黄芪毛状根的大量培养

毛状根（hairyroots）是发根农杆菌（他如加加洲南栩前议）感染双子叶植物后,其R质粒上的TDNA片断整合进植物细胞核基因组中诱导产生的一种特殊表现型,近10年来已发展成一种新的培养系统［‘j。与植物细胞培养相比,毛状根培养具有生长速度快、激素自养、分化程度较高以及遗传性状相对稳定等优点,因此通过毛状根大规模培养,从中提取有价值的植物次生代谢产物具有应用前景。由于近三分之一传统药材的药用部位是根,所以这一培养系统在传统药材生产中具有更重要的意义。自1997年Ache＿。nn［fi首先成功地用发根农杆菌转化高等植物以来…     

黄芪毛状根化学成分分和免疫功能活性的研究

应用大规模培养技术生产的黄芪毛状根、获得１０ｇ／Ｌ的产量。与黄芪干燥根化学在分相比,黄芪毛状根中粗皂甙和可溶性多糖含量较高,黄芪甲甙含量相当,而６种异黄酮,总多糖和生多糖含量较少,证明两种来源的黄芪根质量相似。从免疫功能低下的小鼠免疫功能恢复的实验结果也证实了这一点。本结果显示大规模生产的黄芪毛状根可能成为中药材黄芪的一种新来源。     

黄芪UGPase cDNA克隆、分析及其在大肠杆菌中的表达

在已报道的UGPase的植物cDNA序列基础上,从膜荚黄芪( Astragalus membranaceus (Fisch.) Bunge)毛状根中分离了此酶的cDNA.此cDNA全长为1 831 bp,推测编码分子量为51.5 kD、等电点为6.01的由471个氨基酸残基组成的多肽.将此cDNA的开放阅读框载入质粒pET28(a)+并转入大肠杆菌( Escherichia coli ) BL21.SDS-PAGE表明此酶已经在 E.coli 中获得大量表达,表达量约为总细菌蛋白的40%.酶活分析表明,转化菌中UGPase 的活性比非转化菌高0.50～3.27倍,证明此cDNA可以在原核生物中获得表达.Northern blot表明UGPase在黄芪的根、茎、叶及毛状根中均有表达,在根及毛状根中表达量较高,证明了此酶主要分布于植物贮藏组织的报道.     

黄芪毛状根化学成分和免疫功能活性的研究

应用大规模培养技术生产的黄芪毛状根,获得10g／L的产量。与黄芪干燥根化学成分相比,黄芪毛状根中粗皂甙和可溶性多糖含量较高,黄苠甲甙含量相当,而6种异黄酮,总多糖和酸性多糖含量较步,证明两种来源的黄苠根质量相似。从免疫功能低下的小鼠免疫功能恢复的实验结果也证实了这一点。本结果显示大规模培养生产的黄芪毛状根可能成为中药材黄芪的一种新来源。     

黄芪UGPase cDNA克隆、分析及其在大肠杆菌中的表达

在已报道的UGPase的植物cDNA序列基础上,从膜荚黄芪（Astragalus membranaceus(Fisch.)Bunge)毛状根中分离了此酶的cDNA。此cDNA全长为1831bp,推测编码分子量为51.5kD,等电点为6．01的由471个氨基酸残基组成的多肽。将此cDNA的开放阅读框载入质粒pET28(a)^ 并转入大肠杆菌（Escherichia coli)BL21。SDS－PAGE表明此酶已经在E.coli中获得大量表达,表达量约为总细菌蛋白的40％。酶活分析表明,转化菌中UGPase的活性经非转化菌高0．50－3．27倍,证明此cDNA 可以在原核生物中获得表达。Northern blot表明UGPase在黄芪的根,茎,叶及毛状根中均有表达,在根及毛状根中表达量较高,证明了此酶主要分布于植物贮藏组织的报道。     

反相高效液相色谱法测定黄芪和黄芪毛状根中大极性异黄酮成分的含量

本文用RP-HPLC法测定了黄芪和黄芪毛状根中四种大极性异黄酮成分,同时比较了硅胶处理法和溶剂萃取法制备样品对含量测定的影响.结果显示:对于极性较大的化合物用前法更精确、可靠、快速、误差小,将为生产工艺提供质量检测的可靠依据.色谱柱采用大连化学物理研究所的YMG C_(18)柱(250mm×4.6mmID;10μm)流动相为:①MeOH:MeCN:H_2O=40:5:60,②MeOH:MeCN:H_2O=28:5:67,流速为1.0ml/min,检测波长:230nm.     

反相高效液相色谱法测定黄芪和黄芪毛状根中大极性异黄酮成 …

本文用ＲＰ－ＨＰＬＣ法测定了黄芪和黄芪毛状根中四种大极性异黄酮成分,同时比较了硅胶处理法和溶剂萃取法制备样品对含量测定的影响。     

重金属超富集植物东南景天毛状根的诱导

为建立重金属超富集植物东南景天(Sedum alfredii)的毛状根诱导体系,采用发根农杆菌(Agrobacterium rhizogenes)A4侵染叶片,研究了预培养时间、侵染时间和共培养时间对毛状根诱导率的影响。结果表明,东南景天叶片外植体的预培养时间为48 h、农杆菌侵染时间为6 min、共培养时间为48 h是适宜的毛状根诱导时间,毛状根的诱导率可达85%。PCR检测表明诱导的毛状根中存在rol B基因片段。这是东南景天首次建立用发根农杆菌诱导毛状根体系。     

萌发后食松幼苗淀粉粒结合与可溶性淀粉合酶的研究

裸子植物食松 (PinusedulisEngelm .)幼苗在萌发后具有淀粉积累的独特特征 ,而淀粉合酶在裸子植物中尚未有研究。这项研究对在室温下暗中萌发的食松幼苗的淀粉合酶进行了提取和研究 ,结果显示 :5 8kD和 91kD的淀粉粒结合蛋白均为豌豆淀粉合酶Ⅱ抗体所识别。5 8kD淀粉粒结合蛋白被纯化出来 ,其N端序列与其他几种被子植物淀粉粒结合的淀粉合酶Ⅰ的N端序列有很高的相似性 ,并与豌豆、大麦、马铃薯淀粉粒结合淀粉合酶Ⅰ有免疫相似性 ,被鉴定为淀粉粒结合的淀粉合酶Ⅰ。从DEAE_Sepharose柱上洗脱出来的可溶性淀粉合酶表现出两个淀粉合酶活性峰 ,表明至少有两类可溶性淀粉合酶同工酶。对可溶性淀粉合酶的前体亲和特性进行了研究。以兔肝糖原为前体时 ,可溶性淀粉合酶的亲和性最高。裸子植物淀粉合酶的酶学特性表现与已研究的被子植物相似。     

Improved Cassava Starch by Antisense Inhibition of Granule-bound Starch Synthase I

Cassava is a poor man's crop which is mainly grown as a subsistence crop in many developing countries. Its commercial use was first as animal feed (also known as tapioca), but has shifted since the late sixties to a source of native starch. The availability of native starches, which on the one hand do not require substantial chemical derivatisation and on the other hand have improved properties, would make cassava also for small farmers a potentially attractive cash crop. Since breeding is difficult in this polyploid, vegetatively propagated, crop a transgenic approach would be ideal to improve certain characteristics. We have created a cassava genotype producing amylose-free starch by genetic modification. The absence of amylose increased the clarity and stability of gels made with the transgenic starch, without requiring treatment with environment-unfriendly chemicals such as epoxides (propylene oxide, ethylene oxide) and acetic anhydride, which are normally used to improve stability. The amylose-free starch showed no changes in particle size distribution, chain length distribution or phosphorous content when compared to amylose-containing starch, but the granule melting temperature was increased by almost 2°C. Furthermore, the amylose-free cassava starch shows enhanced clarity and stability properties. These improved functionalities are desired in technical applications in paper and textile manufacturing, but also in the food industry for the production of sauces, dairy products and noodles.     

Molecular Cloning and Sequence of Potato Granule-Bound Starch Synthase Gene

It is known that tuber-specific expressions of many genes exist in the process of tuber development from stolon in potato (Solanum tuberosum). Study on the regulation of those gene expression will share light on the mechanism of organ-specific gene expression. Potato GBSS (granule-bound starch synthase) gene, which is solely responsive for the pres- ence of amylose in potato tuber, expression is tuber-specific. The paper describes the construction of a genomic library of a Chinese potato cultivar "Dongnong 303" in which 20 clones were isolated using partial GBSS gene sequence ampified by PCR. 5428 bp DNA sequence of one clone (GBSS17-1) was determined, including 1823 bp 5' flanking region. 2964 bp structure gene, and 641 bp 3' flanking region. It is highly homologious with reported GBSS gene sequence. In addition, the 730 bp most upstream sequence of 5' flanking region which was not reported previously contained stem and loop structures. The present result may provide some important information for further study in the molecular mechanism of organ specific gene expression.     

膜荚黄芪与蒙古黄芪幼苗的光能利用及其耐光性研究

以盆栽膜荚黄芪和蒙古黄芪幼苗为试验材料,测量2种黄芪叶片的光合速率、叶绿素荧光参数、光能利用率及耐光性等指标的日变化,以探讨它们的光能利用、热耗散以及耐光性差异.结果表明:(1)随着太阳光合有效辐射的日变化进程推进,2种黄芪的初始荧光(F0)、非光化学淬灭(NPQ)、PSⅡ调节性能量耗散量子产最(YNPQ)先升高后降低...     

Isolation and Structural Analysis of an Acidic Polysaccharide from Astragalus membranaceus （Fisch.） Bunge

A new water-soluble hetero-polysaccharlde, APSID3, was obtained from a hot-water extract of the roots of Astragalus membranaceus （Flsch.） Bunge by DEAE-Sepharose Fast Flow and Sephacryl S-300 chromatography. The molecular weight of APSID3 was estimated to be 5.79 × 10^5 Da. Based on a sugar composlUon analysis, methylatlon analysis, partial hydrolysis and 13C nuclear magnetic resonance experimentation, It was concluded that the minimal repeat unit of APSID3 was composed of one terminal arablnose, one 1,5-1Inked arabinose, one 1,3-1Inked rhamnose, one 1,3,4-1Inked rhamnose, five 1,4-1Inked methyl galacturonates and six 1,4-1inked methyl glucuronates.     

Establishment of Hairy Root Cultures of Astragalus membranaceus and the Extrinsic Factors Affecting Their Growt

Hairy root cultures were established from the leaf explant of Astragalus membranaceus (Fisch.) Bge. infected with Agrobacterium rhizogenes and factors that affect their growth were investigated. It was found that the hairy roots grew best in a phytohonnone-free MS medium at pH 6, in which the most optimal concentration of sucrose was 3 % and NH4NO3 was omitted because of its inhibitory effect to the hairy roots. Light was also inhibitory to them but the effect of low temperature was not apparent. Moreover, there were more crude saponin and soluble polysuccharide contents in the hairy roots than in the natural dry roots. Finally, a discussion on the application and the potenrial of the hairy root technique in the production of the plant roots as an effection Chinese traditional medicine was also involved.