生物多样性 ›› 2020, Vol. 28 ›› Issue (8): 1018-1025. DOI: 10.17520/biods.2019366
刘丽平1, 宋瑞凤1, 张馥1,3, 张秀香1, 彭桂香2,*(), 谭志远1,*()
收稿日期:
2019-11-12
接受日期:
2020-03-03
出版日期:
2020-08-20
发布日期:
2020-06-12
通讯作者:
彭桂香,谭志远
作者简介:
zytan@scau.edu.cn基金资助:
Liping Liu1, Ruifeng Song1, Fu Zhang1,3, Xiuxiang Zhang1, Guixiang Peng2,*(), Zhiyuan Tan1,*()
Received:
2019-11-12
Accepted:
2020-03-03
Online:
2020-08-20
Published:
2020-06-12
Contact:
Guixiang Peng,Zhiyuan Tan
摘要:
高秆野生稻(Oryza alta)是一种重要的种质资源, 其组织内也蕴藏着非常宝贵的功能微生物资源。本实验采用无氮培养基, 从高秆野生稻中分离到43株内生固氮菌, 结合乙炔还原法测定其固氮酶活性。经固氮酶基因(nifH)的PCR扩增检测, 43株内生固氮菌代表菌株均能扩增出固氮酶基因片段。利用IS-PCR DNA指纹图谱和SDS-PAGE全细胞蛋白电泳图谱将获得的菌株聚类为6个类群(I、II、III、IV、V、VI)。对各个类群的代表菌株(ZF3, ZF8, ZF13, ZF15, ZF24, ZF43)进行16S rRNA基因序列测定, 结果表明, 类群I属于土生拉乌尔菌(Raoultella terrigena), 类群II属于类肺炎克雷伯氏菌类肺炎亚种(Klebsiella quasipnmoniae subsp. quasipneumoniae), 类群III属于越南伯克氏菌(Burkholderia vietnamiensis), 类群IV的菌株代表肠秆菌属的一个类群(Enterobacter sp.), 类群V的菌株属于门多萨假单胞菌(Pseudomonas mendocina), 类群VI归属于固氮植物菌(Phytobacter diazotrophicus)。Biolog聚类结果与IS-PCR指纹图谱类型及SDS-PAGE全细胞蛋白聚类结果一致。Biolog板测定结果显示, 来自不同类群的代表菌株对碳源的利用差异显著, 说明野生稻内多样的内生固氮菌从环境中获取碳源和氮素的适应能力较强。
刘丽平, 宋瑞凤, 张馥, 张秀香, 彭桂香, 谭志远 (2020) 高秆野生稻内生固氮细菌多样性. 生物多样性, 28, 1018-1025. DOI: 10.17520/biods.2019366.
Liping Liu, Ruifeng Song, Fu Zhang, Xiuxiang Zhang, Guixiang Peng, Zhiyuan Tan (2020) Diversity of endophytic diazotrophs isolated from Oryza alta. Biodiversity Science, 28, 1018-1025. DOI: 10.17520/biods.2019366.
菌株编号 Strain no. | 部位 Organ | 固氮酶活性 Nitrogenase activity (nmol C2H2/mL·h) | 菌株编号 Strain no. | 部位 Organ | 固氮酶活性 Nitrogenase activity (nmol C2H2/mL·h) |
---|---|---|---|---|---|
ZF1 | 根 Root | 10.82 ± 0.15 | ZF23 | 叶 Leave | 72.63 ± 0.93 |
ZF2 | 叶 Leave | 26.19 ± 0.19 | ZF24 | 叶 Leave | 57.67 ± 0.45 |
ZF3 | 茎 Stem | 15.76 ± 0.31 | ZF25 | 茎 Stem | 59.60 ± 0.41 |
ZF4 | 茎 Stem | 10.72 ± 0.24 | ZF26 | 茎 Stem | 13.18 ± 0.18 |
ZF5 | 茎 Stem | 15.39 ± 0.28 | ZF27 | 茎 Stem | 184.15 ± 1.05 |
ZF6 | 茎 Stem | 20.22 ± 0.39 | ZF28 | 叶 Leave | 67.17 ± 0.79 |
ZF7 | 叶 Leave | 76.00 ± 0.45 | ZF29 | 根 Root | 10.91 ± 0.22 |
ZF8 | 根 Root | 254.31 ± 4.51 | ZF30 | 根 Root | 5.44 ± 0.15 |
ZF9 | 根 Root | 26.09 ± 0.29 | ZF31 | 茎 Stem | 257.42 ± 1.57 |
ZF10 | 茎 Stem | 506.46 ± 3.63 | ZF32 | 叶 Leave | 29.41 ± 0.33 |
ZF11 | 茎 Stem | 1.23 ± 0.11 | ZF33 | 茎 Stem | 1.82 ± 0.17 |
ZF12 | 根 Root | 69.44 ± 0.65 | ZF34 | 茎 Stem | 43.62 ± 0.43 |
ZF13 | 茎 Stem | 17.20 ± 0.16 | ZF35 | 茎 Stem | 87.18 ± 0.55 |
ZF14 | 叶 Leave | 41.82 ± 0.39 | ZF36 | 茎 Stem | 132.52 ± 0.66 |
ZF15 | 茎 Stem | 59.84 ± 0.48 | ZF37 | 根 Root | 90.59 ± 0.48 |
ZF16 | 根 Root | 94.03 ± 0.57 | ZF38 | 茎 Stem | 33.36 ± 0.34 |
ZF17 | 根 Root | 107.85 ± 1.11 | ZF39 | 茎 Stem | 9.67 ± 0.19 |
ZF18 | 茎 Stem | 33.02 ± 0.43 | ZF40 | 根 Root | 98.96 ± 0.51 |
ZF19 | 茎 Stem | 11.74 ± 0.21 | ZF41 | 叶 Leave | 35.50 ± 0.43 |
ZF20 | 茎 Stem | 108.38 ± 0.67 | ZF42 | 茎 Stem | 38.05 ± 0.42 |
ZF21 | 根 Root | 64.74 ± 0.71 | ZF43 | 茎 Stem | 80.49 ± 0.59 |
ZF22 | 茎 Stem | 20.50 ± 0.23 |
表1 高秆野生稻内生固氮菌来源及固氮酶活性测定(固氮酶活性为3次测定的平均值)
Table 1 Origin of isolates and nitrogenase activity from Oryza alta L.The activity of nitrogenase was the average value of three determinations.
菌株编号 Strain no. | 部位 Organ | 固氮酶活性 Nitrogenase activity (nmol C2H2/mL·h) | 菌株编号 Strain no. | 部位 Organ | 固氮酶活性 Nitrogenase activity (nmol C2H2/mL·h) |
---|---|---|---|---|---|
ZF1 | 根 Root | 10.82 ± 0.15 | ZF23 | 叶 Leave | 72.63 ± 0.93 |
ZF2 | 叶 Leave | 26.19 ± 0.19 | ZF24 | 叶 Leave | 57.67 ± 0.45 |
ZF3 | 茎 Stem | 15.76 ± 0.31 | ZF25 | 茎 Stem | 59.60 ± 0.41 |
ZF4 | 茎 Stem | 10.72 ± 0.24 | ZF26 | 茎 Stem | 13.18 ± 0.18 |
ZF5 | 茎 Stem | 15.39 ± 0.28 | ZF27 | 茎 Stem | 184.15 ± 1.05 |
ZF6 | 茎 Stem | 20.22 ± 0.39 | ZF28 | 叶 Leave | 67.17 ± 0.79 |
ZF7 | 叶 Leave | 76.00 ± 0.45 | ZF29 | 根 Root | 10.91 ± 0.22 |
ZF8 | 根 Root | 254.31 ± 4.51 | ZF30 | 根 Root | 5.44 ± 0.15 |
ZF9 | 根 Root | 26.09 ± 0.29 | ZF31 | 茎 Stem | 257.42 ± 1.57 |
ZF10 | 茎 Stem | 506.46 ± 3.63 | ZF32 | 叶 Leave | 29.41 ± 0.33 |
ZF11 | 茎 Stem | 1.23 ± 0.11 | ZF33 | 茎 Stem | 1.82 ± 0.17 |
ZF12 | 根 Root | 69.44 ± 0.65 | ZF34 | 茎 Stem | 43.62 ± 0.43 |
ZF13 | 茎 Stem | 17.20 ± 0.16 | ZF35 | 茎 Stem | 87.18 ± 0.55 |
ZF14 | 叶 Leave | 41.82 ± 0.39 | ZF36 | 茎 Stem | 132.52 ± 0.66 |
ZF15 | 茎 Stem | 59.84 ± 0.48 | ZF37 | 根 Root | 90.59 ± 0.48 |
ZF16 | 根 Root | 94.03 ± 0.57 | ZF38 | 茎 Stem | 33.36 ± 0.34 |
ZF17 | 根 Root | 107.85 ± 1.11 | ZF39 | 茎 Stem | 9.67 ± 0.19 |
ZF18 | 茎 Stem | 33.02 ± 0.43 | ZF40 | 根 Root | 98.96 ± 0.51 |
ZF19 | 茎 Stem | 11.74 ± 0.21 | ZF41 | 叶 Leave | 35.50 ± 0.43 |
ZF20 | 茎 Stem | 108.38 ± 0.67 | ZF42 | 茎 Stem | 38.05 ± 0.42 |
ZF21 | 根 Root | 64.74 ± 0.71 | ZF43 | 茎 Stem | 80.49 ± 0.59 |
ZF22 | 茎 Stem | 20.50 ± 0.23 |
图1 高秆野生稻43株内生固氮菌(菌株编号及特征见表1)的IS-PCR指纹图谱聚类(UPGMA)
Fig. 1 Dendrogram obtained by UPGMA method showing the similarity of IS-PCR patterns of 43 endophytic diazotrophs isolated from Oryza alta . The strain numbers and characters of isolates see Table 1.
图2 基于16S rRNA基因序列的高秆野生稻各类群代表菌株的Neighbor-joining树(分支数字代表自展支持率, 标尺代表核苷酸碱基差异为2%, 随机抽样计算次数为1,000)
Fig. 2 Neighbor-joining tree based on the 16S rRNA gene sequences of representative strains of each group isolated from Oryza alta. Branch numbers represent the support rate of self-development. Scales represent the difference of nucleotide bases, which is 2%. The number of random sampling calculations is 1,000.
类群 Groups | 代表菌株 Representative strain | 最相似菌株名称(GenBank登录号) Closely related strain (GenBank accession number) | 相似性 Similarity (%) |
---|---|---|---|
I | ZF3 | 土生乌拉尔菌 Raoultella terrigena ATCC33257T (NR_114503) | 99.79 |
II | ZF8 | 类肺炎克雷伯氏菌肺炎亚种 Klebsiella quasipneumoniae subsp. quasipneumoniae 01A030T (HG933296) | 99.86 |
III | ZF13 | 越南伯克霍尔德氏菌 Burkholderia vietnamiensis LMG10929T (AF097534) | 99.93 |
IV | ZF15 | 肠杆菌 Enterobacter sp. R4-368 (CP005991_s) | 99.59 |
V | ZF24 | 门多萨假单胞菌 Pseudomonas mendocina NBRC14162T (BBQC01000018) | 99.03 |
VI | ZF43 | 固氮植物菌 Phytobacter diazotrophicus LS8T (DQ821583.2) | 99.37 |
表2 高秆野生稻各类群代表菌株16S rDNA序列相似性分析结果
Table 2 Similarity of 16S rDNA sequences of representative strain of each group isolated from Oryza alta
类群 Groups | 代表菌株 Representative strain | 最相似菌株名称(GenBank登录号) Closely related strain (GenBank accession number) | 相似性 Similarity (%) |
---|---|---|---|
I | ZF3 | 土生乌拉尔菌 Raoultella terrigena ATCC33257T (NR_114503) | 99.79 |
II | ZF8 | 类肺炎克雷伯氏菌肺炎亚种 Klebsiella quasipneumoniae subsp. quasipneumoniae 01A030T (HG933296) | 99.86 |
III | ZF13 | 越南伯克霍尔德氏菌 Burkholderia vietnamiensis LMG10929T (AF097534) | 99.93 |
IV | ZF15 | 肠杆菌 Enterobacter sp. R4-368 (CP005991_s) | 99.59 |
V | ZF24 | 门多萨假单胞菌 Pseudomonas mendocina NBRC14162T (BBQC01000018) | 99.03 |
VI | ZF43 | 固氮植物菌 Phytobacter diazotrophicus LS8T (DQ821583.2) | 99.37 |
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