产苦马豆素疯草内生真菌Undifilum oxytropis原生质体的制备和再生

以产苦马豆素内生真菌Undifilum oxytropis NX-FEL001为供试菌株,研究了培养方式、菌龄、酶解时间和酶解温度等,对U.oxytropis原生质体制备和再生的影响。结果表明,U.oxytropis菌丝在质量分数为10g/L纤维素酶+10g/L蜗牛酶+1g/L溶壁酶组成的复合酶溶液,35℃恒温,80r/min振荡孵育3h左右,原生质体释放量最高;原生质体在YCM培养基上,22℃培养9d可以再生形成菌落,培养15d其再生率可达8.5%;原生质体在YCM培养基上再生后其菌落颜色与野生型U.oxytropis不一致,呈现白色;菌丝形态与野生型U.oxytropis相一致,经检测再生后菌丝仍然能够产生苦马豆素。U.oxytropis原生质体的制备,将为进行疯草内生真菌的遗传转化和基因操作研究提供重要工具,为进一步开展疯草内生真菌合成苦马豆素的分子调控机制奠定了基础。

内蒙古棘豆属Oxytropis细胞分类学研究

本文是对内蒙古１４种棘豆属植物的染色体研究，结果如下：染色体基数ｘ＝８．有二倍体和四倍体两个倍性，核型组成由中部和近中部着丝点染色体组成，最后一对（或二对）具中部随体，核型公式有３种类型：２ｎ＝２ｘ＝１６＝８ｍ（２ＳＡＴ）＋８ｓｍ，２ｎ＝２ｘ＝１６＝１０ｍ（２ＳＡＴ）＋６ｓｍ，２ｎ＝２ｘ＝１６＝１２ｍ（２ＳＡＴ）＋４ｓｍ，其中ＯｘｙｔｒｏｐｉｓａｃｉｐｈｙｌｌａＯ．ｏｃｈｒａｎｔｈａ，Ｏ．ｇｒａｃｉｌｌｉｍａ，Ｏ．ｓｑｕａｍｍｕｌｏｓａ，Ｏ．ｄｉｖｅｒｓｉｆｏｌｉａ，Ｏ．ｌａｔｉｂｒａｃｔｅａｔａ，Ｏ．ｌｅｐｔｏｐｈｙｌ－ｌａ，Ｏ．ｈｉｒｔａ属首次报道。

产苦马豆素疯草内生真菌Alternaria SectionUndifilum oxytropis的诱变筛选

【背景】疯草是黄芪属和棘豆属有毒植物的统称,疯草内生真菌Alternaria Section Undifilum oxytropis是从疯草中分离获得的一种具有产生苦马豆素(swainsonine,SW)能力的真菌。因其产物SW一方面体现出良好的抑制肿瘤细胞生长、浸润和转移作用及潜在的抗艾滋病病毒等多种药用活性,另一方面牛羊因大量误食疯草能导致中毒,对草原畜牧业健康发展造成了严重的危害,受到研究人员的广泛关注。但由于疯草内生真菌Alternaria Section Undifilum oxytropis产苦马豆素生物合成机制尚不清楚,严重制约了后续通过生物发酵大量获得SW用以抗肿瘤机制研究与临床应用,以及通过基因工程手段对疯草进行脱毒育种,使疯草成为牛羊可食用、无毒的天然牧草。【目的】利用有效的研究手段,阐明Alternaria Section Undifilum oxytropis产苦马豆素生物合成机制。【方法】以Alternaria Section Undifilum oxytropis为出发菌株,分别采用紫外辐照诱变、亚硝基胍化学诱变、紫外辐照与亚硝基胍复合诱变的3种诱变方式,在不同诱变时间、诱变剂量等条件下对其进行了诱变筛选研究。通过测定不同诱变方式和作用条件下对菌株的致死率,经发酵培养、连续5代传代培养、并采用α-甘露糖苷酶活性分析法检测诱变菌株产苦马豆素含量变化,优化确定了不同诱变方式下最佳诱变条件,并将优势突变菌株接种马铃薯葡萄糖琼脂培养基(potato dextrose agar,PDA)和改良察式液体培养基,连续培养32 d,测定并绘制了突变菌株D4和UD1的生长周期曲线。【结果】经上述3种诱变方式处理后,分别获得1株产苦马豆素含量变化较大、能稳定连续传代培养的突变菌株U4、D4、UD1。其优化的诱变条件,紫外辐照为辐照处理160 s,亚硝基胍化学诱变为亚硝基胍诱变剂量6μL、诱变处理时间5 min,紫外辐照与亚硝基胍复合诱变为紫外辐照20 s,亚硝基胍诱变剂量2μL,诱变处理时间5 min。突变菌株U4、D4、UD1较原始菌株,菌落形态偏小、中间凸起,菌落颜色呈微粉色或白色,且其产苦马豆素含量均有显著变化,其中U4突变菌株产苦马豆素量平均增加16.02%(P<0.01)、D4突变菌株产苦马豆素量平均降低23.58%(P<0.01),UD1突变菌株SW产量平均增加21.87%(P<0.01),但D4、UD1突变菌株生长周期与出发菌株一致,均为24 d。【结论】通过紫外辐照诱变、亚硝基胍化学诱变、紫外辐照与亚硝基胍复合诱变方法,成功筛选出产苦马豆素含量变化较原始菌株差异较大的3株突变菌株U4、D4、UD1,这为后续利用高通量测序等分子生物学手段,在分子水平上阐释疯草内生真菌Alternaria Section Undifilum oxytropis关键基因、关键酶在其生物合成苦马豆素机制关系方面奠定研究基础。

疯草内生真菌Undifilum oxytropis次级代谢产物分析

为阐明疯草内生真菌Undifilum oxytropis次级代谢产物的化学组成,为其生物活性研究和开发利用提供依据,以本实验室保存的疯草内生真菌Undifilum oxytropis为研究菌株,采用察氏液体培养基对该菌发酵培养30d,收集菌丝体和发酵液,按照植物化学成分传统预试方法进行预试。在此基础上用不同极性有机溶剂梯度萃取得到菌丝体和发酵液石油醚相、氯仿相、乙酸乙酯相和正丁醇相,经薄层色谱检测及气相检测方法对其次级代谢产物进行初步分析。系统预试表明,Undifilum oxytropis发酵产物中含有生物碱、蛋白质、酚类与鞣质、黄酮类、醌类、挥发油和油脂等物质;薄层色谱检测发现,Undifilum oxytropis发酵产物中至少含有5种化合物,且菌丝体和发酵液正丁醇相均有苦马豆素及其类似物存在;气相色谱分析Undifilum oxytropis发酵液中苦马豆素含量为5.17×10^(-3)g/L。疯草内生真菌Undifilum oxytropis发酵产物含有苦马豆素等多种次级代谢产物。

A New Flavonoid from Oxytropis kansuensis

A new flavonoid 1 rhamnocitrin-3-O-（6＂-femloyl）-β-D-galactopyranoside-4＇-O-β-D- glucospyranoside called kansuensisoside A together with a known flavonoid 2 （rhamnocitrin- 3-O-β-D-galactopyranoside-4＇-O-β-D-glucospyranoside） were isolated from the extract of the whole plant of Oxytropis kansuensis Bunge. The structure of 1 was elucidated by spectroscopic methods including 2D-NMR techniques （COSY, HMQC, HMBC） and HR-ESIMS.

2D NMR Spectroscopic Studies of the Toxic Alkaloid from Oxytropis Glabra D C.

The Oxytropis, legume genus, is native in the northwest of China, and some of its members have been reported to be toxic to grazing livestock. In the course of studying toxic reason of this legume, a toxic alkaloid was isolated from Oxytropis glabra D C. grown in the Inner Mongolian Autonomous Region. The LD+（50） of （—）-thermopsine（Ⅰ） was shown to be 89.98 mg/kg（in mice）. This paper reports an approach to the structure elucidation of（-）-thermopsine by combining 2D NMR[;H-;H and;H-;C （one-bond and long-range） COSY] and other physicochemical methods. The stereochemistry of compound Ⅰ is also discussed.

Preparation of Polyclonal Antibody Against Swainsonine and Its Effects in Treatment of Oxytropis glabrapoisoned Rabbits

This study was conducted to optimize immunization program to gain the serum against swainsonine （SW） with high titer and to analyze the effects of polychinal antibody in treatment of loco disease of rabbits. Eighteen rabbits were randomly separated into control group, poisoning group and curing group, with six rabbits in each group. Rabbits in poisoning group and curing group were fed 10 g/（kg ~ d） powder of Oxytropis glabra DC every morning for 70 d till the rabbits in poisoning group began to die. From the 21＇t to 24＇h d of O. glabra administration, each of the rabbits in curing group was injected with 1 mL of polyclonal anti- body against SW every day. Blood samples were collected before O. glabra challenge, and then once every 7 d for the serological and immunological evaluation of serum. The resuhs showed that the serum alkaline phosphatase （AKP） activity and SW content in poisoning groups were significantly higher than those in control group on the 7th d （P 〈0.05 ） ; the serum aspartate transaminase （AST） and lactate dehydrogenase （LDH） activity in poisoning groups were significantly higher than those in control group on the 14th d （P 〈 0.05 ）, while the serum ct - mannosidase （AMA） and rate of E-rosette formation in poisoning groups were signifi- cantly lower than those in control group （ P 〈 0.05 ） ; the serum blood urea nitrogen （BUN）, creatinine （ CRE ） and glucose （ GLU ） concentration in poisoning groups were significantly higher than those in control group on the 21＇t d （P 〈 0.05 ）. By injecting serum against swainsonine, the serum AKP, LDH, AST, alanine transaminase （ALT） activity and contents of BUN, CRE, GLU, SW were reduced and serum AMA and rate of E-rosette formation were increased in curing group, by comparing with those in poisoning group. These results in this experiment indicated that polyclonal antibody against SW had good value of preventing from O. glabra DC.

In Vitro Antioxidative Activities of Flavonoid from Oxytropis glabra DC

Anti-lipid peroxidation activity, reducing power and chelating power of flavonoid extracts from Oxytropis glabra DC, as well as their elimination effect on DPPH, superoxide anion and hydroxyl radicals were determined in the paper, so as to evaluate in vitro antioxidative activities of flavonoid extracted from O. glabra. The results showed that flavonoid from O. glabra had fairly strong anti-lipid peroxidation. The antioxidation effects on rapeseed oil successively were vitamin C 〉 sample 〉 vitamin E 〉 BHT 〉 CK; the effects on cottonseed oil successively were sample 〉 vitamin C 〉 BHT 〉 vitamin E 〉 CK; the effects on sunflower oil successively were sample 〉 vitamin C 〉 vitamin E 〉 BHT 〉 CK; the effects on lard successively were BHT 〉 vitamin C 〉 sample 〉 vitamin E 〉 CK. The samples had stronger clearance rate against DPPH, superoxide anion and hydroxyl radicals than control; the reducing power of sample was weaker than Vitamin C and stronger than Vitamin E ; chelating power of sample was weaker than EDTA. Flavonoid from O. glabra had the value for further development and utilization as natural antioxidant.

Effect of Oxytropis glabra DC. Poisoning on α-Mannosidase(AMA) Expression in Mice Brain Tissue

The effect of Oxytropis glabra DC. on α-mannosidase( AMA) expression in mice brain tissue was explored to reveal the toxicity mechanism of O. glabra. Forty mice were randomly divided into four groups,namely control group,experimental group I,experimental group II and experimental group III. The mice in three experimental groups were fed with O. glabra at the doses of 1,5 and 10 g per kilogram weight,respectively. After challenge for 63 d,mice brains were collected to detect changes in distribution and expression of AMA in different brain regions. The results showed that O. glabra poisoning led to declined AMA mRNA expression in mice brain tissue,but the mice in experimental group I had no significant difference with those in control group( P > 0. 05). The AMA mRNA expression in cerebellum,cerebrum and thalamus of mice in experimental groups II and III were significantly lower than that in control group( P < 0. 05),but the AMA mRNA expression in hippocampus and brainstem in three experimental groups had no significant difference with that in control group( P > 0. 05). AMA had very weak expression in hippocampus and brainstem,but it had expressions in other regions,and the expression was positively correlated with the number of neurons and granulosa cells. The results showed that different doses of O. glabra reduced AMA mRNA expression in mice brain tissue,while cerebellum,cerebrum and thalamus were the main target function areas.

Effects of Alkaloids in Oxytropis glabra on Mouse T Lymphocytes

The effects of different doses of swainonine（ SW）,thermopsine（ Ts） and their mixture on transformation of mouse T lymphocytes were studied,in order to figure out the poisoning mechanism of Oxytropis glabra. Totally 240 mice were randomly grouped,and orally administrated with different concentrations of SW,Ts and their mixture. Four mice were randomly selected from each group at the 7th,14th,21st and 35th days,and lymphocytes were separated from anti-coagulated blood collected from eyeball. After Con A stimulation,T lymphocyte proliferation was detected by MTT assay. The results showed that low dose of SW promoted proliferation of mouse T lymphocyte; medium and high dose of SW also promoted proliferation of mouse T lymphocyte within a short period,but would seriously suppress its proliferation after a long period. Low dose of Ts almost had no impact on proliferation of T lymphocyte,while high dose also suppressed its proliferation.A similar result with SW was observed on alkaloid mixture. This indicated that different doses of SW,Ts and their mixture affected proliferation of T lymphocyte in different degrees.

产苦马豆素疯草内生真菌U.oxytropis的发酵培养及苦马豆素检测方法的建立

通过对生长初期疯草内生真菌U.oxytropis菌丝复苏后连续培养32d,每隔4d取样1次,并设3个平行培养组,测定其不同培养时期菌丝干重变化,利用超声减压旋蒸法提取菌丝和发酵液中苦马豆素(SW),并分别利用薄层层析法、气相色谱法和α-甘露糖苷酶抑制法对菌丝、发酵液中SW含量进行测定。结果显示,经测定疯草内生真菌U.oxytropis菌丝干重质量,确定疯草内生真菌U.oxytropis生长周期为24d;薄层层析法、气相色谱法和α-甘露糖苷酶抑制法都可快速对菌丝和发酵液中提取的SW进行定性、定量检测,并比较3种检测方法,发现气相色谱法检测限低、灵敏度更高。本试验完成了疯草内生真菌U.oxytropis生长周期的测定,并建立了菌丝、发酵液中SW的有效、快速提取及检测方法,为下一步产苦马豆素疯草内生真菌U.oxytropis生物合成机理的研究奠定基础。

Novel associations between rhizobial populations and legume species within the genera Lathyrus and Oxytropis grown in the temperate region of China

Fifty rhizobial isolates of Lathyrus and Oxytropis collected from northern regions of China were studied in their genotypic characterization based upon analyses of ARDRA, 16S-23S IGS PCR-RFLP, TP-RAPD, MLEE, sequences of 16S rDNA gene and housekeeping genes of atpD, recA and glnII. The results demonstrated that most of the Lathyrus rhizobia belonged to Rhizobium and most of the Oxytropis rhizobia belonged to Sinorhizobium. A novel group of Rhizobium sp. I and S. meliloti were identified as the main microsymbionts respectively associated with Lathyrus and Oxytropis species in the collection area, which were new associations between rhizobia and the mentioned hosts. This study also provides new evidence for biogeography of rhizobia.

Ecological role of physical dormancy in seeds of Oxytropis racemosa in a semiarid sandland with unpredictable rainfall

Aims Seed dormancy and the soil seed bank are crucial to plant regeneration strategy,especially in semiarid ecosystems with unpredictable precipi-tation.the aim of this study was to investigate how seed dormancy is controlled by environmental factors and how it is correlated with the soil seed bank and regeneration of the perennial legume Oxytropis race-mosa,a dominant perennial herb in Mu Us Sandland of semiarid China.Methods Germination and imbibition experiments on fresh intact and scarified seeds of O.racemosa were used to identify physical dormancy(PY)in seeds of this species.Soil seed bank dynamics,timing of seedling emer-gence and the fate of buried seeds in the natural habitat were investigated.Important Findings PY was broken by mechanical scarification or wet heat/ice water cycles but not solely by dry heat or wet heat treatment.the soil seed bank exhibited seasonal changes in the number of seeds,which was highest in September and lowest in July.Seeds buried at different sand depths gradually lost dormancy;20-42%of the seeds remained dormant after 20 months of burial.Dormancy break occurs grad-ually throughout the year.Our results indicate that O.racemosa exhibits hardcoatedness heterogeneity that spreads germination of a seed cohort between seasons and years in the semiarid environ-ment,where the amount of precipitation during the growing season is highly variable.

SOME NOVEL TRITERPENOID SAPONINS FROM Oxytropis PLANTS

In a previous note, we reported three saponins Ⅰ, Ⅱ and Ⅲ from Oxytropis glabra DC. As a continuing study, five more novel triterpenoid saponins Ⅳ, Ⅴ, Ⅵ, Ⅶ and Ⅹ were isolated from Oxytropis plants. Saponins Ⅳ to Ⅶ was from Oxytropis glabra DC, and Ⅹ from Oxytropis bicolor Bunge. The present note deals with the structures of these saponins.

CHEMICAL CONSTITUENTS OF Oxytropis glabra DC

In our previous paper, two new triterpenoid saponins extracted from Oxytropis ochrocephala Bunge have been reported. This note deals mainly with the structures of other two novel triterpenoid saponins Ⅱ and Ⅲ, isolated from the aerial parts of Oxytropis glabra DC (Leguminosae), a poisonous plant to livestock, distributed

小花棘豆中毒对家兔脏器指标的影响

为探讨小花棘豆(Oxytropis glabra DC)对家兔脏器指标的影响,试验挑选健康22只,分为两组,其中,试验组12只[按照10 g/(d·kg体重)的剂量饲喂小花棘豆干粉];对照组10只(饲喂青干草和少量精料),试验期为70 d,试验结束后取其脏器,计算脏器指数。结果显示,与正常对照相比,试验组家兔脑指数、肝脏指数、肾脏指数、肾上腺指数、睾丸指数、卵巢指数和肺脏指数增大,心脏指数、脾脏指数和胰脏指数下降,部分指标差异达到显著或极显著(P<0.05或P<0.01),与小花棘豆中毒相关性最强的指标为脑指数;但小花棘豆中毒对家兔胃肠道指标无明显影响。结果表明,小花棘豆中毒导致家兔肝、肾、脑等实质性器官损伤。

宁夏黄花棘豆中产苦马豆素内生真菌的分离及鉴定

对宁夏黄花棘豆中产苦马豆素内生真菌进行分离和鉴定,为进一步研究疯草内生真菌合成苦马豆素的分子调控机理提供菌种和奠定基础。分离并纯化黄花棘豆植物组织中的真菌,采用气相色谱质谱联用法对各菌株菌丝中的苦马豆素进行分析,筛选能产生苦马豆素的内生真菌;紫外线照射和黑暗交替培养诱导内生真菌产孢;提取真菌DNA,扩增和测定真菌甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GPD)、内部转录间隔区(internal transcribed space,ITS)和线粒体核糖体小亚基(mitochondrial small subunit,mt SSU)基因序列,进行同源性比对和遗传进化分析,对其进行种属分类和命名。本研究从宁夏黄花棘豆中共分离到5株真菌,其中菌株NX-FEL001菌丝中含有苦马豆素,经诱导培养,该菌株能产生分生孢子及分生孢子梗等结构;序列同源性和系统发育分析结果表明,该菌株与Undifilum真菌的亲缘关系最近,根据形态结构和分子进化分析结果将菌株NX-FEL001鉴定为Undifilum oxytropis。

小花棘豆中毒对和田羊内脏器官α-甘露糖苷酶的影响

探讨小花棘豆(Oxytropis glabra DC)中毒对和田羊内脏器官α-甘露糖苷酶(AMA)的影响,进一步揭示小花棘豆的毒性作用机理。将12只和田羊随机分为3组,即对照组、试验Ⅰ组和试验Ⅱ组。试验组分别按10 g/kg和20 g/kg的剂量饲喂小花棘豆,饲喂至出现典型中毒症状。屠宰后每组随机采集试验羊的肝脏、肾脏、脾脏、心脏和肺脏,检测和田羊内脏器官AMA活性及表达的变化。结果表明,和田羊肝脏、肾脏、脾脏、心脏和肺脏中高尔基体α-甘露糖苷酶Ⅱ(AMA1)和溶酶体α-甘露糖苷酶(AMA2)均有表达。试验Ⅰ组和田羊各器官的AMA2的表达均与对照组差异不显著(P>0.05),试验Ⅱ组和田羊肝脏和肾脏AMA2的表达显著低于对照(P<0.05),但其余器官AMA2的表达均与对照组差异不显著(P﹥0.05);试验Ⅱ组和田羊肝脏和肾脏AMA1的表达极显著低于对照(P<0.01),脾脏AMA1的表达显著低于对照(P<0.05);试验Ⅰ组和田羊肝脏、肾脏AMA1的表达显著低于对照(P<0.05),其余器官AMA1的表达均与对照组差异不显著(P>0.05)。不同剂量小花棘豆均可降低和田羊内脏中AMA活性及其m RNA表达量,肝脏和肾脏是其主要作用的靶区。

小花棘豆中毒对绵羊运氧能力和抗氧化损伤的研究

探讨小花棘豆(Oxytropis glabra DC)对绵羊抗氧化能力的影响,揭示小花棘豆对绵羊的毒性作用机理。将16只绵羊随机分为4组,即对照组、试验Ⅰ组、试验Ⅱ组和试验Ⅲ组,3个试验组分别按照每千克体重5、10、20 g的剂量饲喂小花棘豆,至典型临床症状出现,通过采血检测绵羊红细胞和抗氧化指标的变化。结果显示,与正常对照相比,中毒绵羊血液红细胞数(RBC)、红细胞压积(HCT)、血红蛋白(HGB)、平均红细胞血红蛋白含量(MCH)、平均红细胞体积(MCV)、甘油三酯(TG)、总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、黄嘌呤氧化酶(XOD)活性显著下降,红细胞分布宽度(RDW)、总胆固醇(TC)、丙二醛(MDA)、一氧化氮(NO)、一氧化氮合酶(NOS)、低密度脂蛋白(LDL-C)和极低密度脂蛋白(VLDL-C)含量均显著上升。结果表明,小花棘豆中毒可显著影响绵羊血液运氧和抗氧化能力,并影响绵羊的脂代谢。