黑曲霉几丁质合成酶基因chs形态突变株的构建

Optimization of protoplast formation,regenerationin and transformation in Aspergillus niger

以一株产柠檬酸的黑曲霉菌株为研究材料,为了优化菌株的深层发酵形态,提高柠檬酸产量,采用RNA干扰的方法,使与形态建成相关的几丁质合成酶chs基因沉默。首先将酶切得到的chs基因片段与质粒p JL43-RNAi连接,构建成干扰载体。为提高干扰载体的转化率,对原生质体制备、再生和转化条件进行了优化。然后通过聚乙二醇(PEG)介导,将构建的干扰载体导入黑曲霉的原生质体中,经过转化,筛选出形态突变株,并进行深层发酵。结果表明,在36.5℃培养16 h的幼嫩菌丝最利于原生质体释放;而5 mg/m L的溶菌酶,10 mg/m L的蜗牛酶和10 mg/m L的纤维素酶组成的混合酶系为最优的酶组合;在30℃下,以100 r/min酶解3 h可使原生质体的最大产量达到5.11×106/m L。最优的再生培养基为完全培养基,可以使再生率达到35.33%。获得的3株转化株的chs基因表达量降低,同时在深层发酵过程中,菌球的分支频率降低,分支缩短,离散菌丝减少,柠檬酸产量也分别提高12.33%,24.17%和19.61%。通过分子改造的形态突变株具有良好的产酸性能,对推动柠檬酸发酵工业的进步有重要意义。

For improving the mycelial morphology and citric acid production of A.niger, we construct a gene silencing vector by RNA interference technology to silence the chitin synthase gene.The fragments of chs gene were amplified by PCR,which was ligated to plasmid pJL43-RNAi to generate the RNAi vector.In order to increase the transformation frequency,the effects of some factors on protoplast formation and regeneration from citric acid- producing fungus A.niger were investigated.The silencing vectors were introduced into purificatory protoplasts by a polyethylene glycol（PEG）- mediated transformation method.The results showed that the mycelia incubated for 16 h at 36.5 ℃ were most suitable for protoplast release, which digested by enzyme combination of 5 mg/mL lysozyme,lO mg/mL snailase and 10 mg/mL cellulase for 3 h as 100 r/min at 30 ℃,and a high yield of protoplasts （5.11 × 106/mL） were obtained.In addition, the maximum regeneration rate was 35.33 %, while the complete medium as the regeneration media. After conversion, three morphological mutant strains are smoother and have less dispersed mycelia,which were distinct from the original strain in morphology.The transformants chs-t, chs-2 and chs-3 where citric acid production rise by 12.33% ,24.17% and 19.61% ,respectively.The morphological mutants of chs exhibited the excellent production potential during submerged culture,which had important significance for the development of the citric acid fermentation industry.