CN107142217A - 一种用于减少农作物黄曲霉毒素含量的生物农药 - Google Patents
一种用于减少农作物黄曲霉毒素含量的生物农药 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/67—Aspergillus flavus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B9/00—Preservation of edible seeds, e.g. cereals
- A23B9/16—Preserving with chemicals
- A23B9/24—Preserving with chemicals in the form of liquids or solids
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Abstract
本发明提供了一种不产黄曲霉毒素的黄曲霉菌,所述黄曲霉菌的保藏编号为CGMCC NO.14122,保藏日期为2017年5月10日,所述黄曲霉菌的AflR基因启动子序列发生了缺失突变,所述缺失突变导致了黄曲霉菌不能表达产生黄曲霉毒素。本发明提供的黄曲霉菌能够与产毒的黄曲霉菌在土壤中展开定植竞争,而且本发明提供的黄曲霉菌在土壤中适应能力强,具有强大的生存和繁殖能力,可以减轻产毒的黄曲霉菌对农作物和食品储藏物的感染,显著降低农作物产物和食品储藏物中黄曲霉毒素的含量。
Description
技术领域
本发明涉及一种黄曲霉菌,属于微生物领域。
背景技术
黄曲霉菌(学名:Aspergillus flavus)或称为黄曲菌、黄曲霉等,是一种真菌。在自然环境中,它是一种常见的霉菌,广泛存在于土壤、灰尘、植物及其果实上,特别是在热带和亚热带的核果类和谷类上更为常见,如花生、核桃、开心果、杏仁、桃仁和李仁、椰丝、芝麻和各种粮食。在储存的花生中也容易污染黄曲霉菌。
黄曲霉菌生命力顽强,是温暖地区常见的占优势的霉菌,其生长温度范围在4—50℃之间,最适生长温度为25—40℃。曲霉比其它霉菌更耐旱,而且环境的酸碱性对其影响不大,在pH2—9的条件下都能生长,不过在pH2.5—6.0之间的酸性条件下,毒素的生成量最大。黄曲霉能在含氧量极低的环境中生长,在缺氧环境中发酵。即使在充填二氧化碳的冷库中,黄曲霉的生长也不受影响。
黄曲霉毒素(Aflatoxin,AFT)是主要由黄曲霉(Aspergillus flavus)寄生曲霉(A.parasiticus)产生的次生代谢产物,是一类化学结构类似的化合物,均为二氢呋喃香豆素的衍生物,主要的分子有B1、B2、G1和G2等,在牛奶中黄曲霉毒素还可以代谢成M1和M2。黄曲霉毒素是一种剧毒物质,毒性比KCN大300倍,远远高于氰化物、砷化物和有机农药的毒性,其中以B1毒性最大,仅次于钚,是目前已知霉菌中毒性最强的。当人或动物摄入量大时,可发生急性中毒,出现急性肝炎、出血性坏死、肝细胞脂肪变性和胆管增生,脾脏和胰脏也有重度的病变。国内已经有多起因饲料黄曲霉毒素引起的畜禽大规模死亡事件报道。
当微量持续摄入,黄曲霉毒素可造成人或动物慢性中毒,其主要变化特征为肝脏出现慢性损伤,如肝实质细胞变性、肝硬化等。出现动物生长发育迟缓,体重减轻,母畜不孕或产仔少等系列症状。肉用和奶用牲畜还会在奶和脂肪组织中蓄积黄曲霉毒素的代谢产物M1和M2,引起人的慢性中毒。
黄曲霉毒素也是世界上最强致癌物之一,其致癌能力比六氯环己烷大300万倍,致癌范围广,能诱发鱼类、禽类,各种实验动物、家畜及灵长类等多种动物的实验肿瘤;黄曲霉毒素主要诱发肝癌,还可诱发胃癌、肾癌、泪腺癌、直肠癌、乳腺癌,卵巢及小肠等部位的肿瘤,还可出现畸胎。
一般烹调加工温度不能将黄曲霉毒素破坏,其裂解温度高达1535℃。在水中溶解度较低,溶于油及一些有机溶剂,如氯仿和甲醇中,但不溶于乙醚、石油醚及乙烷。食品及饲料中所污染的主要是黄曲霉毒素B1。
花生是最容易感染黄曲霉菌的农作物。花生在生长的全过程中,都有可能感染黄曲霉菌,特别是在生长后期,优于温度、湿度的变化,病、虫和鼠的危害等使花生的种皮被破坏后都会加剧黄曲霉菌的污染。花生收获后受气温、气湿及储存条件的影响,更容易招致黄曲霉菌感染。黄曲霉菌在繁殖和新陈代谢过程中,就会产生大量的毒素(主要是黄曲菌毒B1),污染花生及其制品。现已查明,在保管不当的花生与花生油、花生饮料、花生酱中,都有可能存在此毒素。由于黄曲霉菌的污染,花生的生长还会受到抑制,造成花生的减产,据估计减产达到10%左右。以中国的山东省为例,山东省是我国花生的主要产地,产量达到全国产量的三分之一,出口达到90%,而青岛地区的花生种植年均150万亩,年产花生50万吨,占山东省花生产量15%,出口总量的30%,是青岛地区重要的经济作物。但是最近由于国际市场上面临美国和南美的激烈竞争,出口量呈显著下降趋势,农民种植花生的收益也连年下降。而在过去几年,欧盟和日本等主要的花生进口国都提高了花生的进口质量标准,特别是黄曲霉毒素的含量标准,对青岛市的花生出口也造成严重的影响。
花生黄曲霉菌污染的大田防治主要在花生生育后期,在花生荚果发育期间保证水分供给,避免收获前干旱造成种皮的破裂使黄曲霉菌的感染机会增多,避免其它病、虫和鼠害的发生,在结荚期和荚果发育期避免耕造成荚果损伤。在收获后及时晒干荚果,使含水量低于5%,筛选有抗性的花生新品种等。但是由于黄曲霉菌的生存力强,产生的孢子可以抵抗多种恶劣自然条件,并不能彻底避免黄曲霉菌的感染。
2004年美国农业部花生重点实验室在美国乔治亚州的花生种植田中分离出一株不分泌黄曲霉毒素的黄曲霉菌,发现在田间施用后,不仅可以显著降低在花生生长期间的黄曲霉毒素的含量,还可以降低花生储存过程中黄曲霉毒素的产生,在各个阶段黄曲霉毒素的含量都在含量限制范围内(<20Ppb),2007年被美国农业部批准用于花生的黄曲霉毒素防治(商品名为),2009年批准用于玉米的黄曲霉毒素防治。随后,巴西和阿根廷都批准了该生物农药在花生种植中的应有。据测算,的使用每年给美国花生种植农户节省因为黄曲霉菌毒素污染造成的直接损失就有2亿美元,还不包括在饲料中应用对畜牧业的保护。
国内也有从土壤中分离不产毒黄曲霉菌的报道,但是目前为止这些研究仅限于实验室内抑制产毒黄曲霉菌生长的报道,并没有对不产毒菌的分子机理进行研究,也没有开展田间实验研究并取得实际的应用效果。而由于各个地区的土壤条件存在着一定的差异。因此,在需要防治地区选择培养原生的不分泌黄曲霉毒素的黄曲霉菌成为必要。
本发明的目的是在中国山东省的花生主产区筛选到对该地区土壤条件自然适应的可以用于黄曲霉毒素的防治的不分泌黄曲霉毒素的黄曲霉菌。
发明内容
基于上述发明目的,发明人从中国山东省花生产区土壤中分离获得了1000多个黄曲霉菌菌株,经过测定发酵样品中黄曲霉毒素的含量和对菌株进行分子生物学鉴定,鉴定到一种不产毒的黄曲霉菌株,因此,本发明首先提供了一种黄曲霉菌株,所述黄曲霉菌株的保藏编号为CGMCC NO.14122,保藏日期为2017年5月10日,保藏分类命名为黄曲霉(Aspergillus flavus),保藏单位为中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,邮政编码:100101。
其次,本发明还提供了一种黄曲霉菌,所述黄曲霉菌的AflR基因启动子序列发生了缺失突变,所述突变的AflR基因启动子序列由SEQ ID NO.1所示。所述AflR基因是黄曲霉毒素(Aflatoxin,AFT)编码基因转录的调节基因启动子,发生于该位置区域的缺失突变直接导致了所述黄曲霉菌不能表达产生黄曲霉毒素。
第三,本发明还提供了含有上述黄曲霉菌孢子的制剂,所述制剂含有保藏编号为CGMCC NO.14122的黄曲霉或者AflR基因启动子序列为SEQ ID NO.1所示的黄曲霉菌孢子。
最后,本发明还提供了上述制剂在预防农作物和食品储藏物产生黄曲霉毒素中的应用。
在一个优选的技术方案中,所述制剂被制备为悬液形式应用。
在一个优选的技术方案中,所述制剂被制备为喷雾剂形式应用。
本发明提供的黄曲霉菌能够与产毒的黄曲霉菌在土壤中展开定植竞争,而且本发明提供的黄曲霉菌在土壤中适应能力强,具有强大的生存和繁殖能力,可以减轻产毒的黄曲霉菌对种植的花生的感染,显著降低收获和储存后的花生中黄曲霉毒素的含量。使用本发明提供的不产黄曲霉毒素的黄曲霉菌M1株孢子的花生收获后黄曲霉毒素的含量为5Ppb,未使用M1孢子的花生中黄曲霉毒素的含量为11Ppb。储存半年后,使用M1黄曲霉菌孢子的花生黄曲霉毒素的含量为9Ppb,而未使用的花生储存半年后黄曲霉毒素的含量达到了34Ppb,已经超过了限量。证明M1黄曲霉菌可以用于黄曲霉毒素污染的生物防控,且具有很好的防控效果。
附图说明
图1.土壤中分离培养的黄曲霉菌菌落观测图;
图2.黄曲霉菌落的荧光鉴定观测图;
图3.不产毒黄曲霉菌大米的黄曲霉毒素含量HPLC检测曲线图;
图4.黄曲霉菌AflR基因启动子DNA扩增结果电泳图谱;
图5.M1黄曲霉菌AflR基因启动子区与野生型黄曲霉菌启动子区的序列对比示意图;
图6.M2黄曲霉菌AflR基因启动子区与野生型黄曲霉菌启动子区的序列对比示意图
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的保护范围构成任何限制。
实施例1:土壤中黄曲霉菌的分离、鉴定和培养
1.1分离
在花生收获后的田地中取土样,加无菌水充分震荡后,2000转室温离心,取上清20ul涂布于PDA固体培养基(称取洗净去皮马铃薯200g,切碎,加水1000ml煮沸半个小时,纱布过滤后再加20g葡萄糖和20g琼脂,15磅蒸气(121℃)灭菌20分钟左右后取出倒平板,冷却后贮存备用。30℃恒温培养,10~14小时后菌落直径3~4或4~7cm,最初带黄色,然后变为黄绿色,老后颜色变暗,平坦或有放射状沟纹,反面无色或带褐色(参见图1,土壤中分离培养的黄曲霉菌菌落(A.flavus))。用无菌接菌环挑取分隔良好的黄曲霉菌落,置于灭菌的1.5ml离心管中,标记备用。
1.2荧光筛选和鉴定
将初步选取的黄曲霉菌孢子在PDA琼脂培养基上划线培养,30度培养14天后,将培养皿打开,于暗室中的365nm紫外灯下对菌落进行观察,因黄曲霉毒素在紫外灯下发荧光,可用于区分产毒和不产毒的黄曲霉菌。鉴定出2株不产毒的黄曲霉菌株(参见图2,1为不产毒黄曲霉菌落M1。2为产毒黄曲霉菌落,因黄曲霉毒素在紫外灯下发荧光,可用于区分产毒和不产毒的黄曲霉菌)。
1.3培养和生长情况观察
取250克大米置于20×30cm的不锈钢盘中,加100ml水,用铝箔纸密封盘口,120度湿热灭菌15分钟,冷却至室温。取2株不产毒的和1株产毒黄曲霉菌株的孢子,用200微升无菌水悬浮,取100微升均匀涂布于LB琼脂培养基中,37度倒置培养3-4天待菌落变成深绿色,产生明显的分生孢子后,用无菌细胞刮刀小心从培养基的表面刮取黄曲霉菌的分生孢子,重悬于5ml无菌水中,充分震荡混匀,然后在无菌条件下均匀添加到已消毒的大米固体培养基中,然后用4层消毒的无菌纱布覆盖不锈钢盘,置于30度培养箱中培养13-14天。
小心收集长满黄曲霉菌的大米,取小量生长有黄曲霉菌的大米,称重后,加10倍体积的无菌水,在匀浆器中充分匀浆至无明显颗粒状物,对混悬液进行梯度稀释到1:105,取20微升稀释后的黄曲霉菌悬液均匀涂布于PDA琼脂培养基上,30度倒置培养7-10天,进行菌落计数。结果发现,2株不产毒的黄曲霉菌株的克隆数分别为M1为3.7×107cfu/ml,M2为4.4×107cfu/ml,而产毒黄曲霉菌株D1的克隆数为3.2×107cfu/ml,无显著差异,说明产毒和不产毒黄曲霉菌株的生长速率和繁殖速率无显著差异。将剩余的大米置于分孢机的进样口,收集黄曲霉菌的孢子,称重。获得的孢子总量分别为:5.7克(M1)、5.2克(M2)和5.3克(D1),说明产孢量也不存在显著差异。
1.4产毒鉴定
取培养M1和M2不产毒黄曲霉菌株的大米样品,干燥后在研钵中充分研磨,加10ml的甲醇:水(1:1)提取黄曲霉毒素,采用高效液相色谱法检测大米样品中的黄曲霉毒素含量。结果显示,样品中的各种黄曲霉毒素含量检测均为零(图3和表1)。验证了这两株黄曲霉菌在生长过程中不产生黄曲霉毒素。
表1.各种类型黄曲霉毒素的检测结果
实施例2:不产毒与产毒黄曲霉菌的AflR基因启动子DNA序列分析
取少量分离的黄曲霉菌孢子置于陶瓷研钵中,加液氮同时进行研磨破坏孢子外壁,加入1mlTE-葡萄糖溶液(pH8.0),充分洗涤研钵,回收溶液置于离心管中,加入1mlNaOH/SDS溶液,充分混匀,冰浴10分钟,加1ml复性溶液,12000转4度离心10分钟,取500微升上清,加入等体积的酚/氯仿(24:1),震荡混匀,4度12000转离心5分钟,小心吸取上清,加2倍体积的无水乙醇,冰上沉淀10分钟,12000转室温离心10分钟,弃上清,沉淀用500微升75%的乙醇洗涤,室温干燥5分钟后,用100微升无菌水溶解黄曲霉菌基因组DNA。
以黄曲霉菌DNA为模板采用PCR扩增AflR基因的启动子DNA序列,所用引物序列为:
F:5'-CTCATGCAGGTGCTAAAGA-3'
R:5'-GCACAACTCGTACAGCTAT-3'
PCR结果如图4(1为M1不产毒黄曲霉菌,2为M2不产毒黄曲霉菌,3为产毒黄曲霉菌,M为分子量标记)。
对PCR产物进行序列分析。序列分析结果如SEQ ID NO.1和2所示。结果表明,与产毒黄曲霉菌的AflR基因启动子区序列对比,M2有15个位点存在点突变(序列比对见图6),M1菌株除了有27个点突变位点外,还存在3个缺失突变,分别缺失了46个、4个和3个碱基的DNA序列(序列比对见图5)。由于黄曲霉菌的突变率高,因此普通的点突变存在再次突变的可能性,成为回复突变再次恢复产毒,而缺失突变产生回复突变的可能性低,因此发明人选择了具有27个点突变,3个缺失突变的M1菌株作为黄曲霉毒素的生防菌株,并将该菌株保藏,保藏编号为CGMCC NO.14122,保藏日期为2017年5月10日,保藏分类命名为黄曲霉(Aspergillus flavus),保藏单位为中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,邮政编码:100101。
防治实施例
正常田间种植的花生,待花生开花后,果针入地前,在每株花生植株周围施用分离获得的M1黄曲霉菌孢子悬液5ml,对照组正常只施用无菌水5ml,正常施肥和浇水。收获花生后,分别取使用和未使用M1孢子的花生,粉碎后用甲醇:水溶液(1:1)提取黄曲霉毒素,采用ELISA的方法测定花生中的黄曲霉毒素的含量。收获的花生经室温保存半年后,提取黄曲霉毒素,ELISA方法测定黄曲霉毒素的含量。结果证明,使用M1孢子的花生收获后黄曲霉毒素的含量为5Ppb,未使用M1孢子的花生中黄曲霉毒素的含量为11Ppb。储存半年后,使用M1黄曲霉菌孢子的花生黄曲霉毒素的含量为9Ppb,而未使用的花生储存半年后黄曲霉毒素的含量达到了34Ppb,已经超过了限量。证明M1黄曲霉菌可以用于花生的黄曲霉毒素污染的生物防控。
序 列 表
<110> 青岛至成生物技术有限公司
<120> 一种用于减少农作物黄曲霉毒素含量的生物农药
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 337
<212> DNA
<213> Aspergillus flavus
<400> 1
ctcatgcagg tgctaaagat ctagcttgca ggaaacaagt cttttctggg ttctaagccc 60
gcccatgacg gactacgtta tcttgagccc gaggcatgca tgcaggcggg ccagctagct 120
gaacattact tgttggtctt ggtttgcttc gttaaacaag gaacgcacag ctagacaatc 180
cttgggccaa gtcagaaccc ctcagctggt gacaggagtg tacatacatt taggtctaag 240
tgcgaggcaa cgaaaagggc gggctactct cccggagaaa gccttcacat tgtgtgtttt 300
ctttccgctt tcaattgaga attcctgaaa attcctt 337
<210> 2
<211> 390
<212> DNA
<213> Aspergillus flavus
<400> 2
ctcatgcagg tgctaaagat ctagcttgca gaaaccaagt cttttctgaa ctgccattcc 60
actaatgacg gactacgtta tcttgagccc gagccatgca tgcaggcggg ccagctagct 120
gaacagtata tgttggttct tggtctgatt cgtcaacccg atcacacaga tctctgatca 180
cacggatcag cctcggtatg taaacaagga acgcaccgct agacaatcct tgggccaagt 240
cagaacccct cagctggtga caggagtgta catacgtaca tttaggtcta agtgcgaggc 300
aacgaaaagg gcgggctact ctcccggaga aagccttcac attgtgtgtt ttctttccgc 360
tttcaattga gaatatatcc tgaattcctt 390
Claims (6)
1.一种黄曲霉菌,其特征在于,所述黄曲霉菌的保藏编号为CGMCC NO.14122,保藏日期为2017年5月10日,保藏单位为中国微生物菌种保藏管理委员会普通微生物中心。
2.一种不产黄曲霉毒素的黄曲霉菌,其特征在于,所述黄曲霉菌的AflR基因启动子序列发生了缺失突变,所述突变的AflR基因启动子序列由SEQ ID NO.1所示。
3.一种含有权利要求1或2所述黄曲霉菌孢子的制剂。
4.权利要求3所述制剂在预防农作物和食品储藏物产生黄曲霉毒素中的应用。
5.根据权利要求4所述的应用,其特征在于,所述制剂被制备为悬液形式应用。
6.根据权利要求4所述的应用,其特征在于,所述制剂被制备为喷雾剂形式应用。
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