CN117512201B - 用于鉴定绣球菌新菌株JSJ-Spar16-1的引物对及其鉴定方法 - Google Patents
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Abstract
用于鉴定绣球菌新菌株JSJ‑Spar16‑1的引物对及其鉴定方法,属于食用菌分子标记技术领域。所述绣球菌新菌株JSJ‑Spar16‑1保藏编号为CGMCC No.40892,ITS序列如SEQ ID NO:1所示;所述引物对为JSp16‑F/R,其上游引物如SEQ ID NO:2所示,下游引物如如SEQ ID NO:3所示。所述引物对JSp16‑F/R是基于哥伦比亚大学100条ISSR通用引物中U885引物于JSJ‑Spar16‑1菌株扩增筛选到的差异性序列所设计,利用JSp16‑F/R引物对对JSJ‑Spar16‑1菌株的DNA进行PCR扩增,于琼脂糖凝胶中电泳,扩增结果中有206bp片段。本发明可利用此引物对快速鉴定JSJ‑Spar16‑1菌株,不需要其他技术手段的辅助,操作方便、耗时短且准确度较高。
Description
技术领域
本发明属于食用菌分子标记技术领域,具体涉及用于鉴定绣球菌新菌株JSJ-Spar16-1的引物对及其鉴定方法。
背景技术
绣球菌(Sparassis crispa),隶属于真菌界、担子菌门、伞菌纲、非褶孔菌目、绣球菌科、绣球菌属。绣球菌极具开发潜能,是一种珍稀的食药用真菌,含丰富的β-葡聚糖,具有抗肿瘤、增强免疫力、抗氧化等多种生物活性。目前国内认定或鉴定的绣球菌品种仅有两个,分别为“闽绣1号”和闽绣2号,子实体颜色为白色或乳白色,均为福建省农业科学院研究团队选育,栽培及育种资源非常有限。为促进绣球菌产业的发展,申请人研发团队于云南大理、怒江等地进行绣球菌野生资源的采集,经分离培养、小试、中试栽培,选育出绣球菌新菌株JSJ-Spar16-1。JSJ-Spar16-1菌株,其子实体叶片颜色为黄色或浅黄色,叶片边缘呈小波浪状,孢子印浅黄色。该菌株菌丝生长快、抗杂菌能力强,原基健壮、出菇率≥90%,生物学转化率≥68.7%,生长发育周期短,不超过97天,商品性状良好,在育种和产业化生产栽培等方面具有很好的应用前景。
传统的食用菌分类方法已经不能满足需求,且操作较为繁琐、鉴定周期长。而分子生物学技术提供了高效、便捷的鉴定方法,对食用菌的种群鉴定、优良品种选育具有十分重要的意义并开拓了更为广阔的前景。绣球菌栽培菌株较少且分子标记工作鲜有报道,这使得绣球菌遗传育种工作相应滞后,品种选育工作较为困难。为了促进绣球菌产业的快速发展,一方面不断挖掘、收集野生种质资源来进行品种选育工作十分重要,另一方面,对选育特定菌株的分子标记工作也同样重要。因此本发明提供具备产业研究的潜能菌株及其分子标记和鉴定方法,为绣球菌的遗传育种奠定重要的基础,保障绣球菌产业的健康可持续发展。
绣球菌相关参考文献可见:
[1]王萌皓,郝正祺,常明昌等.绣球菌多糖表征及其抗氧化和免疫活性[J].菌物学报,2019,38(05):707-716.
[2]黄年来.中国大型真菌原色图鉴[M].北京:中国农业出版社,1998:40.
[3]吴学谦,李海波,魏海龙等.DNA分子标记技术在食用菌研究中的应用及进展[J].浙江林业科技,2004(02):76-81.
[4]周延清.DNA分子标记技术在植物研究中的应用[M].北京.化学工业出版社,2005.
发明内容
本发明的目的在于解决绣球菌产业栽培菌株少且无快速鉴定方法的问题,提供一株绣球菌新菌株JSJ-Spar16-1的快速鉴定引物对及其鉴定方法。
本发明采取的技术方案如下:
用于鉴定绣球菌新菌株JSJ-Spar16-1的引物对,所述绣球菌新菌株JSJ-Spar16-1保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏单位地址为北京市朝阳区北辰西路1号院3号,保藏日期为2023年10月7日,保藏编号为CGMCC No.40892,分类命名为绣球菌Sparassis sp. ,ITS序列如SEQ ID NO:1所示;所述引物对为JSp16-F/R,其上游引物如SEQ ID NO:2所示,下游引物如如SEQ ID NO:3所示。
本发明所述用于鉴定绣球菌新菌株JSJ-Spar16-1的引物对鉴定方法,所述引物对JSp16-F/R是基于哥伦比亚大学100条ISSR通用引物中U885引物BHB GAG AGA GAG AGA GA于JSJ-Spar16-1菌株扩增筛选到的差异性序列所设计,鉴定方法步骤如下:
(1)于PDA培养基中收集JSJ-Spar16-1的菌丝体,用真菌基因组DNA提取试剂盒提取DNA;
(2)以JSJ-Spar16-1的DNA为模板进行PCR扩增,PCR反应体系30μL,PCR Mix 15.5μL,ddH2O 11μL,上游及下游引物各1.1μL,DNA模板1.3μL;
(3)利用引物对JSp16-F/R对JSJ-Spar16-1的DNA进行PCR扩增,PCR扩增程序为94℃预变性5min,94℃变性30s, 51℃退火45s,72℃延伸1min,35个循环,72℃保温10min,4℃保存;
(4)扩增结束后,将扩增产物于1%的琼脂糖凝胶中进行电泳35min,于紫外分析仪中查看有无扩增片段,扩增结果中有206bp片段即为JSJ-Spar16-1菌株。
本发明提供了绣球菌新菌株JSJ-Spar16-1的分子鉴定引物对及其鉴定方法,通过所述引物对,能够从大量绣球菌菌株中快速鉴定是否为JSJ-Spar16-1菌株,为后续的绣球菌分子育种、遗传图谱等的研究提供了重要的基础,同时也为市场上菌株混乱存在提供了一种高效、操作简单的菌株鉴定技术方案。
附图说明
图1为JSp16-F/R引物对于21株绣球菌菌株的扩增结果。
具体实施方式
下面将结合具体实施例对本发明的内容进行清楚、完整的描述。
用于鉴定绣球菌新菌株JSJ-Spar16-1的引物对,所述绣球菌新菌株JSJ-Spar16-1保藏编号为CGMCC No.40892,ITS序列如SEQ ID NO:1所示。该绣球菌新菌株亲本于2022年9月采自云南省大理市老君山镇的野生绣球菌子实体,经系统驯化选育获得。所述引物对为JSp16-F/R,其上游引物为JSp16-F:5,-AAGACTTCCTTCTCCTCTGGA-3,(SEQ ID NO:2),下游引物为JSp16-R:5,- GCTGAGAGAGAGAGAGAAGGGA-3,(SEQ ID NO:3)。
用于鉴定绣球菌新菌株JSJ-Spar16-1的引物对鉴定方法,所述引物对JSp16-F/R是基于哥伦比亚大学100条ISSR通用引物中U885引物BHB GAG AGA GAG AGA GA于JSJ-Spar16-1菌株扩增筛选到的差异性序列所设计。
鉴定方法步骤如下:
(1)将JSJ-Spar16-1菌株及收集到的其他20株绣球菌菌株培养30天后收集菌丝体,用真菌基因组DNA提取试剂盒提取DNA。
(2)以21株绣球菌菌株的DNA为模板进行PCR扩增,PCR反应体系30μL,PCR Mix15.5μL,ddH2O 11μL,上游及下游引物各1.1μL,DNA模板1.3μL,共30μL;
(3)利用引物对JSp16-F/R对21株绣球菌菌株进行PCR扩增,PCR扩增程序为94℃预变性5min,94℃变性30s,引物对的退火温度为51℃,退火时间为45s,72℃延伸1min,35个循环,72℃保温10min,4℃保存;
(4)扩增结束后,于1%的琼脂糖凝胶中进行电泳35min,在紫外分析仪中查看有无扩增片段,扩增结果中有206 bp片段即为JSJ-Spar16-1菌株,反之为其他绣球菌菌株,扩增结果见图1。图1中,M为Marker(D2000);1泳道为ddH20作为DNA模板的扩增对照;2~4泳道为JSJ-Spar16-1菌株的扩增结果,有206bp片段,5~24泳道为其他20株绣球菌菌株的扩增结果,无206bp片段。
ITS序列:
TGGGCTTGCGGAGTCGAGCGAGGTTGTAGCTGGCCTTCTCGGAGGCATCGTGCACGCCCTGCCCGTCCCATATCATACCTGTGAACTTTTTGGTAGGCGGGTTTGTGTCGGCCTCGAAAGGGGTCGGCCGGCCCTCCGGCCGTCTTTATATACACACCATACGAGTCTTTAGAATGTTTGTGCGTCTCGACGCATCTTATATATAACTTTCAGCGACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAACGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTCGGTATTCCGAGGAGCATGCCTGTTTGAGTGTCATGAAATTATCAACCCCTCCTCCTTCATCGGTGGTGGGGCTTGGACTTGGAGGCTTTGCGGGCTTTTAACGAGTCGGCTCCTCTCAAATGCATTAGCTCGAACCCCTGCGGATCGGCCGTCGGTGTGATATAATGTCTACGTCGTGGTCGTGAGCGTCGGATCGGCTTCTAATGGTCCCCTTTCGGAGGCGGAATTTGAACTTGTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA(SEQ ID NO:1)
JSp16-F/R引物对:
上游引物JSp16-F:5,-AAGACTTCCTTCTCCTCTGGA-3,(NO:2),
下游引物JSp16-R:5,- GCTGAGAGAGAGAGAGAAGGGA-3,(NO:3)。
Claims (2)
1.用于鉴定绣球菌菌株JSJ-Spar16-1的引物对,所述绣球菌菌株JSJ-Spar16-1保藏编号为CGMCC No.40892,ITS序列如SEQ ID NO:1所示;所述引物对为JSp16-F/R,其上游引物如SEQ ID NO:2所示,下游引物如SEQ ID NO:3所示。
2.权利要求1所述用于鉴定绣球菌菌株JSJ-Spar16-1的引物对的鉴定方法,其特征在于,所述引物对JSp16-F/R是基于哥伦比亚大学100条ISSR通用引物中U885引物BHB GAGAGA GAG AGA GA于JSJ-Spar16-1菌株扩增筛选到的差异性序列所设计,鉴定方法步骤如下:
(1)于PDA培养基中收集JSJ-Spar16-1的菌丝体,用真菌基因组DNA提取试剂盒提取DNA;
(2)以绣球菌菌株JSJ-Spar16-1的DNA为模板进行PCR扩增,PCR反应体系30μL,PCR Mix15.5μL,ddH2O 11μL,上游及下游引物各1.1μL,DNA模板1.3μL;
(3)利用引物对JSp16-F/R对21株绣球菌菌株进行PCR扩增,PCR扩增程序为94℃预变性5min,94℃变性30s,51℃退火45s,72℃延伸1min,35个循环,72℃保温10min,4℃保存;
(4)扩增结束后,将扩增产物于1%的琼脂糖凝胶中进行电泳35min,于紫外分析仪中查看有无扩增片段,扩增结果中有206bp片段即为JSJ-Spar16-1菌株。
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