Dörnte Bastian, Peng Can, Fang Zemin, Kamran Aysha, Yulvizar Cut, Kües Ursula
Molecular Wood Biotechnology and Technical Mycology, Büsgen-Institute, University of Goettingen, Büsgenweg 2, 37077 Goettingen, Germany.
School of Life Sciences, Anhui University, Hefei, 230601 China.
Fungal Biol Biotechnol. 2020 Oct 12;7:15. doi: 10.1186/s40694-020-00105-0. eCollection 2020.
Two reference strains have been sequenced from the mushroom , monokaryon Okayama 7/#130 (OK130) and the self-compatible homokaryon AmutBmut. An adenine-auxotrophy in OK130 () and a -aminobenzoic acid (PABA)-auxotrophy in AmutBmut () offer selection markers for transformations. Of these two strains, homokaryon AmutBmut had been transformed before to PABA-prototrophy and with the bacterial hygromycin resistance marker , respectively.
Gene encodes a bifunctional enzyme with an N-terminal glycinamide ribonucleotide synthase (GARS) and a C-terminal aminoimidazole ribonucleotide synthase (AIRS) domain required for steps 2 and 5 in the de novo biosynthesis of purines, respectively. In OK130, a missense mutation in rendered residue N231 for ribose recognition by the A loop of the GARS domain into D231. The new vector pAde8 complements the auxotrophy of OK130 in transformations. Transformation rates with pAde8 in single-vector and co-transformations with -selection were similarly high, unlike for plasmids which exhibit suicidal feedback-effects in single-vector transformations with complementation of tryptophan synthase defects. As various other plasmids, unselected pAde8 helped in co-transformations of strains with a -selection vector to overcome suicidal effects by transferred . Co-transformation rates of pAde8 in OK130 under adenine selection with nuclear integration of unselected DNA were as high as 80% of clones. Co-transformation rates of expressed genes reached 26-42% for various laccase genes and up to 67% with silencing vectors. The bacterial gene can also be used as another, albeit less efficient, selection marker for OK130 transformants, but with similarly high co-transformation rates. We further show that the defect in AmutBmut is due to a missense mutation which changed the conserved PIKGT motif for chorismate binding in the C-terminal PabB domain to PIEGT in the mutated 4-amino-4-deoxychorismate synthase.
and auxotrophic defects in reference strains OK130 and AmutBmut for complementation in transformation are described. pAde8 is a new transformation vector useful for selection in single and co-transformations of the sequenced monokaryon OK130 which was transformed for the first time. The bacterial gene can also be used as an additional selection marker in OK130, making in combination with successive rounds of transformation possible.
已对两种蘑菇参考菌株进行了测序，即单核体冈山7/#130（OK130）和自交亲和同核体AmutBmut。OK130中的腺嘌呤营养缺陷型（）和AmutBmut中的对氨基苯甲酸（PABA）营养缺陷型（）为转化提供了选择标记。在这两种菌株中，同核体AmutBmut之前已分别转化为PABA原养型并带有细菌潮霉素抗性标记（）。
基因编码一种双功能酶，其N端为甘氨酰胺核糖核苷酸合酶（GARS）结构域，C端为氨基咪唑核糖核苷酸合酶（AIRS）结构域，分别是嘌呤从头生物合成中第2步和第5步所必需的。在OK130中，基因中的一个错义突变使GARS结构域A环用于核糖识别的N231残基变为D231。新的载体pAde8在转化中可互补OK130的营养缺陷型。与在单载体转化中表现出自杀性反馈效应的质粒不同，pAde8在单载体转化和用（）选择的共转化中的转化率同样高。与其他各种质粒一样，未选择的pAde8有助于用（）选择载体对菌株进行共转化，以克服转移的（）产生的自杀效应。在腺嘌呤选择下，OK130中pAde8与未选择DNA的核整合的共转化率高达80%的克隆。对于各种漆酶基因，表达基因的共转化率达到26 - 42%，使用（）沉默载体时高达67%。细菌基因（）也可作为OK130转化体的另一种选择标记，尽管效率较低，但共转化率同样高。我们进一步表明，AmutBmut中的（）缺陷是由于一个错义突变，该突变将突变的4 - 氨基 - 4 - 脱氧分支酸合酶C端PabB结构域中用于分支酸结合的保守PIKGT基序变为PIEGT。
描述了参考菌株OK130和AmutBmut中用于转化互补的（）和（）营养缺陷型。pAde8是一种新的转化载体，可用于首次转化的已测序单核体OK130的单转化和共转化选择。细菌基因（）也可作为OKI30中的另一种选择标记，使得连续两轮转化成为可能。
