发根农杆菌R1000对野葛的遗传转化及其影响因素

初步研究了发根农杆菌R10 0 0对野葛 (Puerarialobata)的遗传转化及其影响因素。结果表明 :经发根农杆菌R10 0 0感染 13d左右可获得毛状根 ,毛状根诱导率及诱导密度分别为 17 9%和 3 9;降低YEB活化液的 pH值 ,或以肌醇 (10 g/L)代替酵母提取成分 ,可明显提高该菌种的致根性 ;叶片预培养 2～ 3d可提前 4d出根     

转复制酶基因抗病毒烟草的研究

将携带烟草花叶病毒(TMV)54KD蛋白基因和黄瓜花叶病毒(CMV)3'端缺失0.9kb的复制酶基因片段的植物表达载体pBICT,通过很痛农杆菌311SE系,利用叶圆盘法转化烟草,在含有卡那霉素的选择培养基上诱导生芽、生根,得再生植株53株。移入大田后,选取5株,提取植物总DNA,Southern杂交检测,结果5株中均有复制酶基因的整合,且转基因植株在大田中表现了良好的抗TMV和CMV能力。      

Phosphorus detection in vitrified bacteria by cryo‐STEM annular dark‐field analysis

Bacterial cells often contain dense granules. Among these, polyphosphate bodies (PPBs) store inorganic phosphate for a variety of essential functions. Identification of PPBs has until now been accomplished by analytical methods that required drying or chemically fixing the cells. These methods entail large electron doses that are incompatible with low‐dose imaging of cryogenic specimens. We show here that Scanning Transmission Electron Microscopy (STEM) of fully hydrated, intact, vitrified bacteria provides a simple means for mapping of phosphorus‐containing dense granules based on quantitative sensitivity of the electron scattering to atomic number. A coarse resolution of the scattering angles distinguishes phosphorus from the abundant lighter atoms: carbon, nitrogen and oxygen. The theoretical basis is similar to Z contrast of materials science. EDX provides a positive identification of phosphorus, but importantly, the method need not involve a more severe electron dose than that required for imaging. The approach should prove useful in general for mapping of heavy elements in cryopreserved specimens when the element identity is known from the biological context.     

根癌农杆菌介导透明颤茵血红蛋白基因(vgb)在短密青霉菌中的克隆表达

利用根癌农杆菌LBA4404介导,将编码透明颤菌血红蛋白的结构基因(Vitreoscilla Hemoglobin gene,vgb)导入到短密青霉ATCC16024中,用于改善菌体对氧的摄取能力,提高霉酚酸(Myhophenoilc Acid,MPA)的发酵水平,降低生产成本。通过潮霉素抗性筛选、PCR分子鉴定和MPA发酵验证等结果表明,vgb基因在短密青霉中成功获得了表达;vgb基因的表达提高了短密青霉的菌体密度,转化子MPA产量达到2.18 g·L?1,比原始菌株提高了27.5%。     

An Efficient Regeneration System and Optimization of the Transformation from the Cotyledonary Node of Jute (Corchorus capsularis L.)

The purpose of this study was to use cotyledonary nodes as explants to establish an efficient regeneration protocol for jute (Corchorus capsularis L.) on Murashige and Skoog (MS) basal medium. Research into the different growth regulators using the orthogonal design L16 (4⁵) revealed that the best shoot induction medium is MS medium containing 8% (w/v) agar and 3% (w/v) sucrose supplemented with 1.0 mg/L 6BA and 0.25 mg/L NAA. The average number of shoots per explant and the explant induction rate were 9.8 and 100%. After 3 weeks, 2–3 cm shoots were rooting on 1/2 MS medium containing 8% (w/v) agar and 3% (w/v) sucrose supplemented with 1.0 mg/L 6-BA + 0.2 mg/L NAA. Moreover, we optimized Agrobacterium-mediated transformation using the GUS gene transient expression system. The best condition for obtaining higher transformation rate consisted of the use of fresh explants to which 100 μM acetosyringone was added for a co-culture time of 10 min, the OD value of Agrobacterium liquid is 0.5 at 600 nm. These data provide an important basis for the application of other trait gene in the improvement of jute fiber quality.     

胡萝卜发根培养甘薯茎线虫体系研究

利用发根农杆菌转化的胡萝卜发根建立了甘薯茎线虫的单寄主培养体系,观察了甘薯茎线虫的行为及生活史。结果表明：在胡萝卜发根上培养甘薯茎线虫,便于研究其行为,可以清楚地观察到线虫在根毛处自由取食及侵入发根内部活动情况;雌虫可以正常产卵,线虫在20～25天内完成正常的生活史。基于观察到的上述结果,初步证实了所构建的胡萝卜发根培养甘薯茎线虫体系的可行性。     

An Improved Protocol for Agrobacterium-Mediated Transformation in Subterranean Clover (Trifolium subterraneum L.)

Subterranean clover (Trifolium subterraneum) is the most widely grown annual pasture legume in southern Australia. With the advent of advanced sequencing and genome editing technologies, a simple and efficient gene transfer protocol mediated by Agrobacterium tumefaciens was developed to overcome the hurdle of genetic manipulation in subterranean clover. In vitro tissue culture and Agrobacterium transformation play a central role in testing the link between specific genes and agronomic traits. In this paper, we investigate a variety of factors affecting the transformation in subterranean clover to increase the transformation efficiency. In vitro culture was optimised by including cefotaxime during seed sterilisation and testing the best antibiotic concentration to select recombinant explants. The concentrations for the combination of antibiotics obtained were as follows: 40 mg L⁻¹ hygromycin, 100 mg L⁻¹ kanamycin and 200 mg L⁻¹ cefotaxime. Additionally, 200 mg L⁻¹ cefotaxime increased shoot regeneration by two-fold. Different plant hormone combinations were tested to analyse the best rooting media. Roots were obtained in a medium supplemented with 1.2 µM IAA. Plasmid pH35 containing a hygromycin-resistant gene and GUS gene was inoculated into the explants with Agrobacterium tumefaciens strain AGL0 for transformation. Overall, the transformation efficiency was improved from the 1% previously reported to 5.2%, tested at explant level with Cefotaxime showing a positive effect on shooting regeneration. Other variables in addition to antibiotic and hormone combinations such as bacterial OD, time of infection and incubation temperature may be further tested to enhance the transformation even more. This improved transformation study presents an opportunity to increase the feeding value, persistence, and nutritive value of the key Australian pasture.     

土壤杆菌中辅酶Q10的提取工艺

研究了溶剂种类、浓度,提取溶剂比例、提取次数、时间、温度,萃取次数与温度,以及柱纯化和重结晶等条件对提取农杆菌中辅酶Q_(10)的影响;进行了最佳工艺优化,确立了生产工艺,并进行了中试验证.结果表明,最佳工艺条件为:用5倍的体积分数95%的乙醇在50℃浸提2遍,每遍1 h,获得提取液;提取液浓缩后经过提取液-甲醇-石油醚(三者体积比为1:2:3)的萃取体系室温萃取3遍,浓缩石油醚相后经过1步硅胶层析柱纯化,2或3步乙醇重结晶.中试结果表明,该工艺获得辅酶Q_(10)的纯度>99%,平均回收率>80%.