Optimization of protoplast formation,regenerationin and transformation in Aspergillus niger
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摘要: 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。Abstract: 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 p JL43- 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 for16 h at 36.5 ℃ were most suitable for protoplast release,which digested by enzyme combination of 5 mg / m L lysozyme,10 mg / m L snailase and 10 mg / m L cellulase for 3 h as 100 r / min at 30 ℃,and a high yield of protoplasts( 5.11 × 106/ m L) 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-1,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.
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Keywords:
- Aspergillus niger /
- protoplasts /
- RNAi /
- chitin synthase /
- morphological mutant
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