异源表达Hvsusiba2水稻对稻田甲烷排放及土壤相关菌群的影响
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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。
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.
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.
引文
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[20]SUN C X,PALMQVIST S,OLSSON H,et al.A novel WRKY transcription factor,SUSIBA2,participates in sugar signaling in barley by binding to the sugar-responsive elements of the iso1promoter[J].The Plant Cell,2003,15(9):2076–2092
[21]张广斌,马静,徐华,等.稻田甲烷产生途径研究进展[J].土壤,2011,43(1):6–11ZHANG G B,MA J,XU H,et al.Advances on methanogenic pathways in rice fields[J].Soils,2011,43(1):6–11
[22]LU Y H,WATANABE A,KIMURA M.Contribution of plantderived carbon to soil microbial biomass dynamics in a paddy rice microcosm[J].Biology and Fertility of Soils,2002,36(2):136–142
[23]方晓瑜,李家宝,芮俊鹏,等.产甲烷生化代谢途径研究进展[J].应用与环境生物学报,2015,21(1):1–9FANG X Y,LI J B,RUI J P,et al.Research progress in biochemical pathways of methanogenesis[J].Chinese Journal of Applied and Environmental Biology,2015,21(1):1–9
[24]邓永翠,车荣晓,吴伊波,等.好氧甲烷氧化菌生理生态特征及其在自然湿地中的群落多样性研究进展[J].生态学报,2015,35(14):4579–4591DENG Y C,CHE R X,WU Y B,et al.A review of the physiological and ecological characteristics of methanotrophs and methanotrophic community diversity in the natural wetlands[J].Acta Ecologica Sinica,2015,35(14):4579–4591
[25]王明星.中国稻田甲烷排放[M].北京:科学出版社,2001:192WANG M X.Methane Emission from Rice Field in China[M].Beijing:Science Press,2001:192
[2]LIU Y C,WHITMAN W B.Metabolic,phylogenetic,and ecological diversity of the methanogenic archaea[J].Annals of the New York Academy of Sciences,2008,1125(1):171–189
[3]魏海苹,孙文娟,黄耀.中国稻田甲烷排放及其影响因素的统计分析[J].中国农业科学,2012,45(17):3531–3540WEI H P,SUN W J,HUANG Y.Statistical analysis of methane emission from rice fields in China and the driving factors[J].Scientia Agricultura Sinica,2012,45(17):3531–3540
[4]陈槐,周舜,吴宁,等.湿地甲烷的产生、氧化及排放通量研究进展[J].应用与环境生物学报,2006,12(5):726–733CHEN H,ZHOU S,WU N,et al.Advance in studies on production,oxidation and emission flux of methane from wetlands[J].Chinese Journal of Applied&Environmental Biology,2006,12(5):726–733
[5]贾仲君,蔡祖聪.水稻植株对稻田甲烷排放的影响[J].应用生态学报,2003,14(11):2049–2053JIA Z J,CAI Z C.Effects of rice plants on methane emission from paddy fields[J].Chinese Journal of Applied Ecology,2003,14(11):2049–2053
[6]MA K,QIU Q F,LU Y H.Microbial mechanism for rice variety control on methane emission from rice field soil[J].Global Change Biology,2010,16(11):3085–3095
[7]CAI Z C,TSURUTA H,RONG X M,et al.CH4 emissions from rice paddies managed according to farmer’s practice in Hunan,China[J].Biogeochemistry,2001,56(1):75–91
[8]YAO H,YAGI K,NOUCHI I.Importance of physical plant properties on methane transport through several rice cultivars[J].Plant and Soil,2000,222(1/2):83–93
[9]LU Y,WASSMANN R,NEUE H U,et al.Impact of phosphorus supply on root exudation,aerenchyma formation and methane emission of rice plants[J].Biogeochemistry,1999,47(2):203–218
[10]AULAKH M S,BODENBENDER J,WASSMANN R,et al.Methane transport capacity of rice plantsⅡ.Variations among different rice cultivars and relationship with morphological characteristics[J].Nutrient Cycling in Agroecosystems,2000,58(1/3):367–375
[11]王增远,徐雨昌,李震,等.水稻品种对稻田甲烷排放的影响[J].作物学报,1999,25(4):441–446WANG Z Y,XU Y C,LI Z,et al.Effect of rice cultivars on methane emissions from rice field[J].Acta Agronomica Sinica,1999,25(4):441–446
[12]傅志强,黄璜,朱华武,等.水稻CH4和N2O的排放及其与植株特性的相关性[J].湖南农业大学学报:自然科学版,2011,37(4):356–360FU Z Q,HUANG H,ZHU H W,et al.Relativity between CH4 and N2O emission and rice plant characteristics[J].Journal of Hunan Agricultural University:Natural Sciences,2011,37(4):356–360
[13]SU J,HU C,YAN X,et al.Expression of barley SUSIBA2transcription factor yields high-starch low-methane rice[J].Nature,2015,523(7562):602–606
[14]YU Y,LEE C,KIM J,et al.Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction[J].Biotechnology and Bioengineering,2005,89(6):670–679
[15]HOOK S E,NORTHWOOD K S,WRIGHT A D G,et al.Long-term monensin supplementation does not significantly affect the quantity or diversity of methanogens in the rumen of the lactating dairy cow[J].Applied and Environmental Microbiology,2009,75(2):374–380
[16]KOLB S,KNIEF C,STUBNER S,et al.Quantitative detection of methanotrophs in soil by novel pmo A-targeted real-time PCR assays[J].Applied and Environmental Microbiology,2003,69(5):2423–2429
[17]DUBEY S K,SINGH A,SINGH R S,et al.Changes in methanogenic population size and CH4 production potential in response to crop phenology in tropical rice field[J].Soil Biology and Biochemistry,2013,57:972–978
[18]NARIHIRO T,SEKIGUCHI Y.Oligonucleotide primers,probes and molecular methods for the environmental monitoring of methanogenic archaea[J].Microbial Biotechnology,2011,4(5):585–602
[19]SUN C X,H?GLUND A S,OLSSON H,et al.Antisense oligodeoxynucleotide inhibition as a potent strategy in plant biology:Identification of SUSIBA2 as a transcriptional activator in plant sugar signalling[J].The Plant Journal,2005,44(1):128–138
[20]SUN C X,PALMQVIST S,OLSSON H,et al.A novel WRKY transcription factor,SUSIBA2,participates in sugar signaling in barley by binding to the sugar-responsive elements of the iso1promoter[J].The Plant Cell,2003,15(9):2076–2092
[21]张广斌,马静,徐华,等.稻田甲烷产生途径研究进展[J].土壤,2011,43(1):6–11ZHANG G B,MA J,XU H,et al.Advances on methanogenic pathways in rice fields[J].Soils,2011,43(1):6–11
[22]LU Y H,WATANABE A,KIMURA M.Contribution of plantderived carbon to soil microbial biomass dynamics in a paddy rice microcosm[J].Biology and Fertility of Soils,2002,36(2):136–142
[23]方晓瑜,李家宝,芮俊鹏,等.产甲烷生化代谢途径研究进展[J].应用与环境生物学报,2015,21(1):1–9FANG X Y,LI J B,RUI J P,et al.Research progress in biochemical pathways of methanogenesis[J].Chinese Journal of Applied and Environmental Biology,2015,21(1):1–9
[24]邓永翠,车荣晓,吴伊波,等.好氧甲烷氧化菌生理生态特征及其在自然湿地中的群落多样性研究进展[J].生态学报,2015,35(14):4579–4591DENG Y C,CHE R X,WU Y B,et al.A review of the physiological and ecological characteristics of methanotrophs and methanotrophic community diversity in the natural wetlands[J].Acta Ecologica Sinica,2015,35(14):4579–4591
[25]王明星.中国稻田甲烷排放[M].北京:科学出版社,2001:192WANG M X.Methane Emission from Rice Field in China[M].Beijing:Science Press,2001:192