信号肽序列修饰的PAP cDNA克隆及高频率转基因烟草的获得

通过RT-PCR从美洲商陆叶片中获得PAPN端氨基酸修饰的cDNA克隆PAP-sp1。构建携带PAP-sp1的植物转化载体pBPAP,利用农杆菌介导将PAP-sp1导入烟草品种K326叶片细胞,通过抗性筛选、组织化学和PCR鉴定获得了转基因烟草植株,按形成转基因不定芽的外植体数统计,烟草K326的转化率高达8.1%。攻毒实验表明,与对照植株相比,转基因烟草株系发病推迟,感病程度较低,开花结果提早。     

悬浮培养中不同理化因子对烟草发状根生长及烟碱合成的影响

悬浮培养中烟草(Nicotiana tabacum)发状根的生长及烟碱合成受到基本培养基浓度、初始pH值、激素种类和浓度等因素的影响.一个5 cm长的根尖,悬浮在40 mL、pH值为6.0、附加1.0 mg·L-1IAA的1/2 MS液体培养基中,28℃、散光条件下培养30天,烟碱产量可达0.241 mg·mL-1.     

悬浮培养中不同理化因子对烟草发状根生长及烟碱合成的影响

悬浮培养中烟草(Nicotiana tabacum)发状根的生长及烟碱合成受到基本培养基浓度、初始pH值、激素种类和浓度等因素的影响。一个5 cm长的根尖, 悬浮在40 mL、pH值为6.0、附加1.0 mg.L－1IAA的1/2 MS液体培养基中, 28 ℃、散光条件下培养30天, 烟碱产量可达0.241 mg.mL－1。     

ipt与PAPⅡ基因在烟草中共表达减轻PAPⅡ细胞毒性的初步研究

利用农杆菌介导方法,分别将PAPⅡ单基因、PAPⅡ基因和异戊烯基转移酶基因(ipt)共表达的双基因(PAPII-pt)导入烟草品种K326叶细胞中,半定量RT-PCR分析了转基因植株中PAPⅡ、ipt和核糖体蛋白大亚基基因(RPL3A)的表达,并进行了TMV攻毒实验.结果表明:双基因PAPⅡ-ipt对烟草的转化频率高达...     

商陆种子抗病毒蛋白的制备及毒性分析

采用一种改进的方法,从商陆属两种植物——商陆(Phytolacca acinosa)和美州商陆(P. americana)种子中分别制备抗病毒蛋白(Pokeweed antiviral protein from seeds,简称PAP-s)。研究证明,两种植物中的PAP-s性质完全相同。经SDS-PAGE和凝胶过滤层析测得该蛋白质为单一肽链,分子量约30kD;聚丙烯酰胺等电聚焦电泳测得等电点为8.4;经Edman降解,测得N-末端氨基酸为Ile.PAP-s在兔网织红细胞裂解液中抑制蛋白质合成ID_(50)为2.6ng/ml(8.6×10~(-11)mol/1);将该蛋白质与脊髓灰质炎(?)型病毒混合接种于猴肾细胞培养液,显示出较强的抗病毒活性,浓度为4.17×10~(-8)mol/1时能抑制98％的病毒增殖。将PAP-s通过异型双功能连接剂SPDP与抗人T细胞单克隆抗体Wu71偶联制备免疫毒素,对T淋巴细胞白血病CEM细胞显示特异杀伤作用,在10~(-9)mol/1时可杀灭76.4％的靶细胞,~(14)C-亮氨酸掺入蛋白质合成抑制试验显示ID_(50)为10~(-10)—10~(-9)mol/l。     

商陆根中抗真菌蛋白的分离和特性研究

从美洲商陆(Phytolacca americana L.)夏天采集的2—4年生宿根中分离了二种抗真菌蛋白,称为PAFP-R_1和PAFP-R_2。分离程序包括用盐溶液提取,经CM-Sephadex离子交换层析、凝胶过滤层析和羟基磷灰石柱层析纯化。在PGA培养基上,0.1mg/ml蛋白明显抑制木霉菌丝的生长;但对细菌的增殖,即使1mg/ml也无抑制作用。用SDS-PAGE测得二者的相对分子量各为13kD和15kD,单多肽链,等电点约为5.8。用酚-硫酸法未测出含糖。二种蛋白均高含半胱氨酸(19mol/mol)。用Edman降解法测得二者的N末端均为Ala。秋天采集的根中,这二种蛋白含量均很低,但富含由二条PAFP-R_1肽链以双硫键联结的二聚体,它无抗真菌活性。冬天宿根中抗真菌蛋白主要成分是Mr为17kD的单肽链蛋白。上述蛋白对于人红血球均无凝集活性,因此不是PWM的成分。以上结果说明,商陆根中有多种具抗真菌活性的蛋白,成分随季节发生变化,它们都是不含糖,高含半胱氨酸,分子量小于20kD的单肽链蛋白。     

除虫菊发状根的诱导及培养条件优化

以除虫菊（Pyrethrum cinerariifolium Trey.）无菌苗为外植体,研究除虫菊发状根的诱导、培养条件优化,并对发状根中的除虫菊素进行检测和生物活性测定。结果显示,乙酰丁香酮能促进除虫菊下胚轴和子叶发状根的诱导,当乙酰丁香酮浓度为150 μmol/L时除虫菊下胚轴和子叶的诱导率为对照的2.29倍和2.66倍,预培养6 d时,下胚轴发状根诱导率为对照的2.25倍,发根农杆菌A4的诱导率均高于ATCC15834,愈伤组织较适合发状根的诱导,愈伤组织侵染后适合在无激素的MS培养基上进行发状根诱导,250 mL三角瓶中添加50 mL MS培养基较适合发状根的生长。对除虫菊发状根进行PCR检测发现,发根农杆菌含有的Ri T-DNA的rolB基因已整合进入发状根基因组中。通过GCMS检测发现,愈伤组织中除虫菊素的6种成分均未检测到,而发状根中检测到瓜菊素Ⅰ、茉酮菊素Ⅰ和茉酮菊素Ⅱ 3种成分,发状根对粘虫的拒食作用明显优于愈伤组织。本研究为通过组织培养方式生产除虫菊素奠定了基础。     

3种农杆菌对茶树发状根诱导的影响

以茶树'福云6号'和'铁观音'成熟种子下胚轴、未成熟种子下胚轴和愈伤组织为材料,以发状根诱导率为指标,探究菌液浓度、农杆菌菌株、外植体类型和预培养时间对发状根诱导的影响.结果表明:(1)菌液浓度OD600在0.4～1.2范围内,'福云6号'成熟种子下胚轴发状根诱导率先升高后降低,ATCC15834在OD 600为0.6...     

美洲商陆抗病毒蛋白的研究

美洲商陆抗病毒蛋白(pokeweed antiviral proteins.PAP)是一种具有多种生物功能活性的蛋白质,其在广谱抗病毒方面有重要的应用价值。综述美洲商陆抗病毒蛋白的分子结构与功能,抗病毒的分子机理及其在植物基因工程中的应用。     

美洲商陆毛状根诱导及其离体培养的影响因素

为了探讨利用美洲商陆毛状根生产其药用成分的可能性,研究了美洲商陆毛状根诱导及其离体培养的影响因素。结果表明,美洲商陆叶片外植体被发根农杆菌ATCC 15834感染约18 d后,从其叶片外植体形态学下端叶脉切口处产生毛状根,其中以预培养1 d,农杆菌感染20 min,共培养4 d时的毛状根诱导率最高,达到70%。PCR扩增和硅胶薄层层析结果显示,发根农杆菌Ri质粒的rol C基因以及冠瘿碱合成酶基因已在美洲商陆毛状根基因组中整合和表达。所获得的美洲商陆毛状根系都能在无外源激素的MS固体培养基上快速自主生长;其中以毛状根根系2的生长速度最快、分生侧根能力最强和根表面的根毛密度最高;毛状根根表面呈紫红色或呈白色。在供试的MS、1/2MS、B5和6,7-V液体培养基中,以无外源激素的MS培养基最适合美洲商陆毛状根根系生长。与无外源激素的MS培养基相比,6,7-V培养基更有利于毛状根中商陆皂苷甲的合成与积累。本文所建立的美洲商陆毛状根诱导及其离体培养的适宜条件为今后利用其毛状根株系的规模培养来生产其药用有效成分商陆皂苷甲奠定了实验和技术基础。     

美洲商陆中新发现的一种抗菌蛋白基因的克隆和表达

自美洲商陆(PhytolaccaamericanaL.)的种子中发现了一种具有38个氨基酸残基的蛋白(PaAFP),它有明显抑制立枯丝核菌(RhizoctoniasolaniKiihn)作用。依此蛋白的氨基酸序列合成引物,从该种子的mRNA中,通过逆转录PCR,获得了这一基因的具有65个氨基酸的前体蛋白cDNA序列(已在GenBank注册),将这一成熟蛋白的cDNA克隆在pGEX4T1上,并转化到大肠杆菌(E.coli),融合蛋白被大量表达。表达产物经谷胱甘肽Sepharose4B亲合层析,融合蛋白被纯化。纯化后的融合蛋白经凝血酶(thrombin)作用,将谷胱甘肽S转移酶降解,从而获得这一抗真菌蛋白     

Cloning and Expression of a New Gene Encoding an Antifungal Protein in Phytolacca americana

protein (Pa-AFP) with molecular weight about 4 kD was purified from the seeds of Phytolacca americana L. , which obviously inhibits the growth of Rhizoctonia solani Kiihn in vitro. The authors isolated mRNA from the seeds of pokeberry and designed a degenerate PCR primer according to the N-terminal sequence of the purified protein. The full-length cDNA encoding Pa-AFP was cloned by RT-PCR and 5'-RACE and sequenced. The deduced amino acid sequence indicates that a preprotein with 65 amino acid residues is firstly translated and then processed to a mature protein with 38 amino acids. The DNA encoding the mature protein was subcloned into expression vector pGEX-4T1, and expressed efficiently in E. coli BL21 as a GST- Pa-AFP fusion protein. The fusion protein was purified by glutathione-Sepharose 4B affinity colmnn chromatography. The purified fusion protein was specifically digested by thrombin and the Pa-AFP was further purified by filtration column chromatography.     

美洲商陆核基因组抗病毒蛋白基因的克隆及序列分析

通过ＰＣＲ扩增,从美洲商陆（Ｐｈｙｔｏｌａｃａａｍｅｒｉｃａｎａ）核基因组中克隆了商陆抗病毒蛋白基因,序列分析表明,该基因含８８５个核苷酸,与已报道的序列比较,核苷酸同源性为９９．３％。     

Comparison of Phytolacain G,a Cysteine Protease from Fruit of Phytolacca americana,with Phytolacain R

The enzymatic properties of phytolacain G, a protease isolated from green fruit of pokeweed, were compared with those of phytolacain R, a protease obtained from ripe fruit. The optimum pH of phytolacain G was 7.5-8.0 at 37°C using casein as the substrate. The enzyme was strongly inhibited by iodoacetic acid and p-chloromercuribenzoic acid, but not by diisopropyl fluorophosphate or EDTA. These results indicated that phytolacain G was a cysteine protease, like phytolacain R. Nine sites of oxidized insulin B-chain were cleaved by phytolacain G during 20 h of hydrolysis. The six sites cleaved by phytolacain G were also cleaved by phytolacain R. The substrate specificity of phytolacain G was broad, but the preference for hydrophobic residues at the P₂ position was similar to the substrate specificity of papain. The amino-terminal sequence of phytolacain G was not identical with that of phytolacain R; however, the amino acid residues conserved in the papain family were also conserved in this enzyme.     

Isolation and Some Properties of the Antimicrobial Peptide (Pa-AMP) from the Seeds of Pokeweed (Phytolacca americana).

An antimicrobial peptide, designated Pa-AMP, was purified by gel filtration on Sephadex G-75 followed by S-Sepharose, Cosmosil-SP, and reverse-phase HPLC from the seeds of pokeweed (Phytolacca americana). Pa-AMP is a basic peptide having an isoelectric point of over 10 and its extinction coefficient at 280 nm of 1% aqueous solution was 7.7. Pa-AMP has a molecular mass of 4 kDa and 3.4 kDa on tricine SDS-PAGE under nonreducing and reducing conditions, respectively. The N-terminal amino acid of Pa-AMP was blocked. The concentrations of peptide required for 50% inhibition (IC₅₀) of the growth of plant pathogenic fungi, Gram-positive, and Gram-negative bacteria were 3 to 41 μg/ml. Differing from other peptides, Pa-AMP inhibited the growth of some Gram-negative bacteria.     

GC—MS法分析比较垂序商陆根不同提取物的脂溶性成分研究

采用GC—MS联用技术分析比较垂序商陆根不同提取物的脂溶性化学成分。色谱柱为：DB-5毛细管柱,载气为氦气(1mL／min),柱温初始温度50℃,先以20℃／min的升温速率升至170℃,再以2℃／min的升温速率升至210℃;质谱用EI离子源。鉴定了8种相对百分含量大于0．5％的化学成分,其中的3种成分(2-甲氧基-4丙烯基苯酚、带状网翼甾醇、邻苯二甲酸二丁酯)与商陆根中的3种脂溶性成分相同;乙醚提取物与CO2超临界萃取物含有的主要化学成分种类相同,但含量不同;石油醚提取物含有的化学成分种类较少,石油醚萃取物所含成分种类相对较多,且两者有较大差异。     

Crystal structure of pokeweed antiviral protein from seeds of Phytolacca americana at 0.25 nm

Crystals of pokeweed antiviral protein (PAP) from seeds of Phytolacca americana with high diffraction ability were grown from high protein concentration (100 mg/mL) solution at high temperature (33℃). The crystal structure was solved by use of molecular replacement method and refined by use of molecular dynamic method at 0 25 nm to an R factor of 18.15% with standard deviations from standard geometry of 0.001 6 nm and 2.04° for bond lengths and bond angles, respectively. Comparison with two other PAPs revealed, near the active center, a sequence and structure variable region, consisting of the loop connecting the fifth β strand with the second α helix and including a proposed active residue, suggesting this loop probably to be related to difference in activity.$$$$     

Crystal structure of pokeweed antiviral protein from seeds of Phytolacca americana at 0.25 nm

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锰胁迫对垂序商陆生长发育的影响

以垂序商陆为试验材料,采用温室溶液培养方法,在生长介质中添加不同浓度(1、5、8、12和15mmol·L-1)的锰离子,研究锰胁迫对垂序商陆生长发育的影响。结果表明:随着生长介质中锰含量的升高,垂序商陆种子的发芽率、发芽势和有丝分裂指数逐渐降低,幼苗根的生长逐渐受到抑制,根系活力呈下降趋势,植物体各器官的生物量减少。当锰胁迫浓度≥12mmol·L-1时,锰对垂序商陆的生长发育产生非常明显的毒害效应,导致种子的发芽、根尖有丝分裂和幼苗根的生长受到严重抑制,生物量显著减少。锰胁迫对垂序商陆幼苗的芽影响较小,而对生殖器官影响较大,严重降低了垂序商陆的结实数量与质量。