黑曲霉PgxB基因缺失菌株的構(gòu)建及特性研究
發(fā)布時(shí)間：2019-06-06 04:30
【摘要】：黑曲霉是一種廣泛存在于自然界的絲狀真菌,分布于土壤、空氣和谷物上,能夠引起食物、谷物和果蔬的霉腐變質(zhì),尤其是多汁類果實(shí),如蘋果、梨、桃子、石榴等的典型采后儲(chǔ)藏侵染菌。植物細(xì)胞壁中的主要成分-果膠主要是由不同酯化程度的半乳糖醛酸以α-1,4-糖苷鍵連接形成的多糖鏈,而果膠酶是植物病原菌突破植物細(xì)胞壁的主要細(xì)胞壁降解酶類,其中多聚半乳糖醛酸酶(polygalacturonase,PG)是主要的成員。作為工業(yè)發(fā)酵微生物,黑曲霉也用于生產(chǎn)果膠酶,應(yīng)用在食品加工行業(yè)。已有研究表明,對(duì)于某些真菌和植物,PG是真菌的致病因子,但在黑曲霉中尚無果膠酶基因的相關(guān)報(bào)道,因此我們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,這對(duì)于黑曲霉果膠酶基因功能研究以及采后果蔬的儲(chǔ)藏有重要意義。研究基因功能最直接的方法就是敲除此基因。我們以黑曲霉(Aspergillus niger MA70.15(ΔkusA,pyrG-))為材料,利用同源重組的敲除原理,以pyrG作為篩選標(biāo)記基因(pyrG編碼蛋白乳清酸核苷-5,-磷酸脫氫酶,pyrG缺陷型菌株不能在不含尿苷的條件下生長,但能抵抗5-氟乳清酸的毒性)敲除pgxB基因。首先我們從曲霉基因庫獲取黑曲霉pgxB基因序列,擴(kuò)增出pgxB上下游側(cè)翼片段AB(548bp)和CD(564bp),將兩者連接后再與質(zhì)粒pC3(5865bp,含有pyrG)連接,形成pC3-ABCD(6959bp)敲除載體;接下來用PEG轉(zhuǎn)化法將敲除載體轉(zhuǎn)化黑曲霉原生質(zhì)體,利用pyrG進(jìn)行雙向篩選,經(jīng)鑒定得到黑曲霉pgxB基因缺失菌株。我們發(fā)現(xiàn)ΔpgxB所產(chǎn)的果膠酶活性比野生型低5.8%,pgxB的缺失顯著降低了黑曲霉對(duì)采后蘋果和梨果實(shí)的致病性(P0.01),其侵染所致的菌斑直徑相對(duì)于野生型減少了20%。進(jìn)一步實(shí)驗(yàn)表明在侵染采后蘋果過程中,pgxB的缺失導(dǎo)致相同功能的其它多聚半乳糖醛酸酶基因(pgaI,pgaII,pgaA,pgaC,pgaD,pgaE)表達(dá)量升高,可能原因?yàn)檠a(bǔ)償pgxB功能的缺失,但pgxB缺失型菌株的致病性仍然顯著減弱。以上結(jié)果表明pgxB是黑曲霉的重要致病因子,在采后蘋果、梨果實(shí)的儲(chǔ)藏過程中發(fā)揮作用。進(jìn)一步研究表明,pgxB的缺失未能影響黑曲霉在PDA和果膠培養(yǎng)基上的菌落生長。我們還發(fā)現(xiàn)黑曲霉野生型與ΔpgxB對(duì)重金屬離子鋁、鋅、鎘、銅的敏感性無顯著差異,具體表現(xiàn)在菌落直徑和生長量方面,說明多聚半乳糖醛酸酶基因pgxB與黑曲霉對(duì)重金屬脅迫的敏感性沒有關(guān)聯(lián)性。然而,重金屬脅迫能夠抑制黑曲霉產(chǎn)果膠酶。對(duì)果膠酶生產(chǎn)外源影響因子的研究表明,果膠能夠誘導(dǎo)果膠酶的合成,并顯著提高黑曲霉果膠酶編碼基因的表達(dá),而葡萄糖和D-半乳糖醛酸(0.1%和1%)抑制果膠酶的合成。黑曲霉在弱酸性(pH5-6)條件下最適合產(chǎn)果膠酶,而5%的檸檬酸能顯著抑制果膠酶的產(chǎn)量。通過以上研究我們得出結(jié)論:pgxB是黑曲霉的重要致病因子,不僅降低果膠酶活性,還減弱了黑曲霉對(duì)蘋果和梨果實(shí)的致病性,但它不參與正常條件下黑曲霉的生長以及對(duì)重金屬脅迫的應(yīng)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.
【學(xué)位授予單位】：合肥工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TQ920.1
本文編號(hào)：2494071
[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.
【學(xué)位授予單位】：合肥工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TQ920.1
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