system: [SYSTEM]

user: 2. 马桑菌DNA提取，PCR扩增方法 DNA提取：取适量样品用液氮充分研磨后快速盛入1.5 ml的Eppendorf 管中，加入700 μL的预热CTAB (2%)提取缓冲液，65℃水浴1 h后12，000 r/min离心20 min， 取上清液加入等体积的苯酚、氯仿、异戊醇混合液(体积比为25:24:1)充分混匀，慢慢摇晃1 h后12，000 r/min 离心20 min，取上清液转管至新的1.5 mL的Eppendorf管中，加入等体积的氯仿、异戊醇混合液(体积比为24:1)慢慢摇晃1 h后12，000 r/min离心20 min，取上清液转管，加入2/3体积预冷的异丙醇置于−20℃冰箱30 min，8000 r/min离心5 min，弃上清液，沉淀用75%的乙醇洗涤2~3次，晾干加入30 μL TE溶液充分溶解，然后用1.0%的琼脂糖凝胶检测DNA样品的浓度和质量，DNA样品置于−20℃保存备用。 PCＲ扩增：利用真菌通用引物ITS1和ITS4对样品进行PCＲ扩增，反应体系为35 μL，包括：2 × HiFiTaqPCRStarMix17.5 μL、引物ITS1和ITS4各0.6 μL、DNA 模板1 μL，用去离子水将体积补至35 μL。反应程序为：94℃预变性2 min，94℃变性1 min，55℃退火1 min，72℃延伸1 min，34个循环，72℃延伸8 min，4℃保存．扩增得到的PCR产物经1.0%琼脂糖凝胶电泳检测扩增结果。 产物送相关基因测序公司测序。 测定了马桑菌Strain HUB034 18S ribosomal核糖体RNA gene和马桑菌Strain YAASM366 18S ribosomal RNA gene。测序结果如下： Download FASTA (complete sequence) FASTA (aligned sequences) GenBank (complete sequence) Continue Cancel GenBankGraphics Next Previous Descriptions Lentinula edodes strain HUB034 18S ribosomal核糖体RNA gene， partial sequence; internal transcribed spacer 1， 5.8S ribosomal RNA gene， and internal transcribed spacer 2， complete sequence; and 28S ribosomal RNA gene， partial sequence Sequence ID: KT750944.1Length: 759Number of Matches: 1 Related Information Range 1: 41 to 746GenBankGraphics Next Match Previous Match First Match Features: Query10 TGGTGGTGGATTGTTGCTGGCCTTTGGGTATGTGCACATCCTCCTCCGATTTCTATTCAT69 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct41 TGGTGGTGGATTGTTGCTGGCCTTTGGGTATGTGCACATCCTCCTCCGATTTCTATTCAT100 Query70 CCACCTGTGCACTTTTTGTAGGAGTTCTTTCATCGGGTTTTTGAA-GGTGCTCATTATGA128 ||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||| Sbjct101CCACCTGTGCACTTTTTGTAGGAGTTCTTTCATCGGGTTTTTGAAGGGTGCTCATTATGA160 Query129GTTACTTGAAAAGACTAGTTGACAAGGCTTCTATGTTCTTATAAACCATTGAAGTATGTT188 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct161GTTACTTGAAAAGACTAGTTGACAAGGCTTCTATGTTCTTATAAACCATTGAAGTATGTT220 Query189ATAGAATGATCTTGTTATTGGGACTTTATTGACCCTTTAAACTTAATACAACTTTCAGCA248 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct221ATAGAATGATCTTGTTATTGGGACTTTATTGACCCTTTAAACTTAATACAACTTTCAGCA280 Query249ACGGATCTCTTGGCTCTCCCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAA308 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct281ACGGATCTCTTGGCTCTCCCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAA340 Query309TTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCCCTCTGGTATTCCGG368 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct341TTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCCCTCTGGTATTCCGG400 Query369AGGGCATGCCTGTTTGAGTGTCATTAAATTCTCAACTTTATAAGTTTTTACTTATCAAAG428 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct401AGGGCATGCCTGTTTGAGTGTCATTAAATTCTCAACTTTATAAGTTTTTACTTATCAAAG460 Query429CTTGGATGTTGGAGGCTTGCAGGCGTTTGTCAGCTCCTCTTAAATTGATTAGTGGGAACC488 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct461CTTGGATGTTGGAGGCTTGCAGGCGTTTGTCAGCTCCTCTTAAATTGATTAGTGGGAACC520 Query489CTGTTTTGTTAGTTCTAACCTTGGTGTGATAATTATCTACATTTTGGTGGAACCTTACAA548 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct521CTGTTTTGTTAGTTCTAACCTTGGTGTGATAATTATCTACATTTTGGTGGAACCTTACAA580 Query549TAATAAAGCTCTATTGGTTTGGGTTGTTGCATTTAGTTTGCTCAATCTGTTCTATTCATT608 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct581TAATAAAGCTCTATTGGTTTGGGTTGTTGCATTTAGTTTGCTCAATCTGTTCTATTCATT640 Query609GGAGCACAAGGGAAGTCCCGCTTTCTAACTGTCTTGATTGACTATATATAACTTATTTGC668 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct641GGAGCACAAGGGAAGTCCCGCTTTCTAACTGTCTTGATTGACTATATATAACTTATTTGC700 Query669TTGACCTCAAATCAGGTAGGATTACCCGCTGAACTTAAGCATATCA714 |||||||||||||||||||||||||||||||||||||||||||||| Sbjct701TTGACCTCAAATCAGGTAGGATTACCCGCTGAACTTAAGCATATCA746 Download FASTA (complete sequence) FASTA (aligned sequences) GenBank (complete sequence) Continue Cancel GenBankGraphics Next Previous Descriptions Lentinula edodes strain YAASM366 18S ribosomal RNA gene， partial sequence; internal transcribed spacer 1， 5.8S ribosomal RNA gene， and internal transcribed spacer 2， complete sequence; and 28S ribosomal RNA gene， partial sequence Sequence ID: KT750950.1Length: 751Number of Matches: 1 Related Information Range 1: 42 to 749GenBankGraphics Next Match Previous Match First Match Features: Query10 TGGTGGTGGATTGTTGCTGGCCTTTGGGTATGTGCACATCCTCCTCCGATTTCTATTCAT69 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct42 TGGTGGTGGATTGTTGCTGGCCTTTGGGTATGTGCACATCCTCCTCCGATTTCTATTCAT101 Query70 CCACCTGTGCACTTTTTGTAGGAGTTCTTTCATCGGGTTTTTGAA-GGTGCTCATTATGA128 ||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||| Sbjct102CCACCTGTGCACTTTTTGTAGGAGTTCTTTCATCGGGTTTTTGAAGGGTGCTCATTATGA161 Query129GTTACTTGAAAAGACTAGTTGACAAGGCTTCTATGTTCTTATAAACCATTGAAGTATGTT188 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct162GTTACTTGAAAAGACTAGTTGACAAGGCTTCTATGTTCTTATAAACCATTGAAGTATGTT221 Query189ATAGAATGATCTTGTTATTGGGACTTTATTGACCCTTTAAACTTAATACAACTTTCAGCA248 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct222ATAGAATGATCTTGTTATTGGGACTTTATTGACCCTTTAAACTTAATACAACTTTCAGCA281 Query249ACGGATCTCTTGGCTCTCCCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAA308 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct282ACGGATCTCTTGGCTCTCCCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAA341 Query309TTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCCCTCTGGTATTCCGG368 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct342TTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCCCTCTGGTATTCCGG401 Query369AGGGCATGCCTGTTTGAGTGTCATTAAATTCTCAACTTTATAAG-TTTTTACTTATCAAA427 |||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||| Sbjct402AGGGCATGCCTGTTTGAGTGTCATTAAATTCTCAACTTTATAAGTTTTTTACTTATCAAA461 Query428GCTTGGATGTTGGAGGCTTGCAGGCGTTTGTCAGCTCCTCTTAAATTGATTAGTGGGAAC487 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct462GCTTGGATGTTGGAGGCTTGCAGGCGTTTGTCAGCTCCTCTTAAATTGATTAGTGGGAAC521 Query488CCTGTTTTGTTAGTTCTAACCTTGGTGTGATAATTATCTACATTTTGGTGGAACCTTACA547 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct522CCTGTTTTGTTAGTTCTAACCTTGGTGTGATAATTATCTACATTTTGGTGGAACCTTACA581 Query548ATAATAAAGCTCTATTGGTTTGGGTTGTTGCATTTAGTTTGCTCAATCTGTTCTATTCAT607 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct582ATAATAAAGCTCTATTGGTTTGGGTTGTTGCATTTAGTTTGCTCAATCTGTTCTATTCAT641 Query608TGGAGCACAAGGGAAGTCCCGCTTTCTAACTGTCTTGATTGACTATATATAACTTATTTG667 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct642TGGAGCACAAGGGAAGTCCCGCTTTCTAACTGTCTTGATTGACTATATATAACTTATTTG701 Query668CTTGACCTCAAATCAGGTAGGATTACCCGCTGAACTTAAGCATATCAA715 |||||||||||||||||||||||||||||||||||||||||||||||| Sbjct702CTTGACCTCAAATCAGGTAGGATTACCCGCTGAACTTAAGCATATCAA749