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摘要:
DNA碱基编辑技术是由CRISPR/Cas系统发展而来，能对基因组碱基进行精准编辑。目前已开发的DNA碱基编辑器包括介导C•G至T•A转换的胞嘧啶单碱基编辑器、介导A•T至G•C转换的腺嘌呤单碱基编辑器、介导C•G至G•C颠换的糖基化酶单碱基编辑器、介导C•G至T•A和A•T至G•C同时转换的双碱基编辑器、介导任意碱基之间转换的引导编辑器以及线粒体DNA编辑器。本文系统总结了上述6种DNA编辑器的原理、优化历程及最新研究进展，着重介绍了应用到植物研究中的碱基编辑器工具及其在作物遗传改良中的应用，并对碱基编辑技术今后的发展进行了展望。
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关键词:
- CRISPR/Cas9 /
- 碱基编辑技术 /
- DNA碱基编辑器 /
- 线粒体DNA /
- 作物改良
Abstract:The base editing technology is developed from the CRISPR/Cas gene editing systems, which can perform accurate base or gene editing at the DNA level. Recent years, six types of novel DNA base editors have been developed for the editing of nuclear and organellar genomes, including the cytosine base editor (CBE), the adenine base editor (ABE), the glycosylase base editor (GBE), the adenine and cytosine dual base editor (DBE), the prime editor (PE) and the mitochondrial genome editor. In this review, we summarize the principles, optimization processes and current advances of the above six DNA editors and focus on their application in crop genetic improvement. Finally, the future development of base editing technology is prospected.
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Keywords:
- CRISPR/Cas9 /
- Base editing technology /
- DNA base editor /
- Mitochondrial DNA /
- Crop improvement
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甲烷(CH4)是一种主要的温室气体，稻田CH4排放量占全球温室气体总产生量的19%[1]。合理的灌溉和施肥等管理技术对稻田CH4减排有重要作用。目前稻田节水灌溉方式有“薄浅湿晒”灌溉、干湿交替灌溉等，干湿交替灌溉稻田的CH4排放量较常规灌溉和“薄浅湿晒”灌溉低[2]。与常规灌溉相比，“薄浅湿晒”灌溉可使稻田CH4排放量显著减少12.6%~44.9%[3]，干湿交替灌溉可使稻田CH4累积排放量显著降低37%[4]。施氮量也会影响稻田CH4排放[5]，但是施氮量对稻田CH4排放量影响的研究结果不一致。有研究表明，相对于不施氮肥，高无机氮处理(249 kg·hm−2)稻田CH4排放量降低15%[6]；但也有研究发现，随着尿素用量的增加，稻田CH4排放量随之增加[5]。除了施氮量，不同生育时期施氮比例也会影响稻田CH4排放，基肥、分蘖肥、穗肥和粒肥施氮比例为4.5∶2∶1.5∶2的稻田CH4排放量低于施氮比例为5∶2.5∶1∶1.5和4∶2∶2∶2的[7]。不同灌溉方式及施氮量条件下，土壤易氧化有机碳、微生物量碳以及甲烷氧化菌可直接影响稻田CH4排放[2]，且稻田CH4排放通量与土壤可溶性有机碳和微生物量碳含量呈正相关关系[8]，而甲烷氧化菌则会降低稻田CH4排放量[9]。本研究通过3种灌溉方式和3种氮肥处理(氮肥用量与基、追肥比例结合)早、晚稻田间试验，研究双季稻不同生育期稻田CH4排放通量、土壤有机碳组分含量以及甲烷氧化菌数量的变化，分析稻田CH4排放通量与土壤有机碳组分含量和甲烷氧化菌数量之间的关系，以期获得稻田CH4减排的水氮管理模式，并揭示土壤有机碳组分含量和甲烷氧化菌数量对稻田CH4排放通量的影响规律。
1. 材料与方法
1.1 试验地点及材料
2016年7月至2017年7月在南宁市灌溉试验站(N22°52′58.33″，E108°17′38.86″)进行双季稻大田试验。试验所用土壤为第四纪红色黏土发育的水稻土，土壤理化特征如下：饱和含水率49.2%，容重1.2 g·cm−3，有机碳26.9 g·kg−1，全氮1.3 g·kg−1，碱解氮113.6 mg·kg−1，有效磷50.0 mg·kg−1，速效钾110.6 mg·kg−1，pH 7.6。试验期间早稻在分蘖期、孕穗期、乳熟期和成熟期的降雨量分别为186.3、83.0、97.7和222.3 mm，晚稻分别为619.2、6.4、81.0和7.8 mm。早稻和晚稻品种均为‘内5优8015’，为广西本地优良三系杂交水稻品种。
1.2 灌溉方式与施氮处理
试验设3种灌溉方式和3种氮肥处理，完全随机设计，共9个处理。每个处理设3个小区，共27个小区。每个小区面积25 m2，小区之间用25 cm厚红砖水泥墙隔离分开，以保证小区之间水分相互不侧渗和各小区独立灌排水，并安装水表记录每次灌溉量。
3种灌溉方式包括常规灌溉、“薄浅湿晒”灌溉和干湿交替灌溉。常规灌溉是在移栽返青期以及分蘖期到乳熟期田间均保持20~30 mm水层，但分蘖后期和成熟期仅保持土壤湿润。“薄浅湿晒”灌溉在移栽返青期保持20~40 mm水层，分蘖前期仅保持土壤湿润，分蘖后期由湿润状态自然落干至土壤干燥状态并继续晒田，孕穗期及乳熟期仅保持10 mm左右水层，成熟期晒田。干湿交替灌溉是在各小区均安装TEN45水分张力计(南京土壤仪器公司)监测土壤水势的变化，移栽后10 d内田间保持10~20 mm水层，7 d后进行干湿交替灌溉，即当土壤水势为−15 kPa时，灌溉20 mm，再自然落干至土壤水势为−15 kPa，再灌溉20 mm，如此循环，至水稻成熟。详细水分控制方法见本研究的前期工作报道[2, 10]。
3种施氮处理包括N1：120 kg·hm−2氮(20%基肥80%追肥)，N2：120 kg·hm−2氮(50%基肥50%追肥)，N3：90 kg·hm−2氮(50%基肥50%追肥)。所用肥料为：尿素(氮质量分数为46%)、过磷酸钙(P2O5质量分数为12%)和氯化钾(K2O质量分数为60%)。各处理P2O5用量为60 kg·hm−2，K2O用量为120 kg·hm−2，其中，全部过磷酸钙和50%氯化钾作基肥，在插秧前1 d耕地时施入土壤中，余下的50%氯化钾分别以分蘖肥和穗肥均按25%的比例施入土壤中。
晚稻试验于2016年7月4日播种，8月1日选取长势均匀的秧苗单株栽培，株行距为20 cm×20 cm，8月16日施分蘖肥，9月13日开始晒田，9月22日复水后施穗肥，11月8日收割。生育期内“薄浅湿晒”灌溉、干湿交替灌溉和常规灌溉平均灌溉量分别为157.8、130.8和227.9 mm。整个生育期为99 d。
早稻试验于2017年3月10日播种，4月12日选取长势均匀的秧苗单株栽培，株行距为20 cm×20 cm，4月16日施分蘖肥，5月27日开始晒田，6月3日复水后施穗肥，7月18日收割。生育期内“薄浅湿晒”灌溉、干湿交替灌溉和常规灌溉平均灌溉量分别为107.3、79.6和157.4 mm。整个生育期为95 d。
1.3 稻田CH4采集与测定
分别于晚稻移栽后37(分蘖期)、63(孕穗期)、77(乳熟期)和98 d(成熟期)，早稻移栽后24(分蘖期)、64(孕穗期)、74(乳熟期)和94 d(成熟期)用静态箱法采集稻田CH4气体[11]。稻田CH4排放通量的测定用Agilent 7890A气相色谱仪(美国安捷伦科技有限公司)，检测器为氢火焰离子检测器(FID)，采用填充柱，当检测器温度升至350 ℃且基线平稳时，开始测样。CH4通量的计算参考王明星[11]的方法。
1.4 土壤采集与指标测定
在晚稻和早稻各生育期采集稻田CH4，同时在各小区按五点法采集0~20 cm耕层土壤样品，测定土壤微生物量碳、可溶性有机碳、易氧化有机碳含量和甲烷氧化菌数量。
微生物量碳含量测定用氯仿熏蒸–硫酸钾浸提法–总有机碳法[12]，可溶性有机碳含量测定用无氯仿熏蒸–硫酸钾浸提–总有机碳法[13]，易氧化有机碳含量测定用高锰酸钾氧化–分光光度计法[14]，甲烷氧化菌数量测定用培养基–滚管法[15]。
1.5 统计分析
数据整理和分析采用DPS 12.26和ExceL 2003软件，结果用3次重复的平均值±标准误表示。用Duncan’s多重比较法分析各处理指标间的差异显著性(P<0.05)，将稻田CH4排放通量与土壤有机碳组分含量和甲烷氧化菌数量进行相关性分析。
2. 结果与分析
2.1 不同处理各生育期双季稻田CH4排放通量
不同处理下双季稻田CH4排放通量随生育期的变化如图1所示，CH4排放通量在分蘖期较高，在孕穗期和乳熟期较低，在成熟期几乎为0。3种灌溉方式下，CH4在晚稻分蘖期集中排放，不同氮肥处理间CH4排放通量为N1>N2>N3。N3处理下，干湿交替灌溉稻田CH4排放通量在晚稻分蘖期、孕穗期和乳熟期较常规灌溉分别降低53.5%、99.4%和99.2%，在早稻分蘖期、孕穗期和乳熟期较常规灌溉分别降低19.9%、29.2%和63.9%。从图1可看出，在N1、N2和N3处理下，干湿交替灌溉双季稻田CH4排放通量均低于“薄浅湿晒”灌溉和常规灌溉，而在相同灌溉方式下，N3处理稻田CH4排放通量较N1、N2降低；因此，干湿交替灌溉N3处理稻田CH4排放通量最低。
![图 1 不同处理各生育期双季稻田CH4排放通量]()
图 1 不同处理各生育期双季稻田CH4排放通量N1：120 kg·hm−2氮(20%基肥80%追肥)，N2：120 kg·hm−2氮(50%基肥50%追肥)，N3：90 kg·hm−2氮(50%基肥50%追肥)；TS：分蘖期，BS：孕穗期，MS：乳熟期，RS：成熟期Figure 1. CH4 emission fluxes from double-cropping paddy field at each growth stage in different treatmentsN1: 120 kg·hm−2 nitrogen (20% basal fertilizer and 80% topdressing), N2: 120 kg·hm−2 nitrogen (50% base fertilizer and 50% topdressing), N3: 90 kg·hm−2 nitrogen (50% basal fertilizer and 50% topdressing); TS: Tillering stage, BS: Booting stage, MS: Milk stage, RS: Ripening stage2.2 不同处理各生育期土壤有机碳组分的变化
由表1可知，不同处理下双季稻田整个生育期土壤微生物量碳在0.08~0.23 g·kg−1范围内变化，以乳熟期较高。在“薄浅湿晒”灌溉下，晚稻分蘖期、孕穗期和乳熟期以及早稻4个生育期N2处理土壤微生物量碳均较高于N1和N3处理。干湿交替灌溉下，晚稻孕穗期和成熟期以及早稻4个生育时期N2处理土壤微生物量碳均高于N1和N3处理，其中早稻孕穗期、乳熟期和成熟期N2处理较N1处理分别增加23.5%、27.8%和20.0%，早稻孕穗期和乳熟期N2处理较N3处理分别增加23.5%和27.8%。常规灌溉下，晚稻土壤微生物量碳在分蘖期、孕穗期和乳熟期以N2处理较高，乳熟期N2处理较N1和N3处理均增加21.0%。早稻分蘖期、乳熟期和成熟期N2处理均高于N1和N3处理，其中乳熟期和成熟期较N1处理分别增加50.0%和75.0%，成熟期较N3处理增加27.3%。3种氮肥处理下，常规灌溉早稻土壤微生物量碳均低于“薄浅湿晒”灌溉及干湿交替灌溉。因此在相同灌溉方式下，N2处理稻田土壤微生物量碳高于N1和N3处理；在相同氮肥处理下，常规灌溉早稻土壤微生物量碳低于“薄浅湿晒”灌溉和干湿交替灌溉。
表 1 不同处理各生育期土壤微生物量碳含量1)Table 1. Soil microbial biomass carbon contents at each growth stage in different treatmentsw/(g·kg−1) 施氮处理
Nitrogen treatment灌溉方式
Irrigation method晚稻 Late rice 早稻 Early rice TS BS MS RS TS BS MS RS N1 BG 0.15±0.01b 0.17±0.01ab 0.17±0.01b 0.17±0.01ab 0.16±0.01ab 0.17±0.01bc 0.18±0.01bc 0.15±0.01b GG 0.17±0.02ab 0.17±0.01ab 0.17±0.01b 0.17±0.01ab 0.17±0.02ab 0.17±0.01bc 0.18±0.01bc 0.15±0.01b CG 0.17±0.04ab 0.19±0.01ab 0.19±0.01b 0.16±0.01ab 0.14±0.02b 0.12±0.01d 0.12±0.01d 0.08±0.01c N2 BG 0.18±0.01ab 0.18±0.01ab 0.19±0.01b 0.17±0.01ab 0.19±0.01a 0.19±0.01ab 0.20±0.01b 0.18±0.01a GG 0.19±0.01ab 0.18±0.01ab 0.18±0.01b 0.20±0.03a 0.20±0.01a 0.21±0.01a 0.23±0.01a 0.18±0.01a CG 0.21±0.02a 0.21±0.02a 0.23±0.02a 0.15±0.02bc 0.17±0.01ab 0.14±0.01cd 0.18±0.01bc 0.14±0.02b N3 BG 0.17±0.01ab 0.18±0.01ab 0.18±0.01b 0.17±0.01ab 0.17±0.02ab 0.17±0.01bc 0.18±0.01b 0.16±0.01ab GG 0.19±0.01ab 0.18±0.01ab 0.17±0.01b 0.17±0.01ab 0.18±0.01a 0.17±0.01bc 0.18±0.01bc 0.16±0.01ab CG 0.17±0.01ab 0.20±0.01ab 0.19±0.01b 0.11±0.02c 0.16±0.01ab 0.14±0.02cd 0.16±0.01c 0.11±0.02c 1)N1：120 kg·hm−2氮(20%基肥80%追肥)，N2：120 kg·hm−2氮(50%基肥50%追肥)，N3：90 kg·hm−2氮(50%基肥50%追肥)；BG：“薄浅湿晒”灌溉，GG：干湿交替灌溉，CG：传统灌溉；TS：分蘖期，BS：孕穗期，MS：乳熟期，RS：成熟期；同列数据后不同小写字母表示处理间差异显著(P<0.05，Duncan’s法)
1) N1: 120 kg·hm−2 nitrogen (20% basal fertilizer and 80% topdressing), N2: 120 kg·hm−2 nitrogen (50% base fertilizer and 50% topdressing), N3: 90 kg·hm−2 nitrogen (50% basal fertilizer and 50% topdressing); BG: “Thin-shallow-wet-dry” irrigation, GG: Alternate drying and wetting irrigation, CG: Conventional irrigation; TS: Tillering stage, BS: Booting stage, MS: Milk stage, RS: Ripening stage; Different lowercase letters in the same column indicate significant differences among different treatments (P<0.05, Duncan’s method)由表2可知，不同处理双季稻田整个生育期土壤可溶性有机碳含量在45.6~106.3 mg·kg−1范围内变化。“薄浅湿晒”灌溉下，晚稻4个生育期及早稻孕穗期、乳熟期和成熟期N3处理土壤可溶性有机碳含量均大于N1和N2处理，其中晚稻乳熟期N3处理较N1和N2处理分别增加23.8%和22.5%。干湿交替灌溉下，晚稻分蘖期、乳熟期和成熟期N3处理土壤可溶性有机碳含量较N1处理分别增加12.0%、12.6%和13.3%，同时较N2处理分别增加12.9%、18.0%和11.8%；早稻乳熟期和成熟期N3处理土壤可溶性有机碳含量较N1处理分别增加36.2%和42.1%，较N2处理分别增加25.4%和36.4%。常规灌溉方式下，晚稻乳熟期和成熟期N3处理土壤可溶性有机碳含量较N1处理分别增加9.6%和21.3%，乳熟期较N1、N2处理分别增加9.6%和6.6%。在3种氮肥处理下，不同灌溉方式对稻田土壤可溶性有机碳含量影响顺序为干湿交替灌溉>“薄浅湿晒”灌溉>常规灌溉，早稻田在分蘖期及孕穗期以干湿交替灌溉土壤可溶性有机碳含量最高，在乳熟期及成熟期以“薄浅湿晒”灌溉土壤可溶性有机碳含量最高。因此，相同灌溉方式下，双季稻田N3处理土壤可溶性有机碳含量均大于N1和N2处理；相同氮肥处理下，干湿交替灌溉土壤可溶性有机碳含量较高。
表 2 不同处理各生育期土壤可溶性有机碳含量Table 2. Soil soluble organic carbon contents at each growth stage in different treatmentsw/(mg·kg−1) 施氮处理
Nitrogen
treatment灌溉方式
Irrigation
method晚稻 Late rice 早稻 Early rice TS BS MS RS TS BS MS RS N1 BG 80.1±5.2d 83.2±3.5abc 82.3±1.2c 61.1±2.0bc 86.9±5.6a 84.8±4.9ab 88.5±6.3abc 64.2±8.6abc GG 92.8±1.2bc 89.5±3.4a 89.0±2.2b 61.1±3.1bc 89.5±11.6a 84.9±21.9ab 77.1±6.4c 54.9±10.4c CG 92.6±5.5bc 73.6±5.4c 73.6±2.0e 45.6±3.2d 85.0±15.5a 74.9±15.9b 83.1±4.9bc 62.9±5.0abc N2 BG 84.6±2.0cd 82.7±4.9abc 83.2±4.4c 58.9±3.1bc 88.2±10.3a 70.9±6.1b 97.2±13.5ab 71.6±4.7ab GG 92.0±0.1bc 84.2±3.1ab 84.9±3.1bc 61.9±1.3bc 94.7±38.3a 87.6±13.9ab 83.7±2.4bc 57.2±1.9bc CG 90.6±3.0bc 76.4±2.2bc 75.7±1.9de 46.9±4.8d 92.5±13.9a 77.6±4.2ab 70.4±8.1c 49.8±5.1c N3 BG 85.4±4.1cd 87.2±6.7a 101.9±3.0a 66.6±5.0ab 71.3±31.4a 87.6±4.8ab 106.3±6.7a 78.3±6.2a GG 103.9±2.4a 89.5±2.3a 100.2±3.9a 69.2±2.5a 102.9±19.2a 99.1±17.2a 105.0±6.2a 78.0±4.7a CG 99.2±6.7ab 79.5±2.0abc 80.7±4.3cd 55.3±3.1c 96.6±9.6a 93.7±3.7ab 89.1±5.8abc 71.1±4.6ab 1) N1：120 kg·hm−2氮(20%基肥80%追肥)，N2：120 kg·hm−2氮(50%基肥50%追肥)，N3：90 kg·hm−2氮(50%基肥50%追肥)；BG：“薄浅湿晒”灌溉，GG：干湿交替灌溉，CG：传统灌溉；TS：分蘖期，BS：孕穗期，MS：乳熟期，RS：成熟期；同列数据后不同小写字母表示处理间差异显著(P<0.05，Duncan’s法)
1) N1: 120 kg·hm−2 nitrogen (20% basal fertilizer and 80% topdressing), N2: 120 kg·hm−2 nitrogen (50% base fertilizer and 50% topdressing), N3: 90 kg·hm−2 nitrogen ( 50% basal fertilizer and 50% topdressing); BG: “Thin-shallow-wet-dry” irrigation, GG: Alternate drying and wetting irrigation, CG: Conventional irrigation; TS: Tillering stage, BS: Booting stage, MS: Milk stage, RS: Ripening stage; Different lowercase letters in the same column indicate significant differences among different treatments (P<0.05, Duncan’s method)由表3可以看出，不同处理晚稻田土壤易氧化有机碳含量在1.08~7.10 g·kg−1范围内变化。N1处理下，晚稻分蘖期和孕穗期以及早稻分蘖期、孕穗期和成熟期常规灌溉土壤易氧化有机碳含量均大于“薄浅湿晒”灌溉和干湿交替灌溉；常规灌溉土壤易氧化有机碳含量在早稻孕穗期较“薄浅湿晒”灌溉增加47.7%，在成熟期较“薄浅湿晒”灌溉和干湿交替灌溉方式分别增加93.5%和61.3%。N2处理下，除晚稻乳熟期外，常规灌溉双季稻各生育期土壤易氧化有机碳含量均大于“薄浅湿晒”灌溉和干湿交替灌溉；常规灌溉土壤易氧化有机碳含量在晚稻孕穗期和成熟期较“薄浅湿晒”灌溉分别增加50.2%和34.5%，在早稻乳熟期增加27.1%。N3处理下，常规灌溉土壤易氧化有机碳含量在早稻分蘖期较干湿交替灌溉增加115.9%，在早稻孕穗期较“薄浅湿晒”灌溉和干湿交替灌溉分别增加38.8%和40.9%；在“薄浅湿晒”和干湿交替灌溉下，不同氮肥处理之间土壤易氧化有机碳含量没有明显变化规律，在常规灌溉下，除早稻孕穗期、乳熟期及成熟期外，土壤易氧化有机碳含量均是N2>N1>N3。因此，相同氮肥处理下，常规灌溉土壤易氧化有机碳含量较“薄浅湿晒”灌溉和干湿交替灌溉增加。
表 3 不同处理各生育期土壤易氧化有机碳含量Table 3. Soil readily oxidizable organic carbon contents at each growth stage in different treatmentsw/(g·kg−1) 施氮处理
Nitrogen
treatment灌溉方式
Irrigation
method晚稻 Late rice 早稻 Early rice TS BS MS RS TS BS MS RS N1 BG 4.53±1.66ab 3.55±0.21ab 4.65±0.58a 5.74±0.60a 2.41±0.39d 3.08±0.29d 4.48±0.42abc 1.70±0.44b GG 1.08±0.13b 3.82±0.34ab 5.30±0.38a 4.70±0.20bc 2.81±0.17d 3.82±0.34bcd 5.11±0.36a 2.04±0.48b CG 6.48±1.65ab 4.33±0.28ab 5.13±0.31a 5.31±0.12ab 3.67±1.17bcd 4.55±0.23abc 5.06±0.26a 3.29±0.61a N2 BG 4.18±2.84ab 3.05±0.25b 5.27±0.40a 4.12±0.37c 4.74±1.33abcd 4.13±0.12abcd 3.95±0.25bc 1.86±0.32b GG 3.08±1.52ab 4.34±0.65ab 5.57±0.35a 4.52±0.10bc 4.62±1.00abcd 4.15±0.24abcd 4.84±0.20ab 2.23±0.34ab CG 7.10±0.79a 4.58±0.66a 5.51±0.32a 5.54±0.18ab 7.25±0.67a 5.12±0.18a 5.02±0.12a 2.71±0.37ab N3 BG 3.68±1.89ab 4.34±0.65ab 5.94±0.51a 4.79±0.40abc 5.65±1.23abc 3.40±0.86cd 5.15±0.08a 1.55±0.21b GG 5.02±1.79ab 4.34±0.25ab 4.76±0.18a 4.10±0.17c 2.96±0.15cd 3.35±0.29cd 3.77±0.56c 1.74±0.19b CG 6.27±1.95ab 3.62±0.18ab 4.59±0.51a 4.73±0.32abc 6.39±0.28ab 4.72±0.41ab 4.24±0.20abc 2.31±0.16ab 1) N1：120 kg·hm−2氮(20%基肥80%追肥)，N2：120 kg·hm−2氮(50%基肥50%追肥)，N3：90 kg·hm−2氮(50%基肥50%追肥)；BG：“薄浅湿晒”灌溉，GG：干湿交替灌溉，CG：传统灌溉；TS：分蘖期，BS：孕穗期，MS：乳熟期，RS：成熟期；同列数据后不同小写字母表示处理间差异显著(P<0.05，Duncan’s法)
1) N1: 120 kg·hm−2 nitrogen (20% basal fertilizer and 80% topdressing), N2: 120 kg·hm−2 nitrogen (50% base fertilizer and 50% topdressing), N3: 90 kg·hm−2 nitrogen (50% basal fertilizer and 50% topdressing); BG: “Thin-shallow-wet-dry” irrigation, GG: Alternate drying and wetting irrigation, CG: Conventional irrigation; TS: Tillering stage, BS: Booting stage, MS: Milk stage, RS: Ripening stage; Different lowercase letters in the same column indicate significant differences among different treatments (P<0.05, Duncan’s method)2.3 不同处理各生育期土壤甲烷氧化菌数量的变化
如图2所示，不同处理晚稻各生育期土壤甲烷氧化菌数量变化范围为1.64×106~12.98×106 cfu·g−1，早稻为1.63×106~23.50×106 cfu·g−1。在N1处理下，常规灌溉土壤甲烷氧化菌数量在晚稻成熟期以及早稻孕穗期、乳熟期和成熟期均大于干湿交替灌溉和“薄浅湿晒”灌溉。在N3处理下，常规灌溉土壤甲烷氧化菌数量在早稻孕穗期和乳熟期较“薄浅湿晒”灌溉分别增加419.2%和457.8%。在N2处理下，干湿交替灌溉土壤甲烷氧化菌数量在双季稻4个生育期均低于“薄浅湿晒”灌溉和常规灌溉，在晚稻分蘖期较“薄浅湿晒”灌溉方式减少76.0%，在孕穗期和乳熟期较常规灌溉方式分别减少77.2%和77.3%；在早稻分蘖期较“薄浅湿晒”灌溉减少76.6%，在孕穗期、乳熟期和成熟期较常规灌溉方式分别减少87.7%、86.2%和57.9%。在N1处理下，“薄浅湿晒”灌溉土壤甲烷氧化菌数量在晚稻分蘖期、孕穗期和乳熟期均大于干湿交替灌溉和常规灌溉，其中在孕穗期较干湿交替灌溉方式增加136.5%；在早稻分蘖期和乳熟期也较干湿交替灌溉和常规灌溉大。在3种灌溉方式下，3种氮肥处理土壤甲烷氧化菌数量没有明显变化规律。因此，相同氮肥处理下，干湿交替灌溉土壤甲烷氧化菌数量较“薄浅湿晒”灌溉和常规灌溉低。
![图 2 不同处理各生育期土壤甲烷氧化菌数量]()
图 2 不同处理各生育期土壤甲烷氧化菌数量N1：120 kg·hm−2氮(20%基肥80%追肥)，N2：120 kg·hm−2氮(50%基肥50%追肥)，N3：90 kg·hm−2氮(50%基肥50%追肥)；TS：分蘖期，BS：孕穗期，MS：乳熟期，RS：成熟期Figure 2. Soil methane oxidizing bacteria population amounts at each growth stage in different treatmentsN1: 120 kg·hm−2 nitrogen (20% basal fertilizer and 80% topdressing), N2: 120 kg·hm−2 nitrogen (50% base fertilizer and 50% topdressing), N3: 90 kg·hm−2 nitrogen (50% basal fertilizer and 50% topdressing); TS: Tillering stage, BS: Booting stage, MS: Milk stage, RS: Ripening stage2.4 双季稻田CH4通量与土壤有机碳组分和甲烷氧化菌的关系
由表4可知，晚稻田CH4排放通量与土壤可溶性有机碳含量之间呈极显著正相关(r=0.55，P<0.01)，早稻田CH4排放通量与土壤可溶性有机碳和土壤易氧化有机碳含量之间呈显著正相关，双季稻田CH4排放通量与土壤微生物量碳和甲烷氧化菌数量之间的关系均不显著；因此，土壤可溶性有机碳是影响稻田CH4排放通量的主要因素。
表 4 双季稻田CH4排放通量与土壤有机碳组分和甲烷氧化菌相关性分析1)Table 4. Correlation analyses of CH4 emission flux with soil organic carbon fraction and methane oxidizing bacteria稻季
Rice season微生物量碳含量
Microbial biomass
carbon content可溶性有机碳含量
Soluble organic
carbon content易氧化有机碳含量
Readily oxidizable organic
carbon content甲烷氧化菌数量
Methane oxidizing
bacteria number晚稻 Late rice 0.31 0.55** 0.25 0.12 早稻 Early rice 0.22 0.34* 0.42* −0.04 1)“*”、“**”分别表示在0.05、0.01水平显著相关
1)“*”, “**” indicate significant correlation at 0.05 and 0.01 levels respectively3. 讨论与结论
3.1 不同灌溉、氮肥处理对稻田CH4排放通量的影响
在水稻整个生育期内稻田CH4集中在分蘖期排放，原因可能是在水稻生长初期，刚淹水后土壤中的有机质剧烈分解，导致土壤中有较多的CH4产生[12]，而后期随着有效氮素的消耗减少，微生物缺乏基质和能源使稻田CH4排放减少。有研究认为，水层深度和CH4排放通量有很强的负相关关系，水层深度通过影响厌氧环境和水温水量控制水稻根系和根系分泌物数量，而这些物质将为CH4产生提供底物[16]。
相同氮肥处理下，相对于干湿交替灌溉，常规灌溉和“薄浅湿晒”灌溉稻田CH4排放通量降低时间较晚；是因为干湿交替灌溉破坏土壤产甲烷菌的生存环境，CH4产生土层随水分降低而下降，待重新灌溉后需一段时间恢复[17]，而且干湿交替灌溉灌溉频率较低，导致稻田CH4排放通量降低时间较早。成熟期土壤中CH4的产生可能与根系渗出物和根系的腐烂有关[17]。
氮肥施入增加土壤中氮素有效性，会相应地提高产甲烷菌所需有机底物有效性，使得产甲烷菌有较多可利用底物，增加CH4排放量[18]。总体来说，在相同灌溉方式下，N3处理稻田CH4排放通量较N1和N2处理低，可能是因为N1和N2处理能刺激更多微生物生长繁殖，改变土壤有机质含量，增加根系分泌物数量等，为CH4生成提供底物[19]。但有研究表明施入尿素使稻田CH4排放通量减少[20]，可能与稻田土壤理化性质有关，因为低氮水平增施氮肥刺激土壤有机底物生成，高氮会形成长期氮肥输入，增加土壤酸度，抑制微生物活性和CH4的产生[21]。
3.2 不同灌溉、氮肥处理对土壤有机碳组分含量的影响
不同灌溉、氮肥处理下土壤微生物量碳含量在分蘖期较低，在孕穗期较高，之后降低，与Banerjee等[22]在单施氮、磷、钾肥处理的结果一致。相同灌溉方式下，N2处理土壤微生物量碳较N3高，史登林等[23]在减量施氮40%条件下有相似的结果；这是因为增施氮肥可以为土壤微生物提供营养物质，促进其生长，进而增加土壤微生物量碳含量[24]。
3种灌溉方式和3种氮肥处理下，双季稻田土壤可溶性有机碳含量在分蘖期、孕穗期及乳熟期没有明显差异，但是在成熟期均减少；原因可能是土壤可溶性有机碳是土壤中微生物生活的重要物质及能量来源，当水稻生长前期淹水灌溉时，土壤中可溶性有机质分解，导致土壤可溶性有机碳含量升高，从而微生物数量增多，相应所需的土壤可溶性有机碳更多，土壤可溶性有机碳含量减少，因而形成一个稳定的体系[25]。在成熟期，追施氮肥较多，再加上甲烷氧化菌呼吸作用增强，充分利用剩余碳素，使土壤可溶性有机碳相对减少。相同灌溉方式下，N3处理双季稻田土壤可溶性有机碳含量大于N1及N2处理，说明相同灌溉方式下，低氮较高氮更有利于提高稻田土壤可溶性有机碳含量，这与前人在减量施氮40%处理下得到的结果相似[23]。相同施氮处理下，虽然不同灌溉方式的稻田土壤可溶性碳含量在不同生育期内有波动，但总体上以干湿交替灌溉较高。
N1处理干湿交替灌溉晚稻田土壤易氧化有机碳含量较少，可能是因为干湿交替灌溉土壤通透性高，微生物活性增强，加之易分解，所以含量较少[26]。N2处理常规灌溉土壤易氧化有机碳含量较高，可能是因为常规灌溉使微生物活性减弱，土壤易氧化有机碳被利用量减少，从而为土壤易氧化有机碳的移动提供便利条件。
土壤可溶性有机碳含量和稻田CH4排放通量有相同的变化趋势，因此，双季稻田CH4排放通量与土壤可溶性有机碳含量呈显著正相关，表明稻田土壤可溶性有机碳含量的高低可以间接反映稻田CH4排放量大小。
3.3 不同灌溉、氮肥处理对土壤甲烷氧化菌数量的影响
干湿交替灌溉土壤甲烷氧化菌数量较“薄浅湿晒”灌溉和常规灌溉少，原因可能是干湿交替灌溉土壤环境不适合甲烷氧化菌生存。N1和N2处理下，总体上“薄浅湿晒”灌溉土壤甲烷氧化菌数量较大，因为“薄浅湿晒”灌溉不仅满足稻田水分需求，且比常规灌溉有良好的土壤通气性，有利于土壤甲烷氧化菌的生存[26]。但也有研究表明CH4氧化过程也可能在厌氧环境中，是产CH4过程的逆向代谢[27]。
3.4 结论
晚稻分蘖期N3(90 kg·hm−2氮，50%基肥和50%追肥)处理干湿交替灌溉稻田CH4排放通量较其他灌溉方式低，而土壤可溶性有机碳含量较其他灌溉方式高。双季稻田CH4排放通量仅与土壤可溶性有机碳含量呈显著正相关，其中，晚稻田相关系数为0.55，早稻田相关系数为0.34。因此，土壤可溶性有机碳含量显著影响双季稻田CH4排放通量，且在供试土壤和栽培管理条件下，干湿交替灌溉N3处理稻田CH4排放通量较低。
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图 1 CBE、ABE和GBE碱基编辑器原理
a：CBE系统，以BE3为例；b：ABE系统，以ABE8e为例；c：GBE系统，以CGBE1为例；染色体单链上红色数字表示该脱氨酶的编辑窗口
Figure 1. Schematic diagrams of CBE, ABE and GBE systems
a: CBE system taking BE3 as an example; b: ABE system taking ABE8e as an example; c: GBE system taking CGBE1 as an example; The numbers in red indicate deamination windows
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图 2 DBE、PE、线粒体碱基编辑器原理
a：DBE系统，以STEME-1为例；染色体单链上红色数字表示脱氨酶的编辑窗口，其中，APOBEC3A的编辑窗口为C1~C17，TadA的编辑窗口为A4~A8；b：PE系统，以PE2为例；c：线粒体碱基编辑系统，以DdCBE为例
Figure 2. Schematic diagrams of DBE, PE and mitochondrial base editors
a: DBE system taking STEME-1 as an example, the numbers in red indicate deamination windows which are C1−C17 in APOBEC3A and A4−A8 in TadA; b: PE system taking PE2 as an example; c: Mitochondrial base editing system taking DdCBE as an example
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