甜柿果树林下种植的马桑菌核糖体RNA的碱基配对

Base Pairing of Ribosomal RNA of Coriaria spp. Grown under Persimmon Fruit Trees

摘要: 马桑菌具有浓郁的香菇香味。在市场上它的经济价值比其他香菇要高很多，在甜柿果树林下种植马桑菌对增加农民的经济收入起到了重要的作用，同时也推动了贫困地区农民的脱贫致富。由于马桑菌生物学特性独特(只有用马桑树椴木或马桑树木屑才能种植出马桑菌)，采用形态分类学又不能将其区别，所以只能用分子检测来区分它们。文章测定了马桑菌Strain HUB034 18S ribosomal核糖体RNA gene和马桑菌Strain YAASM366 18S ribosomal RNA gene。其结果为：马桑菌Strain HUB034 18S ribosomal核糖体RNA gene含有714~746个碱基配对。马桑菌Strain YAASM366 18S ribosomal RNA gene含有715~749个碱基配对。

Abstract: Coriaria has a strong mushroom flavor. In the market, its economic value is much higher than other Lentinus edodes. Planting Coriaria spp. under the sweet persimmon fruit forest has played an important role in increasing the economic income of farmers, but also promoting the poverty-stricken areas of farmers to get rid of poverty and become rich. Because of the unique biological characteristics of Coriaria spp. (only by using Tilia or sawdust of Morus alba), we cannot distinguish them by morphological taxonomy, so we can only distinguish them by molecular detection. The ribosomal RNA gene of strain hub034 18S ribosomal and strain yaasm366 18S ribosomal RNA gene were determined. The results showed that there were 714 - 746 base pairs in the ribosomal RNA gene of strain hub034 18S ribosomal. The 18S ribosomal RNA gene of strain yaasm366 contains 715 - 749 base pairs.

1. 引言

马桑菌是一种生长在马桑树的枯枝上的野生菌经过驯化的品种，该菌具有浓郁的香菇香味 [1] [2]。野生马桑菌的驯化栽培只能用马桑树椴木或马桑树粉碎成木屑栽培才能长出马桑菌子实体 [3]。采用其它树种椴木或木屑栽培马桑菌，营养生长良好，但不能进行生殖生长。因此作者认为该马桑菌是否是香菇属中一新种？采用形态分类学的方法没有办法将它们区别，由于马桑菌的生物学特性与其他香菇品种生物学特性相差很大，因此作者对在甜柿下种植的马桑菌品种的核糖体Strain HUB034 18S ribosomal核糖体RNA gene和马桑菌Strain YAASM366 18S ribosomal RNA gene进行了测定。其目的是为下一步鉴定此马桑菌是否为香菇属中的新种，与香菇属中其他品种的核糖体进行对比，看它们在分子方面有无区别作前期准备工作。

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：

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FASTA (aligned sequences)

GenBank (complete sequence)

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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

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FASTA (complete sequence)

FASTA (aligned sequences)

GenBank (complete sequence)

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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

3. 结果与讨论

测定马桑菌Strain HUB034 18S ribosomal核糖体RNA gene和马桑菌Strain YAASM366 18S ribosomal RNA gene。其结果为：马桑菌Strain HUB034 18S ribosomal核糖体RNA gene含有714~746个碱基配对。马桑菌Strain YAASM366 18S ribosomal RNA gene含有715~749个碱基配对。

由于野生马桑菌在驯化栽培过程中，只能用马桑树椴木或马桑树粉碎成木屑栽培才能长出马桑菌子实体，用其他壳叶树种椴木或木屑种植马桑菌都未能长出马桑菌子实体。因此作者认为通过测定马桑菌Strain HUB034 18S ribosomal核糖体RNA gene和马桑菌Strain YAASM366 18S ribosomal RNA gene与其他香菇属中的这一片段进行比对。看它们有无差异才能确定马桑菌是否是香菇属中的新种？对此有待于对其他香菇品种的核糖体RNA的测定后才能有所定论。

基金项目

课题任务书合同编号：黔科合NZ[2015]3001-6；黔科合NZ[2015]3001-5；黔科合支撑[2016] 2600号；黔科合SZ[2008]3018；贵州省基金项目(黔科合基础[2018]1147)。

NOTES

^*通讯作者。