病性较优的丹参新种质资源。2.太空丹参SP1-1的根系分泌物与抗病性研究采用极性不同的溶剂乙酸乙酯与正己烷为浸提剂,提取CK与SP1-1的土壤根系分泌物,并采用GC-MS对根系分泌物的成分进行分析,结果CK根系分泌物中的化学成分的种类较多且含量明显高于SP1-1,CK与SP1-1根系分泌物中检测到的多种成分,在连作障碍中对作物的生长发育起到不同程度的抑制作用,降低作物的抗病能力,而CK与SP1-1根系分泌物的种类与数量上的差异,必然会对丹参的生长与抗病性产生一定的影响,但根系分泌物中的哪几类成分对丹参的生长发育与抗病性起关键作用还有待作进一步的研究。3.太空丹参SP1-1的细胞学效应与分子标记研究根尖细胞染色体研究表明,CK与SP1-1的染色体数目相同,均为2n=16,且类型基本相似,但在染色体的形态及臂比方面存在差异,说明航天搭载对SP1-1的染色体核型影响较小；采用RAPD分子标记技术,筛选出A1、A13、A15引物并扩增出稳定的、重复性好的A1-450、A1-900、A13-700、A15-1100四个差异片段,对差异片段进行回收、克隆与测序,测序后在GeneBank中采用BLAST工具进行比对,结果SP1-1中扩增出的A1-450与A1-900序列中的部分序列与丹参中的基因序列具有一定的同源性,可能为丹参中调控丹参酮类与丹酚酸类生物合成途径的关键酶基因；而A13-700与A15-1100则分别与牛耳草和拟南芥基因组的部分序列同源性较高。4.太空丹参SP1-1可溶性蛋白及同工酶谱分析电泳分析表明,CK与SP1-1的可溶性蛋白谱带的数目、迁移率及表达量方面存在较大的差异,SP1-1的蛋白质谱带条数为14条,表达量较多,CK为7条；CAT同工酶共有2条酶带,但SP1-1中的2号条带的表达量较强；POD同工酶条带的数量和强度有明显的差异,SP1-1中的POD酶谱中缺少Rf为0.325条带,CK缺少Rf为0.075、0.542条带,且SP1-1中的POD同工酶的5、6、7号三个条带的同工酶谱的强度较强；SOD同工酶中,SP1-1中缺少Rf为0.533条带,CK缺少Rf为0.233条带；SP1-1的同工酶表达量强度较强,以6号条带的谱带强度较强。5.太空丹参SP1-1的光合特性与产量的相关性研究光合色素含量测定表明,SP1-1中的叶绿素、类胡萝卜素的总量在丹参的不同生长阶段均高于CK,叶绿素a在花期、花后期明显地高于CK,不同生长期SP1-1叶片中的Chla.Chlb的比值较CK有不同程度的降低。光合特性研究表明,SP1-1与CK在净光合速率(P。)上的日变化趋势一致,均呈现出双峰曲线形的日变化特征,因而都具有“午休”现象,而在净光合速率(P。)的量值上,SP1-1的Pn值均高于CK。二者叶片的胞间CO2浓度(Ci)呈现“U型”的日变化特征；光响应曲线研究表明,SP1-1的光补偿点(LSP)较高为65.72μmol·m-2·s-1、光饱和点(LSP)较高为754.13μmol·m-2·s-1,均高于CK(LSP为51.36μmol·m-2·s-1,LSP为643.58μmol·m-2·s-1)。丹参产量调查表明,SP1-1的产量较CK提高了近14%,其增产幅度没有显著性差异,但SP1-1在增产方面具有一定的优势与潜力。6.太空丹参SP1-1的综合质量评价研究为了比较CK与SP1-1之间的药材质量之间的差异,对CK、SP1-1药材中的主要活性成分丹酚酸B、二氢丹参酮Ⅰ、丹参酮Ⅱ A的含量以及石油醚提取物、丹参多糖、微量元素等指标进行检测,结果表明,与CK比较,SP1-1中的丹酚酸B、丹参酮ⅡA的含量分别提高13%与50%；多糖含量提高了近16%；CK与SP1-1中的低极性部分的成分种类相似,但相对含量存在较大的差异。从多项检测指标分析表明,SP1-1的药材内在质量优于CK。为了与不同产地的丹参药材进行综合分析,采用高效液相色谱法构建了13批丹参样品的指纹图谱,通过统计分析软件对丹参指纹图谱数据进行聚类分析,结果表明,CK在分类上与安徽丹参的亲缘关系较近,而SP1-1在聚类分析图上与四川丹参距离最近,说明,SP1-1在化学特征上与CK产生了明显的差异7.太空丹参SP1-1对血瘀证大鼠的药效学试验研究采用肾上腺素法复制大鼠急性血瘀模型,结果急性血瘀模型组大鼠血液流变性表现出“浓、粘、凝、聚”的状态。CK与SP1-1在改善全浆粘度、全血粘度(50/s、100/s、200/s)中高切变率、全血(低切、高切)还原粘度以及红细胞刚性指数方面,与模型组比较具有显著性的差异(P<0.05)；二者也可不同程度地降低全血粘度低切变率(1/s、5/s)、全血(高切、低切)相对指数、红细胞聚集指数、红细胞变形指数等指标,但与模型组比较没有显著性差异。与CK组比较,SP1-1对全浆粘度、全血粘度中切(50/S)、低切(5/S)、全血高切、低切相对指数、红细胞聚集、红细胞刚性指数等方面的作用较强。更多还原

【Abstract】 In this paper, Space Danshen SP1-1was bred through the space inducing mutation via the Chinese seven manned spacecraft "Shen-Zhuo7". In order to investigate the mutagenic effects of space environment on Danshen, the comparative research of the botanical characteristics, resistance, DNA molecular marker, physiology and biochemistry, photo synthetic effect and yield, inherent quality and pharmacodynamics were studied between CK and SP1-1, which was lay the foundation for selecting and breeding high yield, superior quality and a strong resistance Danshen germplasm resources.The main results as follows:1. Study on botanical characteristics of SP1-1The botanical characteristics of root, stem, leaf and flower were observed between CK and SP1-1in this study, the main character of SP1-1is included dwarf aerial stem and thicker, stem section space shorter, leaf blade thickness, leaf surface collapse, leaf larger, dark green, root epidermal reddish-brown, the botanical characteristics differences of CK and SP1-1was more obvious. Common disease of root surveying showed that a lower incidence of root rot disease and root knot nematode disease were observed in SP1-1, the results showed that SP1-1had excellent botanical characters and stronger disease resistance to root disease, which was belong to an excellent Danshen germplasm resource.2. Study on root exudates between SP1-1and CKThe rhizosphere soil of CK and SP1-1were extracted with different solvents i.e. Hexane and ethyl acetate, the GC/MS technique was applied to analyze the root exudates, the results showed that the kinds and contents of root exudates in CK were significantly higher than SP1-1. The constituents of root exudates could inhibit the growth in continuous cropping obstacles and reduce the disease resistance of crops. Therefore, the difference root exudates between CK and SP1-1must affect the growth and disease resistance of Danshen, further research would study to investigate which components of root exudates played an important role to affect the the growth and disease resistance in the process of Danshen planting.3. Study on cytological effects and molecular marker of SP1-1The chromosome configuration of CK and SP1-1root tips were observed, as the results showed that the chromosome number was2n=16, Chromosome type was similar, but there were difference between the morphology and arm ratio.RAPD molecular marker technology was used to analyse the genetic diversity of CK and SP1-1, random RAPD primers A1、A13、A15amplified stable and reproducible differential fragments A1-450,A1-900,A13-700and A15-1100. The four differential fragments were cloned and sequenced, the sequence data has been submitted to GenBank to compare with BLAST tools, the BLAST analysis showed that partial sequence of A1-450and Al-900amplified from SP1-1has a certain similarity with SMCCR,SMCCR and SMGGPPS genes, which was the regulatory key enzyme genes of tanshinone and salvianolic acid biosynthesis pathway in Salvia miltiorrhiza. Partial sequence of A13-700and A15-1100has higher similarity with Boea hyqrometrica and Arabidopsis thaliana gene respectively.4. Analysis of soluble protein, CAT, POD and SOD isozyme of SP1-1Polyacryamide gel electrophoresis (PAGE) was used for analyzing the soluble protein, CAT, POD and SOD isozyme of CK and SP1-1. The result showed that, the number, Rf and expression level of soluble protein bands had difference comparing to CK, there are14bands in SP1-1and7bands in CK about soluble protein. there are2bands of CAT in SP1-1and CK, but expression level of No.2band in SP1-1was increased than CK; the number and expression level of POD isozyme in CK and SP1-1showed obvious differences, the band of Rf (0.325) in SP1-1and the band of Rf (0.075,0.542) in CK were lacked, the expression level of No.5,6,7bands in SP1-1were higher than CK.5. Study on the Correlation between photosynthetic characteristics and yield of SP1-1Study on photosynthetic showed that the total content of chlorophyll a,b, chl(a+b)and carotenoids of SP1-1was higher than CK in different growth stage. The content of chla in SP1-1was higher than that of CK in flowering and late-flowering stage significantly(P<0.05). The ratio of chla/b of SP1-1was lower than CK by increasing the content of chlorophyll b in SP1-1.The diurnal change of net photosynthetic rate (Pn) of SP1-1and CK showed a typical double peaked curves and significant midday depression of photosynthesis. The net photosynthetic rate (Pn) of SP1-1was higher than CK. For the intracellular CO2(Ci), their daily variation curves were shapes like letter "U". Responses of photosynthetic rate to light showed that the light compensation point (LCP,65.72μmol·m·s-1) and the light saturation point (LSP,754.13μmol·m-2·s-1) of SP1-1was higher than CK (LCP,51.36μmol·m-2·s-1; LSP,643.58μmol·m-2·s-1). The yield survey of showed that the yield of SP1-1increased about14%than CK, the yield increase is not significant difference between CK and SP1-1, however, SP1-1has a certain advantages and potential to increase yield.6. Study on evaluation of comprehensive quality of SP1-1In order to compare the difference of drug quality between CK and SP1-1, the content of salvianolic acid B, dihydrotanshinone I, tanshinone II A, petroleum extraction, polysaccharide and trace elements were measured in this study. The results showed that the contents of salvianolic acid B, tanshinone ⅡA and polysaccharide in SP1-1was increased by13%,50%and16%respectively compared with CK. The kinds of petroleum extraction were similar, but the relative content has obvious difference. Comprehensive analysis showed that the inherent quality of the SP1-1was superior to CK. In this study, HPLC fingerprint technology was developed to detect13batches Danshen samples from different places. The fingerprint data were managed by related statistical softwares. SP1-1had relatively distant relationship from CK groups, the result showed that the characteristics of chemical composition of SP1-1showed obvious differences compared to CK.7. Pharmacodynamic study of SP1-1in blood stasis ratsIn this study, the urgent blood stasis syndrome model of rats by injecting adrenaline was duplicated. The model groups all displayed status of concentration, viscosity, aggregation and coagulability. Compared with the model group, the decoction of CK and SP1-1could reduce plasma viscosity, whole blood middle, high shear viscosity(50/s、100/s、200/s), blood reduced whole blood low and high shear viscosity, index of erythrocyte rigidly significantly(P<0.05). At the same time, the decoction of CK and SP1-1could reduce the parameters such as whole blood viscosity (low sheers), index of erythrocyte aggregation, index of erythrocyte deformabilty, and there was no significant difference when they compared with the model group. Compared with CK group, SP1-1could reduce plasma viscosity, whole blood (middle, low) shear viscosity, the relative index of whole blood high and low shear, erythrocyte aggregation and erythrocyte rigidly in different degree.更多还原