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1.
辅酶Q10产生菌的抗性筛选及发酵条件优化 总被引:1,自引:0,他引:1
以根癌土壤杆菌(Agrobacterium tumefaciens)WSHAT12为出发菌株,通过硫酸二乙酯诱变,获得遗传稳定性好的抗L-乙硫氨酸(Eth)突变株WSH-E01,通过进一步的诱变处理,获得L-乙硫氨酸和维生素K3(VK3)双抗性突变株WSH-V01,以双抗性突变株WSH-V01为出发菌株,再进行诱变处理,获得一株X-gal利用能力提高的突变株WSH-X01,与出发菌株WSHAT12相比,突变株WSH-X01的辅酶Q10产量提高幅度达50.6%,同时,对突变株WSH-E01的发酵条件进行优化。出发菌株WSHAT12、突变株WSH-E01、WSH-V01和WSH-X01在优化后的发酵条件下辅酶Q10产量分别达到23.1mg/L、26.8mg/L、29.5mg/L和34.8mg/L。 相似文献
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陈金卿 《氨基酸和生物资源》2016,38(1):53-57
以实验室保存的类球红细菌(Rhodobacter sphaeroides)JDW61为出发菌株,考察了紫外、紫外结合氯化锂和亚硝基胍对菌株产生辅酶Q10能力的诱变效应,并结合辅酶Q10的合成途径设计了快速筛选辅酶Q10高产菌株的模型,获得一株辅酶Q10产量提高的突变株CP222,该菌株摇瓶发酵的辅酶Q10产量为276.14mg·L-1,较出发菌株提高了190%,并且遗传性能稳定。 相似文献
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微波结合紫外诱变选育辅酶Q_(10)高产菌株 总被引:2,自引:0,他引:2
以根瘤土壤杆菌LNUB335为出发菌株,以维生素K3和NaN3双抗性为筛选标记,在根瘤土壤杆菌中首次利用紫外线及微波联合诱变处理,获得1株生产性能比LNUB335显著提高的突变株ARN007,其CoQ10产量为12.01mg/L,较出发菌株提高68.67%,每克干细胞含CoQ102.46mg,较出发菌株提高38.20%。通过传代实验证明该突变株的遗传性稳定,可作为进一步研究的实验菌株。 相似文献
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采用紫外线、半导体激光及紫外线与半导体激光复合作用的方法,诱变产辅酶Q10红酵母菌SY-3,以提高辅酶Q10的产量。结果表明,紫外线和半导体激光单独作用,诱变效果不佳,而二者的复合作用却能产生很好的效果。用紫外照射120 s再经半导体激光辐射8 min,得到一株叠氮钠和维生素K3双抗性突变株,产辅酶Q10的量达到157.7 mg/L,比原始菌株提高了88.1%,并具有良好的遗传稳定性。 相似文献
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对辅酶Q10生产菌株鞘氨醇单胞菌YZ0803的发酵条件进行优化,确定发酵时间为90 h,250 mL摇瓶装液量为30 mL。培养基组成(质量分数,下同):葡萄糖1.5%,淀粉2.5%,黄豆饼粉2.5%,(NH4)2SO40.5%,NaCl0.03%,K2HPO40.02%,MgSO40.005%。优化后的辅酶Q10产量达到192 mg/L,比采用基础培养基的产量(138mg/L)提高了39.13%。 相似文献
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[背景]洛蒙德链霉菌S015能生物合成具有广谱抗菌活性的吩嗪类化合物洛蒙真菌素。[目的]因S015菌株的洛蒙真菌素产量较低,将S015菌株经复合诱变育种和基因工程改造,提高洛蒙真菌素产量。[方法]建立洛蒙真菌素产生菌的高通量筛选方法,对出发菌株S0 15进行常压室温等离子体(atmospheric and room temperature plasma,ARTP)技术和紫外复合诱变,筛选得到高产菌株;并在高产菌株上敲除洛蒙真菌素的前体分支酸竟争途径中的关键基因trpE1、trpE2,再过表达全局调控基因afsR。[结果]利用洛蒙真菌素在紫外波长375 nm处的特征吸收峰,以及洛蒙真菌素浓度和375 nm处吸光度值的正相关关系,建立了基于24孔深孔板发酵和酶标仪快速检测的高通量筛选方法。经过6轮ARTP和紫外复合诱变及高通量筛选,从4 320株突变株中筛选得到遗传稳定的高产菌株M6,其洛蒙真菌素的产量为61.33 mg/L,是S015菌株的7.35倍;M6菌株的分支途径基因trpE1、trpE2双敲株的洛蒙真菌素产量为81.89 mg/L,是S015菌株的9.82倍;在该基因工程菌株中过表达全局调控基因afsR,产量为109.53 mg/L,是S015菌株的13.13倍。[结论]建立的高通量筛选方法可以有效筛选高产洛蒙真菌素的突变株,并且操作简单快速。通过ARTP和紫外复合诱变,结合高产株M6的基因工程改造,能进一步提升洛蒙真菌素的产量。 相似文献
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The embryo sacs and female cells could be isolated from the unfertilized ovules of Nicotiana tabacum L. var.
macrophylla which were treated in a solution containing 1.5 % cellulase R- 1O, 1% macerozyme R-10, 10%
mannitol, 10 mmol/L CaCI:, pH 5.8 for 3 h followed by given slight pressure with a micropipette. The central
cells could be kept viable for 10 h and the egg cells for 3 h in 10% mannital. Sometimes, the in situ fusion
products of egg cell and synergid protoplasts could be obtained and kept viable for at least 5 h. The high
concentration (20 mg/L) of 2, 4-D was used in enzyme solution to induce the division of the unfertilized central
cells and other megagametophytic cells in subsequent culture. Treatment of 2,4-D together with enzymatic
maceration of ovules was proved to be better than its direct treatment of isolated embryo sac or its component
cells. Isolated embryo sacs were cultured in microchambers (Millicell-CM PICM 012 50 MILLIPORE) feeded
with divided mesophyll protoplasts of Nicotiana rustica L. The medium was KMSp medium supple- mented with
1% glucose, 0.1 mol/L mannitol, 0.1 mol/L sorbitol, 0.25 mol/L sucrose, 1 mg/L BA, 6% to 10% coconut water,
and 0.15% low gelling agarose. Division of central cells, antipodal cells and the in situ fusion products of egg cell
and synergid protoplasts were induced. The unfertilized central cell was for the first time to be induced in vitro to
develop into small cell clusters. 相似文献
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烟草未受精中央细胞及其它胚囊细胞的离体分裂 总被引:1,自引:0,他引:1
自70年代中期以来,未传粉子房和胚珠的离体培养已在多种植物中取得成功,得到的单倍体植株来源于胶囊中的卵细胞、助细胞以及反足细胞。而分离的未受精胚囊及其成员细胞的离体培养虽屡经尝试,迄今只有Kranz等诱导了玉米未受精卵细胞分裂形成小愈伤组织,至于中央细胞与其它雌配子体细胞则无离体分裂的报道。本文报道大叶烟草未受精中央细胞首次培养成细胞团及其它胚囊细胞启动离体分裂的实验结果。 相似文献
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Heracleum moellendorffiz Hance is a herb belonging to Umbelliferae used in traditional medicine in China. The young stem-nodes were induced for callus formation on MS medium containing 1 mg/L 2,4-D. After subcultured for about five months, the embryogenic calli were used for cell suspension culture. The protoplasts were prepared from this suspension by digestion with enzyme mixture containing 1. 5% cellulase Onozuka R-10 +0. 3% macerozyme R-10 + 0. 5% snailase + 5 mmol CaCl2 + 0. 6 mol/L mannitol, at pH 5.8, and cultured in modified MS and modified N6 media with 0.3 % agarose. They divided after 3 days and developed into small cell colonies after about 2 weeks. From this time on, the glucose concentration in the culture media was decreased to 0. 2 mol/L,which led to futher growth of the colonies to small calf . After a period of proliferation on solid medium with 0. 5 mg/L 2,4-D, the calli were transferred to a medium with 0. 1 mg/L zeatin on which somatic embryos differentiated and developed to plantlets 相似文献
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Calli were induced and suspension cell lines were set up from ovule of Malus pumila Mill. Protoplasts (5.40 × 106/g fr. wt) were isolated from suspension cell lines in enzyme mixture solution containing 2.0% Onozuka R-10, 0.5% pectinase, 0. 65 mol/L mannital, 0.01 mol/L CaC12, 0.7 mmol/L KH2 PO4, 0.3% dextran sulfate potassium salt, at pH 5.8 for 6 h at 26℃. The cell clumps were formed from protoplasts cultured in modified MS, K8p, D2 media. Calli were formed on MS solid medium containing 2.0 mg/L IAA, 2.0 mg/L NAA, 0.1 mg/L BA. Shoots were differentiated on differentiated medium after several changes of the medium. Eventually, shoots rooted and developed into whole plantlets on a rooting medium. 相似文献
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Calll were initiated from the seedling segment of Peucedanum praeruptorum Dunn and subcultured on the MS agar medium with 0.5 mg/L 2,4-D. Cell suspension culture with a lot of embryogenic cell clumps was obtained in liquid medium. Protoplasts were isolated from the cell clumps in enzyme mixture solution containing 1.5% Onozuka R-10, 0.3% Macerozyme R-10, 0.5% helicase, 5 mmol/L CaCl2 and 0.6 mol/L mannital, at pH 5.6 and shaking for 5- hours at 25℃. Helicase is necessary for isolation. After purified by washing, the protoplasts were cultured in liquid medium containing 1 mg/L 2,4-D +0.5 mg/L zeatin. First cell division was observed after four days. Large cell clumps were formed after thirty days. Microcalli of 1 mm in size was formed after about fifty days, and continued to grow on the MS solid medium containing 0.5 mg/L 2,4-D and 200 mg/L casein hydrolysate, and later differentiated into embryoids when transferred to MS agar medium with 0.1 mg/L zeatin. Eventually, embryoids developed into whole plantlets on the MS solid medium without phytohormones. 相似文献
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银杏雌配子体发育及原生质体分离与培养的研究 总被引:2,自引:0,他引:2
Results of observation showed that the female gametophyte of Ginkgo biloba was at the coenocytic stage from March 30ty to May 30ty. A density of 6-8 x 10(5) protoplasts/ml with a viability of 87.3% was achieved when the female gametopytes collected from May 8th to 15th were treated with 0.5% cellulase Onzuka R-10, 0.5% Pectolyase for 4-5 hours. The thin-layer liquid Murashige and Tuker medium modified by omitting ammonium ions and supplementing with glutamine 1000 mg/L, Vc 5 mg/L, benzyladenine 1.0 mg/L and naphthaleneacetic acid 3.0 mg/L was used for the protoplast culture and multiple cell colonies were obtained. 相似文献
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Kitamura M Terakawa K Inoue H Hayashida T Suto K Morimoto Y Yasuoka N Shibata N Higuchi Y 《Journal of biochemistry》2007,141(4):459-468
Mutants of flavin mononucleotide-binding protein (FMN-bp) were made by site-directed mutagenesis to investigate the role of carboxyl-terminal Leu122 of the pairing subunit in controlling redox potentials, binding the prosthetic group, and forming the tertiary and quaternary structure. We compared the oxidation-reduction potentials, FMN-binding properties, and higher structures of wild-type FMN-bp and four mutant proteins (L122Y, L122E, L122K and L122-deleted). We found that the redox potentials were affected by mutations. Also, the affinities of L122E, L122K and L122 deletion mutant apoproteins for FMN were lower than for the wild-type apoprotein, whereas the affinity of L122Y for FMN was increased. Analytical ultracentrifugation showed that the dissociation constants for dimerization of L122E and L122K were larger than for wild-type FMN-bp, whereas the dissociation constants for L122Y and the deletion mutant were lower than for the wild type. Finally, we determined the higher structures of L122Y, L122E and L122K mutants by X-ray crystallography. Our results show that the mutation of Leu122 in FMN-bp changes midpoint potentials, dissociation constants for FMN, and dimer formation, indicating that this residue is important in the pairing subunit. 相似文献
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玉米、小麦、水稻原生质体制备条件优化 总被引:3,自引:0,他引:3
玉米Zea mays L.、小麦Triticum aestivum L.、水稻Oryza sativaL.是三大重要粮食作物,对其原生质体制备条件的优化具有重要意义.以玉米(综3)、小麦(中国春)、水稻(日本晴)10日龄幼苗为材料,研究了叶肉细胞原生质体分离过程中的酶浓度、酶解时间和离心力大小等因素对产量和活力的影响.结果表明:酶浓度和酶解时间对原生质体产量影响显著,随着酶解液浓度和酶解时间的提高,原生质体产量增加,但细胞碎片同时增多.水稻经真空处理后,原生质体产量大幅度提高.通过正交实验设计得出如下结果:玉米叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5%,离析酶0.5%,50 r/min酶解7h,100×g离心2 min收集,原生质体产量为7×106/g FW;小麦叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5%,离析酶0.5%,50 r/min酶解5h,100×g离心2 min收集,原生质体产量为6×106/g FW;水稻叶肉细胞原生质体分离的最佳条件为:纤维素酶2.0%,离析酶0.7%,50 r/min酶解7h,1 000×g离心2 min收集,得到的原生质体产量为6×106/g FW.通过二乙酸荧光素染色发现原生质体活力均在90%以上.用PEG-Ca2+介导法将含有绿色荧光蛋白的质粒转化入原生质体,转化率可达50% ~80%. 相似文献
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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. 相似文献