单贞,
陈在杰
福建省农业科学院生物技术研究所/福建省农业遗传工程重点实验室 福州 350003
基金项目: 国家自然科学基金项目31670416
福建省科技项目2015N0037
福建省属公益类项目2018R1019-1
福建省农业科学院科技项目A2015-02
详细信息
作者简介:苏军, 主要从事水稻分子研究。E-mail:sj@fjage.org
中图分类号:Q89;Q78计量
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被引次数:0
出版历程
收稿日期:2018-01-23
录用日期:2018-05-15
刊出日期:2018-09-01
Effects of heterogous expression of Hvsusiba2 rice on methane mitigation and related micro-organism abundance in paddy fields
SU Jun,,SHAN Zhen,
CHEN Zaijie
Biotechnology Institute of Fujian Academy of Agricultural Sciences/Fujian Provincial Key Laboratory of Genetic Engineering for Agriculture, Fuzhou 350003, China
Funds: the National Natural Science Foundation of China31670416
Fujian Science and Technology Project2015N0037
Fujian Public Scientific Research Institution Foundation2018R1019-1
the Science and Technology Project of Fujian Academy of Agricultural SciencesA2015-02
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Corresponding author:SU Jun, E-mail: sj@fjage.org
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摘要
摘要:Hvsusiba2是调控大麦淀粉合成和光合产物分配的转录因子。前期研究我们将Hvsusiba2导入粳稻（Oryza sativa L. subsp.japonica），Hvsusiba2粳稻稻田甲烷排放显著下降，胚乳淀粉含量显著提高。为进一步明确Hvsusiba2对稻田甲烷排放的影响，本研究我们将Hvsusiba2导入籼稻（O.sativa L. subsp. indica），研究异源表达Hvsusiba2籼稻全生育期甲烷排放和稻田主要甲烷菌及甲烷氧化菌变化。采用静态箱法测定Hvsusiba2水稻稻田甲烷排放通量，结果显示Hvsusiba2稻田全生育期的大部分时段甲烷排放量显著（P < 0.05）或极显著（P < 0.01）低于对照株系。Hvsusiba2水稻甲烷减排率幅度为54.7%~3.8%，减排率最高的时期为幼穗分化期。2个Hvsusiba2水稻株系生长季累计甲烷排放量分别为5 060.16 mg·m-2和5 250.60 mg·m-2，比对照减排30.30%和27.58%。采用荧光定量PCR法检测水稻关键生长期根土6类产甲烷菌和2类甲烷氧化菌以及土壤总细菌的丰度变化。结果显示：在整个生长期内Hvsusiba2水稻根土6类产甲烷菌菌群丰度的总体趋势是前期高、后期低；甲烷古菌（Archaea，ARC）、甲烷鬃菌科（Methanosaetaceae，Mst）和甲烷微菌目（Methanomicrobiales，MMb）3类菌群丰度的高峰出现在分蘖盛期，甲烷八叠球菌科（Methanosarcinaceae，Msc）菌群丰度的高峰出现在幼穗分化穗期，普通产甲烷菌（Methanogens，MET）和甲烷杆菌目（Methanobacteriales，MBT）分蘖期最高。Hvsusiba2水稻产甲烷菌丰度在分蘖期、抽穗期和开花期显著或极显著地低于野生型对照。在大部分测试时间段内Hvsusiba2水稻的2类甲烷氧化菌群丰度比对照有显著（P < 0.05）或极显著（P < 0.01）下降；Hvsusiba2水稻土壤总细菌丰度在水稻的分蘖期、抽穗期和开花期也显著低于野生型水稻。稻田中产甲烷菌的丰度依次是甲烷鬃菌科（Mst）>甲烷古菌（ARC）>普通产甲烷菌（MET）>甲烷微菌目（MMb）≥甲烷八叠球菌科（Msc）>甲烷杆菌目（MBT）；2类甲烷氧化菌中Ⅰ型甲烷氧化菌（MBAC）丰度极显著大于Ⅱ型甲烷氧化菌（TYPEⅡ）。结合之前的研究结果，我们认为Hvsusiba2可能是通过改变水稻光合同化物分配生理，减少向土壤有机质的输送，降低土壤相关菌群的丰度达到稻田甲烷减排的。
关键词:水稻/
Hvsusiba2异源表达/
甲烷减排/
产甲烷菌/
甲烷氧化菌/
菌群丰度
Abstract:A field experiment was conducted to explore the effects of genetically modified rice with Hvsusiba2 gene on paddy field methane mitigation. Hvsusiba2 gene is a transcription factor that acts on the upstream of starch synthesis pathway and is recognized as a key regulator for barley starch accumulation and assimilation distribution. Previous studies have shown that japonica rice (Oryza sativa L. subsp. japonica) integrated with Hvsusiba2 gene significantly reduces methane emission in paddy fields and increases content of seed starch. To further understand gene effects on cutting down of methane emissions under different rice genetic conditions, we introduced Hvsusiba2 into indica rice (O. sativa L. subsp. indica) and then investigated methane emissions from Hvsusiba2 rice field as well as the population size of bacteria associated with methane emissions in paddy fields during the growing season from April to September 2016. The results showed that the range of methane mitigation for the whole season was 54.7%-3.8%, compared with the control (wild rice). The highest mitigation rate was during booting period, reaching 54.7%. Total methane emissions of the two lines of Hvsusiba2 rice were respectively 5 060.16 mg·m-2 and 5 250.60 mg·m-2, while that under wild rice was 7 249.68 mg·m-2 for the period from the first measurement to harvest. Methane reduction rates of the two lines were 30.30% and 27.58%, respectively. The abundance of 6 orders or families of methanogens and 2 groups of methanotrophs in Hvsusiba2 rice fields showed significant (P < 0.05, P < 0.01) decreases almost throughout the entire growing season when Hvsusiba2 rice was compared with wild rice. In addition, total bacteria populations during rice tillering, heading and flowering periods were significantly (P < 0.05, P < 0.01) lower in Hvsusiba2 rice than in wild rice. Population size of 6 methanogens were in the order of:Methanosaetaceae (Mst) > Archaea (ARC) > methanogens (MET) > Methanomicrobiales (MMb) > Methanosarcinaceae (Msc) > Methanobacteriales (MBT). Among these, Methanosaetaceae had the largest community, followed by Archaea. Of the 2 groups of methanotrophs, the abundance of MBAC was much larger than that of TYPE Ⅱ. After comparison of our experimental data with other studies, we concluded that Hvsusiba2 rice mechanism for reducing methane emission more likely regulated carbohydrate flow to ground parts of the plant, reduced assimilates transported to soil and lowered methane-related bacteria abundance, which ultimately reduced methane emissions.
Key words:Rice/
Heterogous expression of Hvsusiba2/
Methanon mitigation/
Methanogen/
Methanotroph/
Bacteria abundance
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图1播种后不同时间Hvsusiba2籼稻‘86R10-1’和‘86R27-3’稻田不同时段甲烷排放特征
A: 10:00—12:30采集的样品; B: 14:30—17:00采集的样品。每个值是6个生物学重复的平均值。*和**分别表示Hvsusiba2籼稻和对照‘MH86’的差异显著性达5%和1%水平。
Figure1.Characteristics of CH4 emission in different times of a day after different days of sowing in fields of Hvsusiba2 indica rice '86R10-1' and '86R27-3'
Figures A are samples collected at 10:00-12:30. Figures B are samples collected at 14:30-17:00. Each value is the mean of 6 biological replicates. * and ** indicate significance differences between Hvsusiba2 indica rice and wild rice 'MH86' at 5% and 1% levels, respectively.
下载: 全尺寸图片幻灯片
图2Hvsusiba2籼稻‘86R10-1’和‘86R27-3’不同生育期根土总细菌丰度
每个值是3个生物学重复的平均值; *和**分别表示Hvsusiba2籼稻和对照‘MH86’的差异达5%和1%水平。
Figure2.Abundances of total bacteria in Hvsusiba2 indica rice '86R10-1' and '86R27-3' soil during growing season
Each value is the mean of 3 biological replicates. * and ** mean significant differences between Hvsusiba2 indica rice and wild rice 'MH86' at 5% and 1% levels, respectively.
下载: 全尺寸图片幻灯片
图3表达Hvsusiba2籼稻‘86R10-1’和‘86R27-3’全生育期稻田根土6类产甲烷菌丰度变化
每个值是3个生物学重复的平均值; *和**分别表示Hvsusiba2籼稻和对照‘MH86’的差异达5%和1%水平。
Figure3.Abundances of 6 orders or families of methanogens in soil of Hvsusiba2 indica rice '86R10-1' and '86R27-3'during growing season
Each value is the mean of 3 biological replicates. * and ** mean significant differences between Hvsusiba2 indica rice and wild rice 'MH86' at 5% and 1% levels, respectively.
下载: 全尺寸图片幻灯片
图4Hvsusiba2籼稻‘86R10-1’和‘86R27-3’全生育期稻田土壤产甲烷菌的相对丰度(产甲烷菌/总细菌)
每个值是3个生物学重复的平均值; *和**分别表示Hvsusiba2籼稻和对照‘MH86’的差异达5%和1%水平。
Figure4.Relative abundances of methanogens in total bacteria in Hvsusiba2 indica rice '86R10-1' and '86R27-3' paddy soil during growing season
Each value is the mean of 3 biological replicates. * and ** mean significant differences between Hvsusiba2 indica rice and wild rice 'MH86' at 5% and 1% levels, respectively.
下载: 全尺寸图片幻灯片
图5Hvsusiba2籼稻‘86R10-1’和‘86R27-3’稻田土壤2类甲烷氧化菌丰度变化
每个值是3个生物学重复的平均值; *和**分别表示Hvsusiba2籼稻和对照‘MH86’的差异达5%和1%水平。
Figure5.Abundances of 2 groups of methanotrophs in Hvsusiba2 indica rice '86R10-1' and '86R27-3' soil during growing season
Each value is the mean of 3 biological replicates. * and ** mean significant differences between Hvsusiba2 indica rice and wild rice 'MH86' at 5% and 1% levels, respectively.
下载: 全尺寸图片幻灯片
图6Hvsusiba2籼稻‘86R10-1’和‘86R27-3’全生育期稻田土壤产甲烷菌相对丰度(产甲烷菌/甲烷氧化菌)
每个值是3个生物学重复的平均值; *和**分别表示Hvsusiba2籼稻和对照‘MH86’的差异达5%和1%水平。
Figure6.Relative abundances of methanogens in total methanogens in Hvsusiba2 indica rice '86R10-1' and '86R27-3' paddy soil during growing season
Each value is the mean of 3 biological replicates. * and ** mean significant differences between Hvsusiba2 indica rice and wild rice 'MH86' at 5% and 1% levels, respectively.
下载: 全尺寸图片幻灯片表1产甲烷菌(群)和甲烷氧化菌(群)16S rRNA荧光定量PCR引物
Table1.Primers targeting the 16S rRNA genes of methanogens and methanotrophs
| 细菌Bacteria | 引物名称Primer | 引物序列(5'-3') Sequence (5'-3') | 备注Note |
| Archaea (ARC)[14] | ARC787F ARC1059R | ATTAGATACCCSBGTAGTCC GCCATGCACCWCCTCT | 产甲烷菌 Methanogens |
| Methanogens (MET)[15] | Met630F Met803R | GGATTAGATACCCSGGTAGT GTTGARTCCAATTAAACCGCA | 产甲烷菌 Methanogens |
| Methanobacteriales (MBT)[14] | MBT857F MBT1196R | CGWAGGGAAGCTGTTAAGT TACCGTCGTCCACTCCTT | 产甲烷菌 Methanogens |
| Methanosarcinaceae (Msc)[14] | Msc380F Msc828R | GAAACCGYGATAAGGGGA TAGCGARCATCGTTTACG | 产甲烷菌 Methanogens |
| Methanosaetaceae (Mst)[14] | Mst702F Mst862R | TAATCCTYGARGGACCACCA CCTACGGCACCRACMAC | 产甲烷菌 Methanogens |
| Methanomicrobiales (MMB)[14] | MMB282F MMB832R | ATCGRTACGGGTTGTGGG CACCTAACGCRCATHGTTTAC | 产甲烷菌 Methanogens |
| Methylobacter/Met-hylosarcina (MBAC)[16] | A189F Mb606R | GGNGACTGGGACTTCTGG ACRTAGTGGTAACCTTGYAA | 甲烷氧化菌 Methanotrophs |
| Methylosinus (TYPEⅡ)[16] | II223F II646R | CGTCGTATGTGGCCGAC CGTGCCGCGCTCGACCATGYG | 甲烷氧化菌 Methanotrophs |
| Bacteria[17] | Eub338F Eub518R | ACTCCTACGGGAGGCAGCAG ATTACCGCGGCTGCTGG | 土壤总细菌Bacteria |
下载: 导出CSV表2产甲烷菌(群)和甲烷氧化菌(群)荧光定量PCR扩增程序
Table2.qPCR programs for 6 methanogens and 2 methanotrophs
| 步骤 Steps | 产甲烷菌Methanogens | 甲烷氧化菌Methanotrophs | |||||
| 温度 Temperature | 时间 Time | 循环数 Cycles | 温度 Temperature | 时间 Time | 循环数 Cycles | ||
| 预变性 Pre-denaturation | 95 ℃ | 7 min | 94 ℃ | 5 min | |||
| 变性Denature | 95 ℃ | 40 s | $\left. \begin{array}{l}\\\\\\\\\\\\\\\end{array} \right\}40$ | 94 ℃ | 45 s | $\left. \begin{array}{l}\\\\\\\\\\\\\\\end{array} \right\}40$ | |
| 退火 Annealing | N | 1 min | M | 20 s | |||
| 延伸 Extend | 72 ℃ | 40 s | 72 ℃ | 45 s | |||
| 溶解曲线 Solubility curve | 95 ℃ | 15 s | 95 ℃ | 15 s | |||
| 60 ℃ | 1 min | 60 ℃ | 1 min | ||||
| 95 ℃ | 15 s | 95 ℃ | 15 s | ||||
| 60 ℃ | 15 s | 60 ℃ | 15 s | ||||
| N:产甲烷菌退火温度, ARC为60 ℃, Msc为60 ℃, Mst为61 ℃, MMb为66 ℃, MBT为58 ℃, Met为60 ℃。M:甲烷氧化菌退火温度, MBAC为58 ℃, TYPEⅡ为65 ℃。N and M: PCR annealing temperature for methanogens and methanotrophs, for ARC it is 60 ℃, for Msc it is 60 ℃, for Mst it is 61 ℃, for MMb it is 66 ℃, for MBT it is 58 ℃, for Met it is 60 ℃, for MBAC it is 58 ℃, for TYPEⅡ it is 65 ℃. | |||||||
下载: 导出CSV表36类甲烷菌在Hvsusiba2籼稻田土壤总甲烷菌中的比例
Table3.Proportions of 6 methanogens in Hvsusiba2 indica rice soil
| 甲烷菌 Methanogen | MH86 | 86R10-1 | 86R27-3 | |||||
| 数量 Amount [×106 copies·g-1(DWS)] | 占比 Proportion (%) | 数量 Amount [×106 copies·g-1(DWS)] | 占比 Proportion (%) | 数量 Amount [×106 copies·g-1(DWS)] | 占比 Proportion (%) | |||
| 古细菌域甲烷菌Archaea | 133.02 | 12.57 | 80.39 | 11.37 | 79.47 | 14.22 | ||
| 甲烷微菌目 Methanomicrobiales | 2.36 | 0.22 | 1.53 | 0.22 | 2.18 | 0.39 | ||
| 甲烷杆菌目 Methanobacteriales | 0.16 | 0.01 | 0.11 | 0.01 | 0.083 | 0.01 | ||
| 甲烷鬃菌科 Methanosaetaceae | 904.19 | 85.44 | 611.97 | 86.56 | 465.98 | 83.36 | ||
| 甲烷八叠球菌 Methanosarcinaceae | 2.52 | 0.24 | 1.35 | 0.19 | 1.707 | 0.30 | ||
| 普通甲烷菌 Methanogens | 16.04 | 1.52 | 11.68 | 1.65 | 9.597 | 1.72 | ||
下载: 导出CSV表42类甲烷氧化菌在Hvsusiba2籼稻田土壤中的比例
Table4.Proportions of 2 methanotrophs in Hvsusiba2 indica rice soil
| 甲烷氧化菌 Methanotroph | MH86 | 86R10-1 | 86R27-3 | |||||
| 数量 Amount [×106 copies· g-1(DWS)] | 占比 Proportion (%) | 数量 Amount [×106 copies· g-1(DWS)] | 占比 Proportion (%) | 数量 Amount [×106 copies· g-1(DWS)] | 占比 Proportion (%) | |||
| Ⅰ型甲烷氧化菌 Methytobacter / Met-hylosarcina | 15 487.75 | 99.91 | 8 172.73 | 99.87 | 1 121.29 | 98.89 | ||
| Ⅱ型甲烷氧化菌 Methylosinus | 14.12 | 0.09 | 10.80 | 0.13 | 12.61 | 1.11 | ||
下载: 导出CSV参考文献
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