黑曲霉PgxB基因缺失菌株的构建及特性研究

发布时间：2019-06-06 04:30

【摘要】：黑曲霉是一种广泛存在于自然界的丝状真菌,分布于土壤、空气和谷物上,能够引起食物、谷物和果蔬的霉腐变质,尤其是多汁类果实,如苹果、梨、桃子、石榴等的典型采后储藏侵染菌。植物细胞壁中的主要成分-果胶主要是由不同酯化程度的半乳糖醛酸以α-1,4-糖苷键连接形成的多糖链,而果胶酶是植物病原菌突破植物细胞壁的主要细胞壁降解酶类,其中多聚半乳糖醛酸酶(polygalacturonase,PG)是主要的成员。作为工业发酵微生物,黑曲霉也用于生产果胶酶,应用在食品加工行业。已有研究表明,对于某些真菌和植物,PG是真菌的致病因子,但在黑曲霉中尚无果胶酶基因的相关报道,因此我们研究黑曲霉外切多聚半乳糖醛酸酶基因pgxB在致病性中的作用,这对于黑曲霉果胶酶基因功能研究以及采后果蔬的储藏有重要意义。研究基因功能最直接的方法就是敲除此基因。我们以黑曲霉(Aspergillus niger MA70.15(ΔkusA,pyrG-))为材料,利用同源重组的敲除原理,以pyrG作为筛选标记基因(pyrG编码蛋白乳清酸核苷-5,-磷酸脱氢酶,pyrG缺陷型菌株不能在不含尿苷的条件下生长,但能抵抗5-氟乳清酸的毒性)敲除pgxB基因。首先我们从曲霉基因库获取黑曲霉pgxB基因序列,扩增出pgxB上下游侧翼片段AB(548bp)和CD(564bp),将两者连接后再与质粒pC3(5865bp,含有pyrG)连接,形成pC3-ABCD(6959bp)敲除载体;接下来用PEG转化法将敲除载体转化黑曲霉原生质体,利用pyrG进行双向筛选,经鉴定得到黑曲霉pgxB基因缺失菌株。我们发现ΔpgxB所产的果胶酶活性比野生型低5.8%,pgxB的缺失显著降低了黑曲霉对采后苹果和梨果实的致病性(P0.01),其侵染所致的菌斑直径相对于野生型减少了20%。进一步实验表明在侵染采后苹果过程中,pgxB的缺失导致相同功能的其它多聚半乳糖醛酸酶基因(pgaI,pgaII,pgaA,pgaC,pgaD,pgaE)表达量升高,可能原因为补偿pgxB功能的缺失,但pgxB缺失型菌株的致病性仍然显著减弱。以上结果表明pgxB是黑曲霉的重要致病因子,在采后苹果、梨果实的储藏过程中发挥作用。进一步研究表明,pgxB的缺失未能影响黑曲霉在PDA和果胶培养基上的菌落生长。我们还发现黑曲霉野生型与ΔpgxB对重金属离子铝、锌、镉、铜的敏感性无显著差异,具体表现在菌落直径和生长量方面,说明多聚半乳糖醛酸酶基因pgxB与黑曲霉对重金属胁迫的敏感性没有关联性。然而,重金属胁迫能够抑制黑曲霉产果胶酶。对果胶酶生产外源影响因子的研究表明,果胶能够诱导果胶酶的合成,并显著提高黑曲霉果胶酶编码基因的表达,而葡萄糖和D-半乳糖醛酸(0.1%和1%)抑制果胶酶的合成。黑曲霉在弱酸性(pH5-6)条件下最适合产果胶酶,而5%的柠檬酸能显著抑制果胶酶的产量。通过以上研究我们得出结论:pgxB是黑曲霉的重要致病因子,不仅降低果胶酶活性,还减弱了黑曲霉对苹果和梨果实的致病性,但它不参与正常条件下黑曲霉的生长以及对重金属胁迫的应答。
[Abstract]:Aspergillus niger is a kind of filamentous fungus which is widely present in nature, and is distributed on soil, air and grain, and can cause mildew and rot of food, grain and fruit and vegetable, especially juicy fruit, such as apple, pear, peach, pomegranate and other typical postharvest storage and infection bacteria. the main components in the plant cell wall-the pectin is mainly the polysaccharide chain formed by connecting the galacturonic acid with different degree of esterification with the cis-1,4-sugar linkage key, and the pectase is the main cell wall degrading enzyme of the plant pathogenic bacteria which break through the cell wall of the plant, wherein the polygalacturonase (polygalactone, PG) is the main member. As an industrial fermentation microorganism, Aspergillus niger is also used for the production of pectase and is applied in the food processing industry. Studies have shown that PG is a pathogenic factor for fungi for certain fungi and plants, but there is no relevant report of pectase gene in Aspergillus niger, so we study the role of the exonuclease gene pgxB of Aspergillus niger in the pathogenicity. It is of great significance to study the function of the pectinase gene of Aspergillus niger and the storage of postharvest fruits and vegetables. The most direct way to study gene function is to knock out the gene. We use the knock-out principle of homologous recombination as the material, using the knock-out principle of homologous recombination, and the pyrG is used as the screening marker gene (pyrG-encoded protein lactalate-5,-phosphate dehydrogenase, and the pyrG-deficient strain can not be grown under the condition that no urine is present, But the pgxb gene can be knocked out against the toxicity of 5-fluoroorotate acid). First of all, we obtained the sequence of the Aspergillus niger pgxB gene from the Aspergillus gene bank, and amplified the upstream and downstream flanking fragments AB (548 bp) and CD (564 bp) of pgxB, and then ligated the two to the plasmid pC3 (5865 bp, containing pyrG) to form the pC3-ABCD (6959 bp) knockout vector; then, the knockout vector was transformed into the Aspergillus niger protoplast by the PEG transformation method, The strain of pgxB gene of Aspergillus niger was obtained by two-way screening with pyrG. We found that the activity of pectase produced by pgxB was 5.8% lower than that of wild-type, and the deletion of pgxB significantly reduced the pathogenicity of Aspergillus niger to postharvest apples and pears (P0.01), and the bacterial plaque diameter due to its infection decreased by 20% with respect to wild-type. Further experiments show that the deletion of pgxB in the process of postharvest apple leads to an increase in the expression of other polygalacturonase genes (pgaI, pgaII, pgaA, pgaC, pgaD, pgaE) of the same function, possibly due to the loss of the function of the pgxB, However, the pathogenicity of the pgxB-deleted strain is still significantly reduced. The results show that pgxB is an important pathogenic factor of Aspergillus niger and plays a role in the storage of postharvest apples and pears. Further studies have shown that the deletion of pgxB does not affect the colony growth of the Aspergillus niger on the PDA and the pectin medium. We also found that the sensitivity of Aspergillus niger wild-type and pgxB to heavy metal ions, such as aluminum, zinc, iron and copper, showed no significant difference in the sensitivity of pgxB and Aspergillus niger to heavy metal stress in the aspects of colony diameter and growth. However, heavy metal stress can inhibit that production of pectase from Aspergillus niger. It is shown that pectin can induce the synthesis of pectase and significantly increase the expression of pectase-encoding gene of Aspergillus niger, while glucose and D-galacturonic acid (0.1% and 1%) inhibit the synthesis of pectase. Aspergillus niger is most suitable for pectinase production under the condition of weak acid (pH 5-6), while 5% of citric acid can significantly inhibit the production of pectase. It is concluded that pgxB is an important pathogenic factor of Aspergillus niger, which not only reduces the activity of pectase, but also reduces the pathogenicity of Aspergillus niger to the fruit of apple and pear, but it does not participate in the growth of Aspergillus niger under normal conditions and the response to heavy metal stress.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ920.1

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