竹黄菌原生质体的制备与再生分析

为了提高竹黄菌(Shiraia bambusicola P.Hennings)中原生质体用于遗传转化时的转化效率,研究竹黄菌原生质体制备及再生的最佳条件,建立竹黄菌原生质体的高效遗传转化体系。本研究以竹黄菌为材料,采用正交试验分析方法,对影响竹黄菌原生质体制备及再生的主要因素,不同酶解时间、酶解温度、渗透压稳定剂及菌龄、不同再生培养基等进行了系统的研究。结果表明,以0.6 mol/L Na Cl为稳渗剂,用组合酶(质量分数1%裂解酶和质量分数1.5%崩溃酶按4:6体积比混合)在33℃条件下对菌龄为6 d的菌丝体酶解2 h,原生质体产量最高,达到10.57×10~6个/m L;以0.6 mol/L蔗糖为稳渗剂,用组合酶(质量分数1%裂解酶和质量分数1.5%崩溃酶按4:6体积比混合)在29℃条件下对菌龄为4 d的菌丝体酶解2 h,原生质体再生率最高,为0.293%;最适合竹黄菌原生质体再生的培养基是TB3再生培养基。     

α-麦角隐亭产生菌的原生质体诱变育种

为了提高在一般培养条件下不易产生分生孢子的黑麦麦角菌(Claviceps purpurea)的产碱率,对原生质体诱变的可行性进行了探讨。实验表明,对α-麦角隐亭(α-Ergokryptine)产生菌的原生质体采用紫外线(UV)照射9分钟,γ-射线(~(60)Co)10万伦琴/20分钟辐射,亚硝基胍(NTG)6mg/ml 60分钟进行诱变处理,均可获得较稳定的高产突变株。经过上述三种方法的交替诱变处理,α-麦角隐亭的产率由原始菌株的40mg/L,提高到524mg/L,增加了12倍,说明用原生质体进行诱变处理,配合田间复壮,对提高C.purpurea α-麦角隐亭的产率是可行的。     

金龟子绿僵菌原生质体的制备和再生及其羟化酶活性研究

对金龟子绿僵菌(Metarhizium anisopliae)原生质体的制备和再生的影响因素进行实验,并在此基础上考察了甾体底物对原生质体羟化酶的诱导作用。结果表明,原生质体制备的合适条件是:42 h的菌丝体用纤维素酶(10 mg/mL)和蜗牛酶(5 mg/mL)的混合酶在含有0.8 mol/L甘露醇的pH 5.8磷酸缓冲液中,28℃震荡(80 r/min)酶解3 h,原生质体产量可达到6.12×10~7/mL,在含有0.6 mol/L KCl的双层马铃薯培养基上再生率达到7.79%。经过6 h底物诱导的菌丝体制备的原生质体细胞色素P450的表达量比没经过诱导的菌丝体制备的原生质体高约40%,证明该菌羟化酶系统的可诱导性。由于没有细胞壁的阻碍经过底物诱导的原生质体能够高效的将底物转化为产物,且副产物相对较少。     

疏绵状嗜热丝孢菌原生质体的制备与再生

以疏绵状嗜热丝孢菌(Thermomyces lanuginosus)为供试菌株,研究了菌龄、酶的种类及浓度、酶解时间、酶解温度和稳渗剂对原生质体制备的影响及稳渗剂对原生质体再生的影响。结果表明,制备嗜热丝孢菌原生质体比较适宜的条件为:PDB液体培养基培养28 h,以0.7 mol/L NaCl为稳渗剂,0.15 mol/L的溶壁酶,30℃酶解4 h。原生质体再生以0.7 mol/L蔗糖作稳渗剂为最佳。     

蜂头层束梗孢原生质体制备及再生条件研究

从蜂头虫草(Cordyceps Sphecocephala)上分离的无性型蜂头层束梗孢(Hym enostilbe sphecophala)为出发菌株,进行原生质体制备及再生条件的研究。将培养48h的菌丝体用3%溶壁酶于28℃酶解3.5h,原生质体产量可达2.5×107个/mL。原生质体在0.6mol/L硫酸镁的黄豆粉培养基上再生率最高,为0.46%。在50℃热灭活30分钟,原生质体再生率为零。     

大丽轮枝菌原生质体的制备及再生

为建立大丽轮枝菌(Verticillium dahliae)原生质体的制备和再生体系,采用单因素分析法分析了大丽轮枝菌摇培时间、裂解酶浓度、酶解时间和温度、渗透压稳定剂的种类和浓度及pH对原生质体释放的影响;从再生培养基、培养基Agar浓度和酶解时间对原生质体的再生条件进行了优化;并对优化条件下获得的原生质体进行了GFP瞬时表达。结果表明,将分生孢子培养18 h收集菌丝体,以1.2 mol/L的KCl作为渗透压稳定剂,在pH 6.0的条件下,加入10 mg/m L的裂解酶,30℃酶解4 h时,获得的原生质体产量最高,达到3.3×10~7个/mL;在Agar浓度为0.5%的TB3再生培养基中进行原生质体再生,再生率最高,可达22.45%;GFP瞬时表达结果表明,优化条件下获得的原生质体可用于遗传转化材料。     

尖顶羊肚菌原生质体的分离及再生

培养在液体培养基中的尖顶肚菌(Morchella conica Pers)菌丝体可以分离出大量的原生质体。在组合的2号酶液中,在30℃下经4.5小时处理的产量最高(为6.2×10~6个原生质体/100mg·ml)。此法分离得的原生质体再生力很强,液体培养18小时即可再生成菌丝。再生的细胞进行植板培养后形成了菌丝体,并获得了从原生质体再生的菌株。     

蓝色犁头霉原生质体的制备与再生

研究了氢化可的松生产菌蓝色犁头霉原生质体的形成与再生。通过对溶解酶系统的选择,影响原生质体形成的因素如渗透压稳定剂、酶浓度、菌龄、菌丝培养基和培养方式等因素进行考察,发现以0．4mol/L NH4Cl做为稳定剂、2．5mg/mL溶壁酶和5mg/mL纤维素酶组成的混合酶液溶解菌丝,4h后原生质体量可达10^6cell/mL。通过显微镜观察原生质体的形成过程以及在高渗培养基上的再生情况,再生率为15．6％。     

姬松茸原生质体形成和再生的研究

报道了溶壁酶系统、酶浓度、不同菌龄、脱壁促进剂、渗透压稳定剂和酶解温度对姬松茸原生质体释放率及不同再生培养基、渗稳剂种类、菌丝酶解时间和单双层平板对原生质体再生的影响。结果表明 ,菌龄为3～ 5d的菌丝以 1.5 %溶壁酶、0 .5 %蜗牛酶和 0 .5 %纤维素酶组成的酶系统在 30℃以KCl为渗透压稳定剂时 ,形成率为 1.4～ 1.5ⅹ 10 7/mL酶液 ;以蔗糖为渗透压稳定剂 ,菌丝酶解 1.5～ 3h ,以SMY和MYP为再生培养基 ,姬松茸原生质体再生率为 1.1‰～ 1.3‰。     

羊肚菌原生质体制备与再生*

羊肚菌原生质体制备的最佳条件为以培养3d的菌丝体为材料,0.6mol/L蔗糖溶液为稳渗剂,1.5%浓度的溶壁酶30℃条件下酶解3个小时,可得原生质体产量最高为3.35×106个/100mg.以0.6mol/L的蔗糖溶液做稳渗剂,CYM再生培养基上得到原生质体再生率为0.171%.这一研究结果为羊肚菌通过原生质体技术进行菌株的遗传改良提供了重要技术参数.     

Identification of fatty acids in submerged cultures of Claviceps species

Abstract Fatty acids were identified in the submerged mycelium of Claviceps purpurea, Claviceps paspali, Claviceps fusiformis and Claviceps sp. SD-58 by gas chromatography-mass spectrometry (GC-MS) and used for a chemotaxonomical study. A close relatedness was found between Claviceps sp. SD-58 and C. fusiformis . The composition of fatty acids in high-production mutants differed substantially from that of the parent strains. Fatty acid analysis is thus probably only applicable in the taxonomy of non-mutated isolates of Claviceps cultured under standard conditions.     

Isolation,Culture and Plant Regeneration of Protoplasts from an Embryogenic Callus Tissue of Gossypium hirsutum

Protoplasts were isolated and cultured from hypocotyl embryogenic callus tissue of Gossypium hirsutum L. cv. "Lumian 6". The highest yields of viable protoplasts were obtained from a vigorous embryogenic callus 7 to 9 d old subcultured on MS medium supplemented with 2 mg/L IAA and 1 mg/L KT using a solution of 1% cellulase Onozuka R-10, 1% pectinase, 0.7 mmol/L KH2PO4, 2.5 mmol/L Ca2+ , and 0.5 mol/L osmoticum (mannitol), at pH 5.8 and at a temperature of 30 ℃. After separation and purification (in 21% sucrose floatation medium), the protoplasts were laid up in a quiet liquid protoplast culture medium containing K3 salts, NT vitamins with 0.1 mg/L 2,4-D, 0.2 mg/L KT and 0.45 mol/L glucose for 10 to 15 min. The protoplasts were fractioned into an upper and a lower layer in the centrifugal tube. Most of the protoplasts in the lower layer were smaller, round and rich in cytoplasts in which contain many granular substances. When this kind of protoplasts were cultured in the thin liquid protoplast culture medium with a density of 1 x l0s to 5 x los protoplasts/mL, the division and the callus formation of the regenerated cells were easily observed. The first divisions occurred in 3 days and small cell clusters could be seen after 2 to 3 weeks in the culture. At this moment, the addition of the protoplast culture medium with decreased osmoticum once or twice is needed for the continuous protoplasts division to form calli. Regenerated calli, 3 to 5 mm in diameter, were transferred in succession on MS medium with 2 mg/L IAA and 1 mg/L KT for the initiation of embryogenesis. The embryoids germinated on the hormonefree MS medium and a number of plantlets were obtained. It seems that using vigorous embryogenic callus and decreasing osmoticum are the two critical factors for plant regeneration of cotton protoplasts.     

酿酒酵母和产朊假丝酵母原生质体的形成、再生及属间融合

使用由亚硝基胍诱变所得到的营养缺陷型作为单倍体融合亲株的核基因标记,同时也采用线粒体球红霉素抗性突变株的小菌落形式作为融合亲株的线粒体基因标记。酿酒酵母(Saccharomyces cerevisiae)和产朊假丝酵母(Candida utilis)两亲株原生质体的制备是用对数生长早期的细胞在蜗牛酶和0.7M KCl及β-巯基乙醇或二巯基苏糖醇的作用下完成的。二者的原生质体的形成率在30—60分钟内达到90—99％。原生质体再生率,酿酒酵母最高为29—35％,产朊假丝酵母为7.5％。两亲株的原生质体在35％PEG(M.W.6,000),10mM CaCl_2条件下被诱导融合。在基础培养基上,长出以营养互补为标记的融合菌株。融合频率为10~(-5)—10~(-6)。试验表明,这些融合菌株具有杂种的性质。其中一株杂合子在同化D-木糖、纤维二糖等的能力上比亲株明显增强。     

酿酒酵母染色体DNA样品制备和CHEF分析

等高锁状均匀电场（contolllsclampedhomogeneouselectncfield,CHED电泳技术与分子杂交、酵母人工染色体（yeastartificialchromosome,YAC）等分子生物学技术的有机结合,推动了真菌电泳核型、染色体作图和染色体DNA长度多型性（chromosomalDNAlellgthpolymorphism,CLP...     

Plant Regeneration from Protoplasts of Peucedanum Terebinthaceum (Fisch.) Fisch. ex Turcz

Calll with many embryogenic cell colonies were produced from segments of seedlling of Peucedanum terebinthaceum (Fisch.) Fisch. ex Turcz. which were cultured on the 1/2MS agar medium (with half quantity of macronutrients) containing 1 mg/l 2,4-D. Cell suspension culture with high percentage of embryogenic cell colonies was established from the calli shaking in liquid medium. The cell suspension culture was used for protoplast preparation. Protoplasts were obtained with the enzyme mixture containing 1.5% Onozuka R-10, 0.3% Macerozyme R-10, 0.5% Snailase, 5 mmol/l CeCl2, 1 mmol/l KH2PO4, 0.6 mol/l mannital at pH 5.8 and 25℃. Cultured in a modified MS liquid medium containing 1 mg/l 2,4-D+ 0.5 mg/l zeatin, the protoplasts emered division after four days, and formed cell colonies of 0.5–1mm after about forty days. When transfered to 1/2 MS liquid medium supplemented with zeatin (0.5 mg/l), the cell colonies differentiated in to embryoids, then developed into plantlets with many green leaves and roots on the 1/2 MS agar medium devoid of phytohormones.     

Comparison of ergot alkaloid biosynthesis gene clusters in Claviceps species indicates loss of late pathway steps in evolution of C. fusiformis

The grass parasites Claviceps purpurea and Claviceps fusiformis produce ergot alkaloids (EA) in planta and in submerged culture. Whereas EA synthesis (EAS) in C. purpurea proceeds via clavine intermediates to lysergic acid and the complex ergopeptines, C. fusiformis produces only agroclavine and elymoclavine. In C. purpurea the EAS gene (EAS) cluster includes dmaW (encoding the first pathway step), cloA (elymoclavine oxidation to lysergic acid), and the lpsA/lpsB genes (ergopeptine formation). We analyzed the corresponding C. fusiformis EAS cluster to investigate the evolutionary basis for chemotypic differences between the Claviceps species. Other than three peptide synthetase genes (lpsC and the tandem paralogues lpsA1 and lpsA2), homologues of all C. purpurea EAS genes were identified in C. fusiformis, including homologues of lpsB and cloA, which in C. purpurea encode enzymes for steps after clavine synthesis. Rearrangement of the cluster was evident around lpsB, which is truncated in C. fusiformis. This and several frameshift mutations render CflpsB a pseudogene (CflpsB(Psi)). No obvious inactivating mutation was identified in CfcloA. All C. fusiformis EAS genes, including CflpsB(Psi) and CfcloA, were expressed in culture. Cross-complementation analyses demonstrated that CfcloA and CflpsB(Psi) were expressed in C. purpurea but did not encode functional enzymes. In contrast, CpcloA catalyzed lysergic acid biosynthesis in C. fusiformis, indicating that C. fusiformis terminates its EAS pathway at elymoclavine because the cloA gene product is inactive. We propose that the C. fusiformis EAS cluster evolved from a more complete cluster by loss of some lps genes and by rearrangements and mutations inactivating lpsB and cloA.     

Regeneration of Hybrid Plantlets Via Pollen-Hypocotyl Protoplast Fusion in Brassica spp.

It has been reported that "gameto-somatic hybridization" was induced by fusion of microspore tetrad protoplasts with somatic protoplasts in Nicotiana and Petunia. However, since the success of isolation of pollen protoplasts in recent years, the use of protoplasts at pollen stage as one of the fusion partners in such hybridization is a novel experimentation. Young pollen protoplasts were isolated from the pollen grains of Brassica chinensis at mid-late unicellular to early bicellular stage the pollens for 1.5--2.5 h at 25℃ in a CPW solution containing 0.8 % of eellulase, 0.5 % pectinase, 0.1% pectolyase, 1 3 % mannitol, 1 0 % glucose, 0. 3% potassium dextran sulphate and 3 mmol/L MES. The purified pollen protoplasts were then fused with the hypocotyl protoplasts of B. napus by PEG method. Heterokaryons were identified by means of visualization of the fluorescence from FITC-prela-beled pollen protoplasts. In order to increase heterokaryons and reduce hypocotyls homokaryons, the denstity of hypocotyl protoplasts were lowered and the ratio of the number of hypocotyl vs. pollen protoplasts were adjusted from 1 : 3 to 1 : 6. The fusion products were cultured in a liquid KM8p medium supplemented with 0.4 mol/L glucose, 0.8 mg/L 2, 4-D, 0.25 mg/L NAA. 0. 5 mg/L BA, 500 mg/L glutamine and 3 mmol/L MES where cell division and callus formation took place. The calli, after being transferred to a MS medium supplemented with 2.0 mg/L BA, 3 % sucrose and 0.4 % agarose, differentiated into a few shoots. The shoots were transferred onto a half-strength MS medium supplemented with 2% sucrose, 0.1--0. 2 mg/L NAA, 0.5 mg/L IBA and 20% potato juice for root formation. Finally, three plantlets were regenerated. Chromosome counts by roottip squash method revealed that one plantlet was 2n= 48, corresponding to an allotriploid resulted from a fusion between one pollen protoplast of B. chinensis (2n = 20) and one hypocotyl protoplast of B. napus (2n = 38), and the other two plantlets were 2n = 58, which might be an allotetraploid originated from a fusion between two pollen protoplasts and one hypocotyl protoplast. The isozyme patterns of leaf esterases showed that all the three plantlets had bands characteristic of both parents. This is the first case of success in "gameto-somatic hybridization" by using pollen protoplasts rather than tetrad protoplasts as the haploid partner.     

影响决明无菌苗子叶原生质体分离和培养因素的研究

以决明(Cassia obtusi folia)无菌苗子叶为材料,对酶组合、无菌苗日龄,植物激素组合和培养方法对其原生质体的分离和培养的影响进行了研究。结果表明:用3％的纤维素酶和0.2％Pectinase Y-23的酶组合处理决明无菌苗子叶块8小时可以高效分离出有活力的原生质体;约14日龄的决明无菌苗子叶比较适合于原生质体的分离;适当浓度的2,4-D 有利于原生质体的分离。促进原生质体分裂的理想的植物激素组合为0.4 mg/L 2,4-D,1.0 mg/L NAA and 0.1 mg/L KT;漂浮培养法最有利于原生质体的分裂和发育。找出了适合于决明无菌苗子叶原生质体的分离和培养的酶组合、植物激索组合、有效培养方法和决明无菌苗子叶日龄。这为有效地从决明无菌苗子叶原生质体再生植株奠定了基础。