从烟草悬浮细胞中分离液泡前体的方法研究

通过跟踪含有液泡前体特异标记物的组分, 利用细胞壁酶水解胞壁制造原生质体, 并用真针头挤压的方法裂解细胞, 用蔗糖密度梯度离心的方法分离液泡前体. 通过相差显微镜观察, 发现所分离的液泡前体保持完整的结构. 利用不同细胞器的标记物抗体进行免疫标记检测, 证明所纯化的液泡前体不含其他细胞器, 具有较高的纯度.     

Expression of green fluoscrescent protein gene in Sclerotinia sclerotiorum

Protoplasts of the pathogenic plant fungus,Sclerotinia sclerotiorum,were transformed using the pPGF plasmid,which contains green fluorescent protein gene,under the control of Aspergillus nidulans regulatory sequences. The pPGF plasmid was introduced by PEG/CaCl2 treatment. Positive transformants were harvested with hygromycin B (HYG) resistance as selective marker,and then were observed with green fluorescence phenomena in response to blue light,which suggested that GFP gene was cloned into genome DNA of S. sclerotiorum. The transformants were verified mitotically stable by Southern blotting analysis and passage culturing. This study is developed as an initial step for further research into infection mechanisms of S. sclerotiorum to plants and interactions with bio-control fungus.     

应用基因组重排技术提高普那霉素产量

将普那霉素产生菌——始旋链霉菌ND-23的孢子和原生质体经紫外线诱变后获得高产突变菌株,其中高产突变株SP-S73的产量达到356 mg/L,比出发菌株提高了31%.在上述增产突变株中选取4株作为基因组重排的出发亲本,对它们进行了4轮基因组重排育种,筛选得到了高产重排菌株,其中1株重排菌株G-211的普那霉素产量为832 mg/L,比产量最高的亲本菌株提高了134%,比原始出发菌株ND-23(272 mg/L)提高了206%.通过研究高产菌株和出发菌株ND-23在5 L罐上的发酵过程,发现普那霉素产生菌的生物合成属于非生长耦合型;其高产菌株G-211在合成产物的时期对还原糖和氨基氮的消耗量均大于原始菌株ND-23,这些结果将为高产菌株发酵条件优化和发酵放大研究提供有价值的参考.     

An Efficient and Universal Protoplast Isolation Protocol Suitable for Transient Gene Expression Analysis and Single-Cell RNA Sequencing

The recent advent of single-cell RNA sequencing (scRNA-seq) has enabled access to the developmental landscape of a complex organ by monitoring the differentiation trajectory of every specialized cell type at the single-cell level. A main challenge in this endeavor is dissociating plant cells from the rigid cell walls and some species are recalcitrant to such cellular isolation. Here, we describe the establishment of a simple and efficient protocol for protoplast preparation in Chirita pumila, which includes two consecutive digestion processes with different enzymatic buffers. Using this protocol, we generated viable cell suspensions suitable for an array of expression analyses, including scRNA-seq. The universal application of this protocol was further tested by successfully isolating high-quality protoplasts from multiple organs (petals, fruits, tuberous roots, and gynophores) from representative species on the key branches of the angiosperm lineage. This work provides a robust method in plant science, overcoming barriers to isolating protoplasts in diverse plant species and opens a new avenue to study cell type specification, tissue function, and organ diversification in plants.     

蒽降解遗传重组菌的构建及其对蒽污染水体修复的研究

为了提高外源蒽降解菌An降解蒽的能力和存活能力,采用原生质体电融合技术,在蒽降解菌An和土生菌Tu之间实行原生质体电融合,并从再生的融合子中筛选到具有特定性状的后代菌株。经研究原生质体制备和再生的最优条件、融合子降解蒽的性能以及存活能力的特性,结果表明,融合子既具有较好降解蒽的能力,100 h内融合子F和亲本An对蒽的降解转化率分别为73%、68%;融合子又具有土生菌Tu存活能力高的特性,到降解后期,An菌的数量大大下降,而融合子保持较稳定的数量。     

微波-紫外灭活原生质体融合选育米曲霉菌株的研究

实验对生长速度较快但产酶能力较低的米曲霉CICC2339的原生质体采用700W微波灭活12s,对产酶能力较高但生长速度较慢的米曲霉AS3.951的原生质体采用15W紫外灭活5min,然后对灭活双亲用PEG作融合剂进行原生质体融合,融合率达到了7.6%。通过测定Hc值以及蛋白酶活力大小,最终筛选出编号为CAW202、CAW205和CAW210的三株融合株,其生长速度快且蛋白酶活力还高于产酶能力较高的亲本菌株CICC2339;三株融合株的蛋白酶活力分别为10450U/g、9730U/g、10780U/g,其中融合株CAW202和CAW210的蛋白酶活力比亲本菌株CICC2339有了显著的增加,分别提高了7.8%和11.25%。     

黑曲霉原生质体的制备、再生及转化条件

为了建立原生质体介导的黑曲霉转化系统，研究了菌龄、酶系统、渗透压稳定剂、酶解时间对葡萄糖淀粉酶生产菌株黑曲霉CICIMF0410原生质体形成与再生的影响。结果表明，培养4d的幼嫩菌丝体最适于制备原生质体；综合考虑原生质体形成与再生的情况，作者选用1mol／L山梨醇为最适渗透压稳定剂，1g／dL蜗牛酶-1g／dL纤维素酶-0．1g／dL溶壁酶为最适裂解酶组合，30℃酶解2．5～3h，最适合的原生质体的再生培养基为含0．6mol／LMgSO4的TZ培养基。在PEG和CaCl2存在条件下，以潮霉素B为选择性标记，质粒pBC-Hygro转化原生质体，每微克DNA可获得4～5个转化予。     

氦氖激光对棘孢小单孢菌诱变育种的研究

小单孢菌是具有巨大潜力的抗生素产生菌,可产生包括氨基甙类、大环内脂类、多肽类等多种抗生素。庆大霉素是氨基甙类第二代产品。在获得优质高产的氨基甙类抗生素产生菌的选育中,目前仍是以诱变为主。但是常规方法中都是对孢子及菌丝进行诱变,正突变率较低,不易筛选到产量增高的菌株,因而有必要进行改进。本实验采用氦氖激光辐照小单孢菌的原生质体,获得了提高产量的菌株,现将结果报道如下。     

平菇原生质体释放过程的电镜扫描研究

本文用电子显微镜扫描的方法,对平菇菌丝释放原生质体的过程进行了探讨,摸索出了一套从玻璃纸培养的菌丝获取原生质体,并进行电镜扫描的理想研究方法,进一步证实了原生质体顶端释放,侧面释放和断裂释放的三种释放方式,并对原生质体的形状,表面结构进行了详细的描述.在电镜下,原生质体呈倒卵形,光滑圆球形或表面有齿状或皱褶突起的圆球形.