潘争艳^,1,2, 刘博¹, 蒋洪波¹, 姚继攀¹, 白元俊^,1, 徐正进^,21辽宁省水稻研究所,沈阳110101
²沈阳农业大学农学院,沈阳110161

Effect of Panicle Neck Blast on Grain Yield and Stem Node Metabolites at the Rice Filling Stage

PAN ZhengYan^,1,2, LIU Bo¹, JIANG HongBo¹, YAO JiPan¹, BAI YuanJun^,1, XU ZhengJin^{,2 1}Liaoning Rice Research Institute, Shenyang 110101
²College of Agriculture, Shenyang Agricultural University, Shenyang 110161

通讯作者: 白元俊,E-mail: cycbyj@126.com; 徐正进,E-mail: xuzhengjin@126.com

责任编辑: 岳梅
收稿日期:2020-03-11接受日期:2020-05-8网络出版日期:2020-10-16

基金资助:

国家重点研发计划.2018YFD0200200 辽宁省科学事业公益研究基金.GY-20180046 辽宁省博士后项目.178150

Received:2020-03-11Accepted:2020-05-8Online:2020-10-16
作者简介 About authors
潘争艳,E-mail: pzhyma@126.com。








摘要
【目的】检测自然条件下稻瘟病感病品种茎节的代谢产物,从代谢物角度解析水稻品种感病和产量降低的原因,为粳稻品种感病机理研究和生态防控提供理论依据。【方法】以稻瘟病感病品种辽星1号和粳型恢复系C418为材料,灌浆期调查穗颈瘟发病率,同时采用气相色谱-质谱（GC-MS）非靶向代谢组学方法检测感病茎节和正常茎节的代谢产物,利用主成分分析（PCA）感病茎节与正常茎节之间代谢物组成,利用偏最小二乘法判别分析（PLS-DA）的变量权重值（VIP>1.0）进行两者之间差异代谢产物种类的筛选,比较两者糖类物质和氨基酸类物质的差异,从代谢物角度解析水稻品种产量降低的原因。成熟期进行结实率、千粒重、粒长、糙米率等产量和品质性状测定。【结果】辽星1号和C418穗颈瘟发病率分别为57.23%和82.94%。穗颈瘟显著降低一次枝梗整精米率、二次枝梗的结实率、千粒重、长宽比和整精米率,其他性状无显著差异。辽星1号和C418的茎节GC-MS检测均定性到358种代谢产物,感病茎节上调了73.46%和67.88%的代谢产物。PCA和PLS-DA分析可以将感病茎节和正常茎节的代谢产物清楚分开,辽星1号和C418的感病茎节和正常茎节之间具有明显不同的代谢产物表型,与正常茎节相比,辽星1号感病茎节诱导61种代谢产物相对含量显著变化,C418感病茎节诱导47种代谢产物相对含量显著变化,两个品种共有显著变化的代谢产物30种。其中,次生代谢产物香草酸、α-生育酚、脱氢抗坏血酸、三十烷醇、大豆苷元和N-乙酰-d-甘露糖胺的相对含量显著下调,辽星1号分别下调了87.03%、96.29%、77.95%、74.56%、86.48%和82.68%,C418分别下调了99.92%、99.68%、97.26%、86.67%、94.96%和76.74%。正常茎节中辽星1号这6种次生代谢产物的相对含量分别是C418的2.64、2.39、2.39、2.27、3.09和2.56倍;感病茎节上调了63.33%的糖类物质和68.42%的氨基酸类物质,糖类物质上调了1.02—17.92倍,氨基酸类物质上调了1.05—13.12倍,其中C418糖类物质上调的幅度更大。【结论】穗颈瘟发生显著改变了感病品种辽星1号和C418的茎节代谢产物组成,感病品种可能通过香草酸、α-生育酚、脱氢抗坏血酸、三十烷醇、大豆苷元和N-乙酰-d-甘露糖胺相对含量的变化调控茎节对稻瘟病的防御反应。感病茎节通过积累大量的糖类物质和氨基酸类物质,降低其向籽粒转运,抑制了产量形成。
关键词： 水稻;穗颈瘟;代谢产物;糖类;氨基酸;产量

Abstract
【Objective】The objective of this study is to detect the stem node metabolite of susceptible cultivars which suffered the rice blast under the natural condition, analyze the reason for rice cultivars susceptibility and yield reduction, and to provide a theoretical basis for the susceptible mechanism study of japonica rice and ecological control.【Method】With susceptible cultivar which suffered the rice blast Liaoxing1 and japonica rice restorer C418 as the materials, the yield and quality character including seed setting rate, 1000-grain weight, length, brown rice were measured at the maturation stage. The incidence of panicle neck blast was investigated at the filling stage, and GC-MS was carried out to analyze the metabolites of diseased stem nodes and normal stem nodes, the metabolite composition between diseased stem nodes and normal stem nodes was analyzed by principal components analyses (PCA), the variable weights (VIP>1.0) of partial least-squares discriminant analysis (PLS-DA) were used to screen the species of differential metabolites, the difference of carbohydrate and amino acid metabolites was compared, and the reasons for the decrease of rice yield were analyzed from the metabolites.【Result】The incidence of panicle neck blast of Liaoxing1 and C418 was 57.23% and 82.94%, respectively. The panicle neck blast significantly reduced the head rice rate of the primary branch, the seed set rate, 1000-grain weight, length-width ratio and head rice rate of the secondary branch, there was no significant difference in other characters. 358 metabolites were identified in the two cultivars, the metabolites of diseased stem node increased by 73.46% and 67.88%, respectively. The PCA and PLS-DA analysis could clearly identify the metabolites of diseased stem node and normal stem node. The metabolite phenotypes of Liaoxing 1 and C418 were obviously different between the diseased stem node and normal stem node. Compared with normal stem node, the relative content of 61 metabolites induced by Liaoxing 1 diseased stem nodes changed significantly, and the relative content of 47 metabolites induced by C418 diseased stem nodes changed significantly, there were 30 metabolites in same of the diseased stem nodes of these two cultivars. The relative content of secondary metabolites of vanillic acid, alpha tocopherol, dehydroascorbic acid, triacontanol, daidzein and N-acetyl-d-mannosamine significantly decreased by 87.03%, 96.29%, 77.95%, 74.56%, 86.48% and 82.68% in Liaoxing1, respectively. In C418, it decreased by 99.92%, 99.68%, 97.26%, 86.67%, 94.96% and 76.74%, respectively. The relative content of the above six types of secondary metabolites in normal stem node of Liaoxing1 was 2.64, 2.39, 2.39, 2.27, 3.09 and 2.56 times of that in C418, respectively. The panicle neck blast increased 63.33% of carbohydrate metabolites and 68.42% of amino acid metabolites in stem node, the carbohydrate and amino acid metabolites increased by 1.02-17.92 and 1.05-13.12 times, respectively. Among them, the enhanced amplitude of carbohydrate metabolites in C418 was more significant.【Conclusion】The occurrence of panicle neck blast significantly changed the metabolite composition of susceptible cultivars Liaoxing 1 and C418 stem node, the susceptible cultivar may adjust its defensive reaction to the rice blast following the relative amount change of vanillic acid, alpha tocopherol, dehydroascorbic acid, triacontanol, daidzein and N-acetyl-d-mannosamine. By accumulating a large amount of the carbohydrate and amino acid metabolites, the diseased stem nodes reduce their transport to grains and restrain the formation of yield.
Keywords：Oryza sativa;panicle neck blast;metabolite;carbohydrate;amino acid;yield


PDF (1205KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文
本文引用格式
潘争艳, 刘博, 蒋洪波, 姚继攀, 白元俊, 徐正进. 水稻灌浆期穗颈瘟发生对产量及茎节代谢产物的影响[J]. 中国农业科学, 2020, 53(20): 4177-4188 doi:10.3864/j.issn.0578-1752.2020.20.007
PAN ZhengYan, LIU Bo, JIANG HongBo, YAO JiPan, BAI YuanJun, XU ZhengJin. Effect of Panicle Neck Blast on Grain Yield and Stem Node Metabolites at the Rice Filling Stage[J]. Scientia Acricultura Sinica, 2020, 53(20): 4177-4188 doi:10.3864/j.issn.0578-1752.2020.20.007

0 引言

【研究意义】水稻（Oryza sativa）是全球约50%人口的主粮^[1],由稻瘟病菌（Magnaporthe oryzae）引起的稻瘟病是世界各水稻产区普遍存在的一种病害^[2],对水稻造成大幅减产,严重时可减产40%—50%^[3]。稻瘟病在水稻的各个生育时期均可发生,穗颈瘟是其中危害最大的一种^[4]。植物体内代谢产物的变化是生物系统对遗传和环境变异的最终反映^[5],代谢组学（metabonomics）可以对植物体内代谢产物的整体组成进行动态分析^[6]。应用代谢组学方法分析穗颈瘟对水稻茎秆代谢产物的影响,可为解析穗颈瘟感病机理提供理论依据。【前人研究进展】代谢组学是连接最上游基因功能和最下游表观型变化的纽带^[5],已广泛应用于水稻各个领域。ISHIKAWA等^[7]利用代谢组学方法研究发现,氧化胁迫促使水稻细胞的中枢代谢途径耗竭和氨基酸积累;SANA等^[8]利用代谢组学方法研究细菌性白叶枯病菌（Xanthomonas oryzae pv. oryzae）与水稻的互作发现,细菌性白叶枯病菌可以显著影响水稻的苯丙氨酸代谢;PENG等^[9]研究水稻叶鞘和褐飞虱（Nilaparvata lugens）互作发现,褐飞虱对抗/感品种的诱导方式不同,褐飞虱可以诱导感病品种脂肪酸氧化、乙醛酸循环、糖异生和GABA作用的增强,还可以诱导抗病品种糖酵解和莽草酸途径的增强。稻瘟病菌是研究植物病原菌重要的模式生物,在全基因组、转录组以及抗病基因定位与克隆^[10]等方面都对其有大量的研究,代谢组学方法也成功应用到稻瘟病菌与水稻互作的研究中,JONES等^[11]对稻瘟病菌侵染感病型与抗病型水稻叶片的代谢产物进行分析发现,感病型与抗病型水稻叶片差别最大的代谢产物为丙氨酸。水稻茎鞘中贮藏的可溶性糖等非结构碳水化合物（nonstructural carbohydrate,NSC）和氮素是水稻增产的重要物质^[12,13],抽穗前茎鞘中积累的NSC在后期被高效率转运到籽粒中是水稻产量提高的重要因素^[14],魏凤桐等^[15]研究表明,施氮后NSC积累不足和转移效率降低限制了千粒重和结实率的提高。稻瘟病菌侵染茎节后,叶片和茎中的营养物质不能运输到籽粒中^[16],影响水稻植株营养物质的积累、运转和分配^[17],导致水稻减产且无法补救^[18]。【本研究切入点】选用两个感病品种辽星1号和C418,其中辽星1号为高产优质常规稻品种,在辽宁省曾大面积种植,C418为通过“籼粳架桥”技术选育的粳型恢复系,因具有较高的配合力在全国范围内培育出较多强优势组合,随着稻瘟病菌小种变化等原因,辽星1号和C418抗病性逐渐下降。代谢组学是研究水稻响应生物胁迫和非生物胁迫的重要手段^[19],在稻瘟病方面的研究多集中在叶瘟与寄主之间的互作,而自然条件下穗颈瘟发生对茎节代谢产物的变化及其对产量的影响鲜见报道。【拟解决的关键问题】以自然条件下辽星1号和C418感染穗颈瘟的茎节为研究对象,考察产量性状并利用气相色谱-质谱（GC-MS）非靶向代谢组学方法检测茎节代谢产物,分析感病茎节与正常茎节之间的差异代谢产物种类,比较糖类物质、氨基酸类物质相对含量,从代谢物角度解析水稻品种感病和产量降低的原因,为感病机理研究提供理论依据。

1 材料与方法

1.1 供试品种

选用水稻感病品种辽星1号和C418为材料,种子来源于辽宁省水稻研究所。

1.2 样品采集

试验于2019年4—10月在辽宁省水稻研究所试验基地进行。辽星1号和C418生育期田间正常管理,无药剂防治。灌浆期间进行穗颈瘟调查,计算发病率。选择仅有穗颈瘟发生的植株进行标记,无任何病害的正常植株为对照（分别为LX1-CK和C418-CK）,因辽星1号和C418抽穗时期不一致,9月15日对辽星1号穗颈瘟发生的茎节（LX1-disease）和正常茎节进行取样,9月18日对C418穗颈瘟发生的茎节（C418-disease）和正常茎节进行取样,茎节取样后经液氮冷冻研磨成粉末,-80℃冰箱保存备用。两个样品均取样完成后进行代谢物检测。每个处理4次重复,每个重复20穗;另一部分成熟期取样进行产量及品质构成因素测定。

1.3 茎节代谢产物定量、定性分析

茎节代谢产物定量、定性测定委托具有资质的专业第三方检测机构上海鹿明生物科技有限公司完成。

1.3.1 仪器设备 代谢产物测定所需仪器包括,超声波清洗机（SB-5200DT）,旋涡振荡器（TYXH-I）、研磨仪（JXFSTPRP-24/32）、离心干燥器（LNG-T98）、高速冷冻离心机（TGL-16MS）、气浴恒温振荡器（THZ-82A）、真空干燥箱（DZF-6021）、气相色谱-质谱联用仪（7890B-5977B）和色谱柱（HP-5MS（30 m×0.25 mm×0.25 μm））。

1.3.2 样品处理 精密称取60 mg茎秆粉末样本,放入1.5 mL的离心管中,加入40 μL内标（L-2-氯-苯丙氨酸,0.3 mg·mL^-1,甲醇配置）和360 μL冷甲醇,在-20℃冰箱中放置2 min,冰水浴超声提取30 min,加入200 μL的氯仿,漩涡机中涡旋2 min,再加入400 μL的水,漩涡机中涡旋2 min,冰水浴超声提取30 min;-20℃静置30 min后低温离心10 min,取300 μL的上清液装入玻璃衍生瓶中,用离心浓缩干燥器挥干样本,向玻璃衍生小瓶中加入15 mg·mL^-1的甲氧胺盐酸盐吡啶溶液80 μL,涡旋振荡2 min后,于37℃振荡培养箱中放置90 min进行肟化反应。将样本取出后再加入80 μL BSTFA（含1%TMCS）衍生试剂、20 μL正己烷和11种内标（C8/C9/C10/C12/C14/C16,0.8 mg·mL^-1;C18/C20/C22/C24/C26,0.4 mg·mL^-1,均为氯仿配置）10 μL,涡旋振荡2 min后,于70℃反应60 min。取出样本后,在室温放置30 min,进行GC-MS代谢组学分析。每6个样品中插入1个质控（quality control,QC）样本,最后4个样品插入1个质控样本,评价整个实验过程中系统质谱平台的稳定性。

1.3.3 色谱条件 HP-5MS毛细管柱（30 m×0.25 mm×0.25 μm,Agilent J&W Scientific,Folsom,CA,USA）,载气为高纯氦气（纯度不小于99.999%）,流速1.0 mL·min^-1,进样口的温度为260℃。进样量为1 μL,不分流进样,溶剂延迟5 min。柱温箱的初始温度为60℃,以8℃·min^-1程序升温至125℃,5℃·min^-1升温至210℃,10℃·min^-1升温至270℃,20℃·min^-1升温至305℃保持5 min。

1.3.4 质谱条件 电子轰击离子源（EI）,离子源温度230℃,四级杆温度150℃,电子能量70 eV。扫描方式为全扫描模式（SCAN）,质量扫描范围:m/z 50-500。

1.3.5 数据分析 将GC-MS原始数据经软件转换,导入MS-DIAL软件进行峰识别,与公共数据库Fiehn匹配相似度实现化合物定性,每个物质在质谱中的响应信息经过转换得到丰度值即为相对定量值。

1.4 产量及品质性状测定

水稻成熟后取样,测定单穗产量性状、外观品质和碾磨品质,包括结实率、千粒重、粒长、粒宽、糙米率、精米率、整精米率和垩白率等。

1.5 统计分析

运用Excel软件进行产量性状的方差分析。代谢产物数据通过在线软件（http://www.metaboanalyst.ca/）进行主成分分析（principal components analyses,PCA）、偏最小二乘法判别分析（partial least-squares discriminant analysis,PLS-DA）和差异代谢产物等分析。

2 结果

2.1 辽星1号和C418穗颈瘟发生调查

灌浆期对辽星1号和C418穗颈瘟发病率进行调查,结果表明,C418的发病率和发病部位长度均大于辽星1号。辽星1号穗颈瘟发病率为57.23%,穗颈瘟发病部位长度介于1.10—2.35 cm。C418穗颈瘟发病率为82.94%,发病部位长度介于1.52—3.64 cm。

2.2 穗颈瘟发生对辽星1号和C418产量构成因素的影响

对辽星1号和C418产量构成因素进行测定,结果表明穗颈瘟发生降低了辽星1号和C418的结实率和千粒重。辽星1号穗颈瘟发生极显著（显著）降低了一次枝梗结实率、二次枝梗结实率和千粒重,分别降低了2.91%、5.78%和5.41%;一次枝梗的千粒重降低了2.95%,但差异不显著。C418穗颈瘟发生极显著（显著）降低了一次枝梗千粒重、二次枝梗千粒重和结实率,分别降低了5.93%、8.22%和7.44%,一次枝梗结实率降低了3.17%,但差异不显著（表1）。辽星1号和C418穗颈瘟发生的不同穗之间结实率和千粒重变化幅度较大,可能是由于穗颈瘟发生在不同时期造成的。

Table 1
表1
表1穗颈瘟发生对辽星1号和C418产量构成因素的影响
Table 1Effect of panicle neck blast on Liaoxing 1 and C418 yield components

品种 Cultivar	处理 Treatment	一次枝梗 Primary branch			二次枝梗 Secondary branch
		结实率 Seed setting rate (%)	千粒重 1000-grain weight (g)		结实率 Seed setting rate (%)	千粒重 1000-grain weight (g)
辽星1号 Liaoxing1	LX1-CK	95.05±4.05	25.39±0.74		89.59±8.91	22.35±0.79
	LX1-disease	92.28±7.45	24.64±4.05		84.42±17.52	21.15±3.81
	P值P-value	0.0016**	0.11		0.0004**	0.0218*
C418	C418-CK	87.11±5.11	27.54±0.88		81.03±7.04	23.68±1.53
	C418-disease	85.00±10.81	25.91±1.78		75.00±34.95	21.74±3.83
	P值P-value	0.0899	0.0037**		0.0116*	0.0136*

*:P<0.05;**;P<0.01。下同The same as below

新窗口打开|下载CSV

2.3 穗颈瘟发生对辽星1号和C418外观品质和碾磨品质的影响

对辽星1号和C418碾磨品质和外观品质进行测定,结果表明辽星1号穗颈瘟发生显著降低了一次枝梗和二次枝梗稻粒长度,分别降低了1.51%和1.54%,稻粒宽度略有增加,差异不显著。C418穗颈瘟发生显著降低了一次枝梗和二次枝梗稻粒宽度,分别降低3.53%和3.27%,稻粒长度差异不显著。穗颈瘟发生显著（极显著）降低了辽星1号和C418的二次枝梗稻粒的长宽比,分别降低了8.68%和4.83%。同时,穗颈瘟发生显著增加了辽星1号的一次枝梗垩白率,垩白率增加了54.08%（表2）。

Table 2
表2
表2穗颈瘟发生对辽星1号和C418外观品质的影响
Table 2Effect of panicle neck blast on appearance quality of Liaoxing 1 and C418

品种 Cultivar	部位 Part	处理 Treatment	粒长 Length (mm)	粒宽 Width (mm)	长宽比 Length-width ratio	垩白率 Chalky rice rate (%)
辽星1号Liaoxing1	一次枝梗 Primary branch	LX1-CK	7.30±0.03	3.16±0.01	2.31±0.01	5.03±0.30
		LX1-disease	7.19±0.02	3.18±0.01	2.27±0.01	7.75±1.10
		P值P-value	0.0116*	0.6178	0.0525	0.0136*
	二次枝梗 Secondary branch	C418-CK	7.15±0.04	3.04±0.02	2.19±0.01	19.23±1.60
		C418-disease	7.04±0.05	3.08±0.04	2.00±0.05	15.36±10.61
		P值P-value	0.0413*	0.4243	0.0002**	0.0976
C418	一次枝梗 Primary branch	LX1-CK	8.50±0.02	3.12±0.01	2.73±0.01	18.92±0.77
		LX1-disease	8.42±0.16	3.01±0.02	2.80±0.03	15.59±2.56
		P值P-value	0.2781	0.0016**	0.0513	0.1284
	二次枝梗 Secondary branch	C418-CK	8.31±0.03	3.06±0.01	2.07±0.01	29.05±1.22
		C418-disease	8.25±0.14	2.96±0.03	1.97±0.03	25.35±4.43
		P值P-value	0.4419	0.0104*	0.0115*	0.1404

新窗口打开|下载CSV

穗颈瘟发生降低了辽星1号和C418的糙米率、精米率和整精米率。糙米率和精米率与正常差异不显著,整精米率显著（极显著）降低,一次枝梗整精米率分别降低了7.93%和8.79%,二次枝梗整精米率分别降低了5.15%和9.74%（表3）。

Table 3
表3
表3穗颈瘟发生对辽星1号和C418碾磨品质影响
Table 3Effect of panicle neck blast on milling quality of Liaoxing 1 and C418 (%)

品种 Cultivar	处理 Treatment	一次枝梗 Primary branch				二次枝梗 Secondary branch
		糙米率 Brown rice	精米率 Milled rice	整精米率 Head milled rice		糙米率 Brown rice	精米率 Milled rice	整精米率 Head milled rice
辽星1号Liaoxing1	LX1-CK	81.26±0.30	71.19±2.25	69.99±2.21		80.55±3.00	69.81±1.84	67.22±2.25
	LX1-disease	80.03±4.54	69.07±2.08	64.44±12.41		79.16±1.63	68.38±6.05	63.76±4.21
	P值P-value	0.1964	0.0688	0.0179*		0.1797	0.5054	0.0061**
C418	C418-CK	78.44±0.95	66.47±9.88	62.96±13.91		76.75±3.02	64.88±1.68	63.06±6.15
	C418-disease	74.98±13.96	62.41±9.48	57.43±14.50		72.47±4.42	60.93±6.90	56.92±8.05
	P值P-value	0.1517	0.0741	0.0081**		0.0956	0.0941	0.0444*

新窗口打开|下载CSV

2.4 穗颈瘟发生对辽星1号和C418茎节代谢产物的影响

采用GC-MS对辽星1号和C418感病茎节和正常茎节代谢产物进行检测,均定性到358种代谢产物,辽星1号茎节感病后上调的代谢产物有263种,占检测到的代谢产物总数的73.46%;C418茎节感病后上调的代谢产物有243种,占检测到的代谢产物总数的67.88%。两组分析均上调的代谢产物有220种,占检测到的代谢产物总数的61.45%。

采用PCA分析来评估所有样本的整体分布和整个分析过程的稳定性。如图1-A所示,PCA分析的两个主成分解释了总方差信息的75.0%。所有的质控样品（QC）聚在一起,表明所有检测具有良好的分析稳定性和实验重现性。在PCA模型中,辽星1号和C418的感病茎节和正常茎节在第一个成分中被很好地分离,表明处理之间的代谢差异显著。

图1

新窗口打开|下载原图ZIP|生成PPT
图1穗颈瘟发生茎节与正常茎节之间代谢产物PCA和PLS-DA分析

A:所有样品PCA分析PCA analysis of all samples;B:辽星1号处理间 PLS-DA分析PLS-DA analysis of Liaoxing1;C:C418处理间的 PLS-DA分析PLS-DA analysis of C418
Fig. 1PCA and PLS-DA analysis of the metabolites between diseased stem nodes and normal stem nodes



对检测到的代谢产物进行PSL-DA分析,辽星1号和C418的感病茎节和正常茎节的代谢产物均沿着第一主成分清楚的分开,分别解释了总变异的78.9%（图1-B）和82.0%（图1-C）。辽星1号的模型累积解释率R²=0.9947,Q²=0.9879;C418模型累积解释率R²=0.99726,Q²=0.99063,数值均>0.5,表明模型的拟合准确性较好。结果表明,辽星1号和C418的感病茎节和正常茎节之间具有明显不同的代谢产物表型,穗颈瘟发生严重影响了茎节的代谢物组成。

2.5 感病茎节与正常茎节之间差异代谢产物分析

利用PLS-DA分析的变量权重值（VIP>1.0）进行感病茎节与正常茎节之间差异代谢产物分析,明确促使分离的代谢产物。与正常茎节相比,辽星1号感病茎节诱导61种代谢产物显著变化,其中49种代谢产物的相对含量显著上调;C418感病茎节诱导47种代谢产物显著变化,其中24种代谢产物的相对含量显著上调。两组处理共有的差异代谢产物30种,β-谷甾醇、二甲基尿酸、α-生育酚、正壬醇、香草酸和磷酸己酸在两组分析的VIP值均>2.0,表明这6种代谢产物在感病茎节和正常茎节的代谢产物分离中起到重要作用。

根据代谢产物相对含量的变化可以将30种代谢产物分为4组（图2）。第1组19种代谢物,包括高丝氨酸、别嘌呤醇、D-红鞘氨醇、阿拉伯糖醇、山梨醇、β-谷甾醇、半乳糖-6-磷酸、乙酰氨基葡萄糖、菊粉糖、羟基氨基甲酸酯、葡萄糖-6-磷酸、D-果糖-1,6-二磷酸、磷酸己糖酸、二甲基尿酸、2-单油酸、葡萄糖酸、尿囊酸、N-乙酰半乳糖胺和5-甲基尿苷。与正常茎节相比,辽星1号和C418穗颈瘟发生导致这19种代谢产物相对含量上调,分别上调了3.83—2 763.97和10.01—18 989.02倍。第2组9种代谢物,包括甲基α-呋喃硫醚、2,3-二羟基-2-丁烯二酸、亚甲基二磷酸盐、脱氢抗坏血酸、香草酸、N-乙酰-d-甘露糖胺、三十烷醇、α-生育酚和大豆苷元。这一组代谢产物具有两个特点,一个是与正常茎节相比,辽星1号和C418穗颈瘟发生导致这组代谢产物相对含量下调,分别下调了73.87%—96.29%和73.02%—99.68%;二是正常茎节中这组代谢产物辽星1号的相对含量大于C418的相对含量。第3组和第4组分别有一种代谢物,分别是1,5-脱水葡萄糖醇和正壬醇,与对照相比,辽星1号和C418感病茎节中1,5-脱水葡萄糖醇分别上调了6.91倍和下调了86.23%,正壬醇分别下调了80.03%和85.22%。由于正常茎节中C418的正壬醇相对含量大于辽星1号的正壬醇相对含量,所以将正壬醇与第2组分开,单独独立为一组。

图2

新窗口打开|下载原图ZIP|生成PPT
图2差异代谢产物层次聚类分析生成的热图

聚类分析采用质心法和欧氏距离法,红色矩形表示代谢物含量显著上调,蓝色矩形代表代谢物含量的显著下调。图3同
Fig. 2Hierarchical cluster analysis of differential metabolites resulting in thermal energy maps

Centroid method and euclidean distance method are used for cluster analysis. The red rectangle indicates that the metabolite content is significantly up-regulated and the blue rectangle indicates the significant down-regulation of the metabolite content. The same as Fig. 3

2.6 穗颈瘟发生对辽星1号和C418茎节糖类物质的影响

GC-MS共检测到糖类物质30种,从热图中可见（图3-A）,与正常茎节相比,辽星1号穗颈瘟发生上调的糖类物质有21种,占糖类物质总数的70%;C418穗颈瘟发生上调的糖类物质有22种,占糖类物质总数的73.33%。两个品种中共有的上调糖类物质19种,占糖类物质总数的63.33%。分别为阿拉伯糖、葡萄糖、异麦芽糖、纤维二糖、D-核糖、蜜二糖、三糖、菊粉糖、酮己糖、D-木糖、β-龙胆糖、槐糖、松三糖、D-果糖、D-甘露糖、D-岩藻糖、哌啶-α-d-果糖、葡庚糖和赤鲜糖。与对照相比,这19种糖类物质在辽星1号和C418感病茎节中分别上调了1.02—6.22和1.07—17.92倍。除赤藓糖和葡庚糖,其余17种代谢产物在C418感病茎节中上调的幅度更大。与对照相比,两个品种感病茎节中共有下调的代谢产物6种,包括左旋葡聚糖、棉子糖、D-塔格糖、D7-葡萄糖、海藻糖和6-脱氧葡萄糖,这6种糖类物质在辽星1号和C418感病茎节中分别下调了2.16%—21.80%和7.15%—99.77%。

图3

新窗口打开|下载原图ZIP|生成PPT
图3糖类物质和氨基酸类物质层次聚类分析生成的热图

A:糖类物质Carbohydrate metabolites;B:氨基酸类物质Amino acid metabolites
Fig. 3Thermal energy maps of hierarchical cluster analysis of carbohydrate metabolites and amino acid metabolites

2.7 穗颈瘟发生对辽星1号和C418茎节氨基酸类物质的影响

GC-MS共检测到氨基酸类物质38种,从热图中可见（图3-B）,与对照相比,辽星1号感病茎节中上调的氨基酸类物质有31种,占氨基酸类物质总数的81.58%;C418感病茎节中上调的氨基酸类物质有29种,占氨基酸类物质总数的76.32%。两个品种共有上调的氨基酸类物质26种,占氨基酸类物质总数的68.42%。上调的氨基酸有L-半胱氨酸甘氨酸、草氨酸、L-天冬氨酸、丙氨酸、β-丙氨酸、L-异亮氨酸、丝氨酸、L-苯丙氨酸、氰基丙氨酸、N-甲基丙氨酸、氧化谷氨酸、高丝氨酸、甘氨酸、L-缬氨酸、L-天冬酰胺、L-蛋氨酸、鸟氨酸、赖氨酸、邻琥珀酰高丝氨酸、L-谷氨酸、谷氨酸、L-酪氨酸、L-谷氨酰胺、L-脯氨酸、亮氨酸和苏氨酸。这些代谢物在辽星1号感病茎节中上调了1.05—7.98倍,在C418感病茎节中上调了1.10—13.12倍。两个品种感病茎节中共有的下调代谢产物4种,包括N-甘氨酰脯氨酸、D3-色氨酸、N-甲基谷氨酸和N-乙酰鸟氨酸,这4种氨基酸在辽星1号感病茎节中下调了8.96%—28.19%,在C418感病茎节中下调了2.78%—32.33%。

3 讨论

次生代谢产物在植物应对生物胁迫中起着重要防御作用^[20]。本研究发现,穗颈瘟发生诱导辽星1号和C418香草酸、N-乙酰-d-甘露糖胺、脱氢抗坏血酸、三十烷醇、α-生育酚和大豆苷元的相对含量显著下调,这6种物质均属于次生代谢产物范畴。生育酚和脱氢抗坏血酸作为抗氧化物质可以保护植物新陈代谢免受应激损伤^[21]。三十烷醇广泛存在水稻等植物的蜡质中^[22,23],能够促进碳氮代谢,加速茎中贮藏的有机物向籽粒运输^[24]。大豆苷元未见水稻方面的报道,但是豆科植物中常见的次级代谢产物^[25],可以提高豆科植物对病原菌的抗性。香草酸作为化感类物质在植物生长发育过程和抗逆中起到一定作用^[26,27]。本研究还发现,辽星1号正常茎节中这6种次生代谢产物的相对含量分别是C418的2.64、2.39、2.39、2.27、3.09和2.56倍,穗颈瘟发病率调查结果与这一结果相反,辽星1号发病率和发病程度均小于C418。推测这6种次生代谢产物的相对含量与水稻茎节感染穗颈瘟有一定关系,稻瘟病菌可能诱导水稻茎节下调这些次生代谢产物相对含量降低茎节对稻瘟病菌的防御反应。

禾谷类作物倒1节间主要发挥物质运输通道的功能^[28],抽穗后水稻茎鞘中70%左右的非结构性碳水化合物（NSC）通过倒1节转运到籽粒中,对最终产量的表观贡献率可达 24%—27%^[29,30]。本研究发现,穗颈瘟发生上调了辽星1号和C418茎节中70%和73.33%的糖类物质,其中阿拉伯糖上调了6.22%和7.45%,菊粉糖上调了5.68%和17.92%,葡萄糖上调了3.0和4.80倍,甘露糖上调了1.39和3.43倍,果糖上调了1.92和2.97倍。这些可溶性糖的上调表明水稻“源”向“库”输入的强度和“库”将糖转化为淀粉的能力降低,同时果糖的上调也预示着茎中蔗糖转化酶异常活跃^[31]。本研究还发现,C418穗颈瘟发生有17种糖类物质上调幅度大于辽星1号,并且,C418穗颈瘟发生二次枝梗结实率和千粒重下降幅度也大于辽星1号,分析原因,可能是感病茎节碳水化合积累,降低了茎节光合作用,茎秆运输到籽粒上的碳水化合物量减少,导致产量下降,尤其C418产量下降更多。

水稻籽粒的形成除了需要累积大量的碳水化合物外还需要大量的有机氮类代谢产物,茎鞘中的氮素对籽粒氮的贡献率达到30%以上^[32]。植物体内游离氨基酸直接或是间接对生物胁迫和水稻生长发育作出响应^[33,34]。本研究表明,穗颈瘟发生上调了辽星1号和C418茎秆中81.58%和76.32%的氨基酸类物质,破坏了氮素在营养器官和生殖器官的分配比例,降低了氮素向穗部的再运转量,导致产量的降低。有研究也表明,大量丙氨酸产生可能导致植物细胞死亡,促进稻瘟病菌侵染^[10],本研究结果与这一结论一致,稻瘟病菌侵染诱导了辽星1号和C418茎节中丙氨酸上调了3.34和3.74倍。

穗颈瘟发生后,辽星1号和C418二次枝梗的结实率和千粒重较一次枝梗下降显著,分析原因可能是强势粒在不同环境下存在遗传稳定性,而弱势粒灌浆受环境因素影响大^[35]。穗颈瘟对精米率和糙米率没有显著影响,但对整精米率影响较大,可能是一方面碳水化合物合成受阻易造成籽粒充实不良^[36],另一方面,穗颈瘟发生造成“源”输出不足,籽粒灌浆速率极慢,充实度差容重极轻,造成整精米率大幅度降低^[37]。

4 结论

穗颈瘟发生显著（极显著）影响了辽星1号和C418的二次枝梗结实率和千粒重,改变了茎节代谢产物组成,穗颈瘟可能通过下调脱氢抗坏血酸、α-生育酚、N-乙酰-d-甘露糖胺等次生代谢产物相对含量降低了水稻茎节对稻瘟病的防御反应。感病茎节通过积累大量的糖类物质和氨基酸类物质,导致碳氮代谢受阻,降低了其向籽粒转运,从而抑制了水稻产量。

参考文献 View Option 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子

[1] 刘国权, 孟昭河, 任艳军, 李春光, 刘永巍, 孟巧霞. 水稻抗稻瘟病研究进展与对策
中国农学通报, 2004,20(1):211-214.
URL [本文引用: 1]
稻瘟病是由稻瘟病菌引起的水稻真菌性病害。稻瘟病使水稻的主要病害之一，严重威胁着水稻生产，不但造成水稻减产10-30%，而且降低水稻品质。本文对水稻抗稻瘟病研究进展及对策进行分析和总结的基础上，提出全面防治稻瘟病的综合策略：首先选择抗稻瘟病较强的品种进行种植，其次运用水稻品种多样性防治稻瘟病技术控制稻瘟病的发生和流行，最后采用化学农药防治稻瘟病。
LIU G Q, MENG Z H, REN Y J, LI C G, LIU Y W, MENG Q X. Study advances and countermeasures on blast resistance of rice
Chinese Agricultural Science Bulletin, 2004,20(1):211-214. (in Chinese)
URL [本文引用: 1]
稻瘟病是由稻瘟病菌引起的水稻真菌性病害。稻瘟病使水稻的主要病害之一，严重威胁着水稻生产，不但造成水稻减产10-30%，而且降低水稻品质。本文对水稻抗稻瘟病研究进展及对策进行分析和总结的基础上，提出全面防治稻瘟病的综合策略：首先选择抗稻瘟病较强的品种进行种植，其次运用水稻品种多样性防治稻瘟病技术控制稻瘟病的发生和流行，最后采用化学农药防治稻瘟病。

[2] DWAN R A, TALBOT N J, EBBOLE D J, FARMAN M L, MITCHELL T K, ORBACH M J, THON M, KULKARNI R, XU J R, PAN H Q, et al. The genome sequence of the rice blast fungus Magnaporthe grisea
Nature, 2005,434(7036):980-986.
DOI:10.1038/nature03449URLPMID:15846337 [本文引用: 1]
Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation.

[3] 王加峰, 刘浩, 王慧, 陈志强. 水稻NBS-LRR类抗稻瘟病蛋白Pik-h的互作蛋白筛选
中国农业科学, 2016,49(3):482-490.
DOI:10.3864/j.issn.0578-1752.2016.03.007URL [本文引用: 1]
【目的】利用酵母双杂交系统，以组成抗稻瘟病基因Pik-h介导的抗病反应途径。【方法】以含抗稻瘟病基因Pik-h的近等基因系IRBL8为材料，取稻瘟病菌(Magnaporthe oryzae)GD0193接种12和24 h后的水稻叶片，等量混合后提取总RNA，按照2个紧密连锁且功能独立的Pikh-1和Pikh-2蛋白为诱饵，在水稻叶片中筛选与之互作的蛋白，以便深入研究抗病基因Pik-h的酵母双杂交试剂盒（Make Your Own “Mate&Plate” Library System）的要求构建水稻叶片靶标cDNA文库。利用快速重组克隆的方法构建pGBKT7-Pikh1和pGBKT7-Pikh2诱饵载体，并分别将它们转化至酵母菌株Y2H Gold，提取细胞总蛋白后利用Western blot检测Pikh1和Pikh2的表达情况，并对这2个诱饵载体自激活和毒性分析后进行酵母双杂交筛选。提取SD/-Ade/-Leu/-Trp/ -His/X-α-Gal筛选平板培养基上呈蓝色的酵母单克隆的质粒，将其分别与对应的诱饵载体共转化酵母菌株Y2H Gold，涂布于筛选平板培养基上进行互作的重复验证，将通过重复验证的质粒测序分析所得的序列比对水稻基因组数据库以确定目的基因，并对这些基因进行gene ontology（GO）注释分析以确定其分子功能、生物过程及细胞组成。【结果】靶标cDNA文库的库容量约为2.2×10⁶，插入片段长度均大于400 bp，表明水稻cDNA文库质量高。诱饵载体pGBKT7-Pikh1和pGBKT7-Pikh2均能在酵母细胞中正确表达出对应的Pikh1及Pikh2蛋白,无自激活活性而且对酵母无毒性作用，符合文库筛选的要求。利用含有诱饵载体的酵母菌株Y2H Gold与靶标文库菌株Y187结合（Mating）的方式筛选，获得13个与Pikh-1相互作用的蛋白、5个与Pikh-2相互作用的蛋白，其中有2个与Pikh-1及Pikh-2同时存在相互作用。这些蛋白包括4个在逆境响应或激素信号转导过程中起到重要作用的（辅）转录因子、3个信号蛋白、4个参与光合作用的叶绿体蛋白、1个含有U-BOX结构域的蛋白及4个未知功能蛋白。【结论】成功构建了适宜于研究抗病基因（R基因）介导反应途径的酵母双杂交cDNA文库，筛选出Pik-h的互作蛋白，为进一步研究Pik-h或其他抗稻瘟病基因介导的抗病机制打下了基础。
WANG J F, LIU H, WANG H, CHEN Z Q. Screening of putative proteins that are interacted with NBS-LRR protein Pik-h by the yeast two-hybrid system
Scientia Agricultura Sinica, 2016,49(3):482-490. (in Chinese)
DOI:10.3864/j.issn.0578-1752.2016.03.007URL [本文引用: 1]
【目的】利用酵母双杂交系统，以组成抗稻瘟病基因Pik-h介导的抗病反应途径。【方法】以含抗稻瘟病基因Pik-h的近等基因系IRBL8为材料，取稻瘟病菌(Magnaporthe oryzae)GD0193接种12和24 h后的水稻叶片，等量混合后提取总RNA，按照2个紧密连锁且功能独立的Pikh-1和Pikh-2蛋白为诱饵，在水稻叶片中筛选与之互作的蛋白，以便深入研究抗病基因Pik-h的酵母双杂交试剂盒（Make Your Own “Mate&Plate” Library System）的要求构建水稻叶片靶标cDNA文库。利用快速重组克隆的方法构建pGBKT7-Pikh1和pGBKT7-Pikh2诱饵载体，并分别将它们转化至酵母菌株Y2H Gold，提取细胞总蛋白后利用Western blot检测Pikh1和Pikh2的表达情况，并对这2个诱饵载体自激活和毒性分析后进行酵母双杂交筛选。提取SD/-Ade/-Leu/-Trp/ -His/X-α-Gal筛选平板培养基上呈蓝色的酵母单克隆的质粒，将其分别与对应的诱饵载体共转化酵母菌株Y2H Gold，涂布于筛选平板培养基上进行互作的重复验证，将通过重复验证的质粒测序分析所得的序列比对水稻基因组数据库以确定目的基因，并对这些基因进行gene ontology（GO）注释分析以确定其分子功能、生物过程及细胞组成。【结果】靶标cDNA文库的库容量约为2.2×10⁶，插入片段长度均大于400 bp，表明水稻cDNA文库质量高。诱饵载体pGBKT7-Pikh1和pGBKT7-Pikh2均能在酵母细胞中正确表达出对应的Pikh1及Pikh2蛋白,无自激活活性而且对酵母无毒性作用，符合文库筛选的要求。利用含有诱饵载体的酵母菌株Y2H Gold与靶标文库菌株Y187结合（Mating）的方式筛选，获得13个与Pikh-1相互作用的蛋白、5个与Pikh-2相互作用的蛋白，其中有2个与Pikh-1及Pikh-2同时存在相互作用。这些蛋白包括4个在逆境响应或激素信号转导过程中起到重要作用的（辅）转录因子、3个信号蛋白、4个参与光合作用的叶绿体蛋白、1个含有U-BOX结构域的蛋白及4个未知功能蛋白。【结论】成功构建了适宜于研究抗病基因（R基因）介导反应途径的酵母双杂交cDNA文库，筛选出Pik-h的互作蛋白，为进一步研究Pik-h或其他抗稻瘟病基因介导的抗病机制打下了基础。

[4] ZHUANG J Y, MA W B, WU J L, CHAI R Y, LU J, FAN Y Y, JIN M Z, LEUNG H, ZHENG K L. Mapping of leaf and neck blast resistance genes with resistance gene analog, RAPD and RFLP in rice
Euphytica, 2002,128:363-370.
DOI:10.1023/A:1021272710294URL [本文引用: 1]
An F₈ recombinant inbred population was constructed using a commercial indica rice variety Zhong 156 as the female parent and a semidwarf indica variety Gumei 2 with durable resistance to rice blast as the male parent. Zhong 156 is resistant to the fungus race ZC₁₅ at the seedling stage but susceptible to the same race at the flowering stage. Gumei 2 is resistant to ZC₁₅ at both stages. The blast resistance of 148 recombinant inbred lines was evaluated using the blast race ZC₁₅. Genetic analysis indicated that the resistance to leaf blast was controlled by three genes and the presence of resistant alleles at any loci would result in resistance. One of the three genes did not have effects at the flowering stage. Two genes, tentatively assigned as Pi24(t) and Pi25(t), were mapped onto chromosome 12 and 6,respectively, based on RGA (resistance gene analog), RFLP and RAPD markers. Pi24(t) conferred resistance to leaf blast only, and its resistance allele was from Zhong 156. Pi25(t) conferred resistance to both leaf and neck blast, and its resistance allele was from Gumei 2. In a natural infection test in a blast hot-spot, Pi25(t) exhibited high resistance to neck blast, while Pi24(t) showed little effect.

[5] FIEHN O. Metabolomics—the link between genotypes and phenotypes
Plant Molecular Biology, 2002,48:155-171.
DOI:10.1023/A:1013713905833URL [本文引用: 2]
Metabolites are the end products of cellular regulatory processes, and their levels can be regarded as the ultimate response of biological systems to genetic or environmental changes. In parallel to the terms `transcriptome' and `proteome', the set of metabolites synthesized by a biological system constitute its `metabolome'. Yet, unlike other functional genomics approaches, the unbiased simultaneous identification and quantification of plant metabolomes has been largely neglected. Until recently, most analyses were restricted to profiling selected classes of compounds, or to fingerprinting metabolic changes without sufficient analytical resolution to determine metabolite levels and identities individually. As a prerequisite for metabolomic analysis, careful consideration of the methods employed for tissue extraction, sample preparation, data acquisition, and data mining must be taken. In this review, the differences among metabolite target analysis, metabolite profiling, and metabolic fingerprinting are clarified, and terms are defined. Current approaches are examined, and potential applications are summarized with a special emphasis on data mining and mathematical modelling of metabolism.

[6] 罗杰, 漆小泉. 水稻产量、抗逆性状代谢组研究
科技创新导报, 2016(1):166.
[本文引用: 1]

LUO J, QI X Q. Metabolome-based analysis of yield and stress resistant traits in rice
.Science and Technology Innovation Herald, 2016(1):166. (in Chinese)
[本文引用: 1]

[7] ISHIKAWA T, TAKAHARA K, HIRABAYASHI T, MATSUMURA H, FUJISAWA S, TERAUCHI R, UCHIMIYA H, KAWAI- YAMADA M. Metabolome analysis of response to oxidative stress in rice suspension cells overexpressing cell death suppressor bax inhibitor-1
Plant and Cell Physiology, 2010,51(1):9-20.
DOI:10.1093/pcp/pcp162URLPMID:19919949 [本文引用: 1]
Bax inhibitor-1 (BI-1) is a cell death suppression factor widely conserved in higher plants and animals. Overexpression of Arabidopsis BI-1 (AtBI-1) in plants confers tolerance to various cell death-inducible stresses. However, apart from the cell death-suppressing activity, little is known about the physiological roles of BI-1-overexpressing plants. In this study, we evaluated the effects of AtBI-1 overexpression on the rice metabolome in response to oxidative stress. AtBI-1-overexpressing rice cells in suspension displayed enhanced tolerance to menadione-induced oxidative stress compared with vector control cells, whereas AtBI-1 overexpression did not influence the increase of intracellular H(2)O(2) concentration or inhibition of oxidative stress-sensitive aconitase activity. Capillary electrophoresis-mass spectrometry (CE-MS)-based metabolome analysis revealed dynamic metabolic changes in oxidatively stressed rice cells, e.g. depletion of the central metabolic pathway, imbalance of the redox state and energy charge, and accumulation of amino acids. Furthermore, comparative metabolome analysis demonstrated that AtBI-1 overexpression did not affect primary metabolism in rice cells under normal growth conditions but significantly altered metabolite composition within several distinct pathways under cell death-inducible oxidative stress. The AtBI-1-mediated metabolic alteration included recovery of the redox state and energy charge, which are known as important factors for metabolic defense against oxidative stress. These observations suggest that although AtBI-1 does not affect rice metabolism directly, its cell death suppression activity leads to enhanced capacity to acclimate oxidative stress.

[8] SANA T R, FISCHER S, WOHLGEMUTH G, KATREKAR A, JUNG K H, RONALD P C, FIEHN O. Metabolomic and transcriptomic analysis of the rice response to the bacterial blight pathogen Xanthomonas oryzae pv. oryzae
Metabolomics, 2010,6(3):451-465.
DOI:10.1007/s11306-010-0218-7URL [本文引用: 1]
Bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv. oryzae (Xoo), gives rise to devastating crop losses in rice. Disease resistant rice cultivars are the most economical way to combat the disease. The TP309 cultivar is susceptible to infection by Xoo strain PXO99. A transgenic variety, TP309_Xa21, expresses the pattern recognition receptor Xa21, and is resistant. PXO99△raxST, a strain lacking the raxST gene, is able to overcome Xa21-mediated immunity. We used a single extraction solvent to demonstrate comprehensive metabolomics and transcriptomics profiling under sample limited conditions, and analyze the molecular responses of two rice lines challenged with either PXO99 or PXO99△raxST. LC–TOF raw data file filtering resulted in better within group reproducibility of replicate samples for statistical analyses. Accurate mass match compound identification with molecular formula generation (MFG) ranking of 355 masses was achieved with the METLIN database. GC–TOF analysis yielded an additional 441 compounds after BinBase database processing, of which 154 were structurally identified by retention index/MS library matching. Multivariate statistics revealed that the susceptible and resistant genotypes possess distinct profiles. Although few mRNA and metabolite differences were detected in PXO99 challenged TP309 compared to mock, many differential changes occurred in the Xa21-mediated response to PXO99 and PXO99△raxST. Acetophenone, xanthophylls, fatty acids, alkaloids, glutathione, carbohydrate and lipid biosynthetic pathways were affected. Significant transcriptional induction of several pathogenesis related genes in Xa21 challenged strains, as well as differential changes to GAD, PAL, ICL1 and Glutathione-S-transferase transcripts indicated limited correlation with metabolite changes under single time point global profiling conditions.

[9] PENG L, ZHAO Y, WANG H Y, ZHANG J J, SONG C P, SHANGGUAN X X, ZHU L L, HE G C. Comparative metabolomics of the interaction between rice and the brown planthopper
Metabolomics, 2016,12(8):132.
DOI:10.1007/s11306-016-1077-7URL [本文引用: 1]

[10] 王芳权, 陈智慧, 许扬, 王军, 李文奇, 范方军, 陈丽琴, 陶亚军, 仲维功, 杨杰. 水稻广谱抗稻瘟病基因PigmR功能标记的开发及应用
中国农业科学, 2019,52(6):955-967.
DOI:10.3864/j.issn.0578-1752.2019.06.001URL [本文引用: 2]
【目的】稻瘟病是世界上最严重的水稻病害之一。通过功能标记的开发,为加快广谱持久抗稻瘟病基因PigmR在水稻育种中的应用提供依据。【方法】利用Snapgene 2.3.2软件分析PigmR的碱基变异特征,用Oligo 7设计特异功能标记。为了避免因PCR扩增失败引起的假阴性,设计扩增内参基因Actin1的引物作为参照,对功能标记进行优化。用功能标记对水稻亲本材料、丽江新团黑谷的单基因系材料、育种中间材料和南粳53045/谷梅4号BC₁F₃群体株系进行鉴定。稻瘟病鉴定的供试菌株为江苏省稻瘟病代表菌株（2018-4、2018-65、2018-102、2018-222和2018-241）的混合菌。将供试菌株移植RCA培养基上,25℃培养7 d,用黑光灯照射72 h,待稻瘟病菌产生孢子后,再用无菌水洗下,配成10×10倍显微镜下每视野30—40个孢子的悬浮液。于水稻抽穗前3—4 d注射混合菌株,每穗注射1 mL菌液,做好标记。在水稻灌浆饱满后进行抗性调查。【结果】根据PigmR与PigmS、Pigm-R4序列比对及差异分析,设计了8对分子标记引物。通过分子检测,筛选获得的PigmR功能标记GMR-3,能特异扩增来源于谷梅4号的PigmR,获得98 bp产物,而不携带PigmR扩增不出产物。以不同浓度配比优化GMR-3与内参基因Actin1检测引物Actin1-1,发现以0.4 μmol·L ^-1 GMR-3与0.1 μmol·L ^-1 Actin1-1组合浓度扩增出PigmR和Actin1特征条带的效率相当,效果最优,将该标记命名为GMRA。用该标记扩增水稻样品,携带PigmR的样品能扩增出146和98 bp条带,不携带PigmR的样品仅能扩增出146 bp条带。利用GMRA标记检测229份水稻材料,只有谷梅4号能扩增出146和98 bp条带,其他籼稻和粳稻均只能扩增出146 bp条带。进一步对29份丽江新团黑谷的单基因系材料进行检测发现,GMRA标记能有效区分PigmR与Pi9、Piz、Piz-t等同源性较高的基因,有很好的特异性。利用GMRA标记,从240份育种中间材料中筛选到3份携带PigmR的材料,可作为该基因的供体材料。利用该标记进行分子标记辅助选择,将PigmR通过回交转育到优质食味粳稻南粳53045。对南粳53045/谷梅4号BC₁F₃群体单株进行分子标记检测及稻瘟病人工接种鉴定,发现携带PigmR的单株均表现为抗或中抗,不携带PigmR的单株均表现为高感,表明导入PigmR能显著改良南粳53045的穗颈瘟抗性。【结论】PigmR的功能标记能有效用于抗稻瘟病遗传改良和资源筛选。
WANG F Q, CHEN Z H, XU Y, WANG J, LI W Q, FANG F J, CHEN L Q, TAO Y J, ZHONG W G, YANG J. Development and application of the functional marker for the broad-spectrum blast resistance gene PigmR in rice
Scientia Agricultura Sinica, 2019,52(6):955-967. (in Chinese)
DOI:10.3864/j.issn.0578-1752.2019.06.001URL [本文引用: 2]
【目的】稻瘟病是世界上最严重的水稻病害之一。通过功能标记的开发,为加快广谱持久抗稻瘟病基因PigmR在水稻育种中的应用提供依据。【方法】利用Snapgene 2.3.2软件分析PigmR的碱基变异特征,用Oligo 7设计特异功能标记。为了避免因PCR扩增失败引起的假阴性,设计扩增内参基因Actin1的引物作为参照,对功能标记进行优化。用功能标记对水稻亲本材料、丽江新团黑谷的单基因系材料、育种中间材料和南粳53045/谷梅4号BC₁F₃群体株系进行鉴定。稻瘟病鉴定的供试菌株为江苏省稻瘟病代表菌株（2018-4、2018-65、2018-102、2018-222和2018-241）的混合菌。将供试菌株移植RCA培养基上,25℃培养7 d,用黑光灯照射72 h,待稻瘟病菌产生孢子后,再用无菌水洗下,配成10×10倍显微镜下每视野30—40个孢子的悬浮液。于水稻抽穗前3—4 d注射混合菌株,每穗注射1 mL菌液,做好标记。在水稻灌浆饱满后进行抗性调查。【结果】根据PigmR与PigmS、Pigm-R4序列比对及差异分析,设计了8对分子标记引物。通过分子检测,筛选获得的PigmR功能标记GMR-3,能特异扩增来源于谷梅4号的PigmR,获得98 bp产物,而不携带PigmR扩增不出产物。以不同浓度配比优化GMR-3与内参基因Actin1检测引物Actin1-1,发现以0.4 μmol·L ^-1 GMR-3与0.1 μmol·L ^-1 Actin1-1组合浓度扩增出PigmR和Actin1特征条带的效率相当,效果最优,将该标记命名为GMRA。用该标记扩增水稻样品,携带PigmR的样品能扩增出146和98 bp条带,不携带PigmR的样品仅能扩增出146 bp条带。利用GMRA标记检测229份水稻材料,只有谷梅4号能扩增出146和98 bp条带,其他籼稻和粳稻均只能扩增出146 bp条带。进一步对29份丽江新团黑谷的单基因系材料进行检测发现,GMRA标记能有效区分PigmR与Pi9、Piz、Piz-t等同源性较高的基因,有很好的特异性。利用GMRA标记,从240份育种中间材料中筛选到3份携带PigmR的材料,可作为该基因的供体材料。利用该标记进行分子标记辅助选择,将PigmR通过回交转育到优质食味粳稻南粳53045。对南粳53045/谷梅4号BC₁F₃群体单株进行分子标记检测及稻瘟病人工接种鉴定,发现携带PigmR的单株均表现为抗或中抗,不携带PigmR的单株均表现为高感,表明导入PigmR能显著改良南粳53045的穗颈瘟抗性。【结论】PigmR的功能标记能有效用于抗稻瘟病遗传改良和资源筛选。

[11] JONES O A H, MAGUIRE M L, GRIFFIN J L, JUNG Y H, SHIBATO J, RAKEAL R, AGRAWAL G K, JWA N S. Using metabolic profiling to assess plantpathogen interactions: An example using rice (Oryza sativa) and the blast pathogen Magnaporthe grisea
European Journal of Plant Pathology, 2010,129(4):539-554.
[本文引用: 1]

[12] 潘俊峰, 王博, 崔克辉, 黄见良, 聂立孝. 氮肥对水稻节间和叶鞘非结构性碳水化合物积累转运特征的影响
中国水稻科学, 2016,30(3):273-282.
[本文引用: 1]

PAN J F, WANG B, CUI K H, HUANG J L, NIE L X. Effects of nitrogen application on accumulation and translocation of nonstructural carbohydrates in internodes and sheaths of rice
Chinese Journal of Rice Science, 2016,30(3):273-282. (in Chinese)
[本文引用: 1]

[13] 殷春渊, 赵全志, 刘贺梅, 王书玉, 薛应征, 杨海霞, 张华珍. 水稻籽粒非结构性碳水化合物及充实的氮素调控
中国农学通报, 2011,27(30):6-11.
[本文引用: 1]

YIN C Y, ZHAO Q Z, LIU H M, WANG S Y, XUE Y Z, YANG H X, ZHANG H Z. The nitrogen regulation on the non-carbohydrate and grain-plumpness in rice grain
Chinese Agricultural Science Bulletin, 2011,27(30):6-11. (in Chinese)
[本文引用: 1]

[14] YANG J, PENG S, ZHANG Z, WANG Z, VISPERAS R M, ZHU Q. Grain and dry matter yields and partitioning of assimilates in japonica/indica hybrid rice
Crop Science, 2002,42(3):766-772.
[本文引用: 1]

[15] 魏凤桐, 陶洪斌, 王璞. 旱稻297非结构性碳水化合物的生产与产量构成因子的关系
作物学报, 2010,36(12):2135-2142.
[本文引用: 1]

WEI F T, TAO H B, WANG P. Relationship of non-structure carbohydrate production and yield components of aerobic rice, Handao 297
Acta Agronomica Sinica, 2010,36(12):2135-2142. (in Chinese)
[本文引用: 1]

[16] SHIM H S, HONG S J, YEH W H, HAN S S, SUNG J M. Damage analysis of rice panicle blast on disease occurrence time and severity
The Plant Pathology Journal, 2005,21(2):87-92.
[本文引用: 1]

[17] KATSANTONIS D, KOUTROUBAS S D, NTANOS D A, LUPOTTO E. Effect of blast disease on nitrogen accumulation and remobilization to rice grain
Journal of Plant Pathology, 2008,90(2):263-272.
[本文引用: 1]

[18] LIU Q, YANG J Y, ZHANG S H, ZHAO J L, FENG A Q, YANG T F, WANG X F, MAO X X, DONG J F, ZHU X Y, LENG H, LEACH J E, LIU B. OsGF14b positively regulates panicle blast resistance but negatively regulates leaf blast resistance in rice
Molecular Plant-Microbe Interactions, 2016,29(1):46-56.
[本文引用: 1]

[19] 雷刚, 黄英金. 代谢组学在水稻研究中的应用进展
中国农业科技导报, 2017,19(7):27-35.
[本文引用: 1]

LEI G, HUANG Y J. Application progress of metabolomics in rice research
Journal of Agricultural Science and Technology, 2017,19(7):27-35. (in Chinese)
[本文引用: 1]

[20] 阎秀峰, 王洋, 李一蒙. 植物次生代谢及其与环境的关系
生态学报, 2007,27(6):2554-2562.
[本文引用: 1]

YAN X F, WANG Y, LI Y M. Plant secondary metabolism and its response to environment
Acta Ecologica Sinica, 2007,27(6):2554-2562. (in Chinese)
[本文引用: 1]

[21] SADIQ M, AKRAM N A, ASHRAF M, AL-QURAINY F, AHMAD P. Alpha-tocopherol-induced regulation of growth and metabolism in plants under non-stress and stress conditions
Journal of Plant Growth Regulation, 2019,38:1325-1340.
[本文引用: 1]

[22] 李芳芳, 杨娜, 钱猛, 甘立军. 生长素参与三十烷醇诱导的拟南芥侧根发育
南京农业大学学报, 2018,41(3):473-480.
[本文引用: 1]

LI F F, YANG N, QIAN M, GAN L J. Auxin is involved in triacontanol-induced lateral root development in Arabidopsis thaliana
Journal of Nanjing Agricultural University, 2018,41(3):473-480. (in Chinese)
[本文引用: 1]

[23] NAEEM M, KHAN M M A, MOINUDDIN . Triacontanol: A potent plant growth regulator in agriculture
Journal of Plant Interactions, 2012,7(2):129-142.
[本文引用: 1]

[24] 崔睿, 刘喆, 阚侃, 田媛, 崔宝玉. 三十烷醇的生理作用及研究进展
黑龙江科学, 2011,2(6):33-36.
[本文引用: 1]

CUI R, LIU Z, KAN K, TIAN Y, CUI B Y. Research development of triacontanol physiological effects
Heilongjiang Science, 2011,2(6):33-36. (in Chinese)
[本文引用: 1]

[25] 张卓, 刘振库, 黄卓, 马建, 姚丹, 曲静, 王丕武. 大豆查尔酮还原酶基因(Gmchr4) 的克隆与功能鉴定
中国油料作物学报, 2014,36(6):720-727.
[本文引用: 1]

ZHANG Z, LIU Z K, HUANG Z, MA J, YAO D, QU J, WANG P W. Isolation and functional characterization of soybean chalcone reductase gene Gmchr4
Chinese Journal of Oil Crop Sciences, 2014,36(6):720-727. (in Chinese)
[本文引用: 1]

[26] 孙小霞, 王海斌, 何海斌, 陆锦池, 林文雄. 田间旱育条件下不同化感潜力水稻根际土壤酚酸类和萜类物质分析
中国生态农业学报, 2014,22(7):806-812.
[本文引用: 1]

SUN X X, WANG H B, HE H B, LU J C, LIN W X. Analysis of phenolic acids and terpenoids in rhizosphere soils of different allelopathic rice varieties under dry field conditions
Chinese Journal of Eco-Agriculture, 2014,22(7):806-812. (in Chinese)
[本文引用: 1]

[27] 李青, 张名位, 张瑞芬, 魏振承, 邓媛元, 唐小俊, 张业辉, 李武, 马永轩. 5种籼稻品种谷壳中游离态和结合态酚类物质含量及其抗氧化活性比较
中国农业科学, 2012,45(6):1150-1158.
[本文引用: 1]

LI Q, ZHANG M W, ZHANG R F, WEI Z C, DENG Y Y, TANG X J, ZHANG Y H, LI W, MA Y X. Free and bound phenolic contents and antioxidant activity of five varieties of indica rice husk
Scientia Agricultura Sinica, 2012,45(6):1150-1158. (in Chinese)
[本文引用: 1]

[28] LIU G L, MEI H W, YU X Q, ZOU G H, LIU H Y, HU S P, LI M S, WU J H, CHEN L, LUO L J. QTL analysis of panicle neck diameter, a trait highly correlated with panicle size, under well-watered and drought conditions in rice (Oryza sativa L.)
Plant Science, 2008,174(1):71-77.
[本文引用: 1]

[29] PAN J F, CUI K H, WEI D, HUANG J L, XIANG J, NIE L X. Relationships of non-structural carbohydrates accumulation and translocation with yield formation in rice recombinant inbred lines under two nitrogen levels
Physiologia Plantarum, 2011,141(4):321-331.
DOI:10.1111/j.1399-3054.2010.01441.xURLPMID:21175644 [本文引用: 1]
Stem non-structural carbohydrates (NSCs) and its relationship with yield formation was investigated under low nitrogen (LN) and normal nitrogen (NN) treatments, using 46 recombinant inbred lines from Zhenshan 97 x Minghui 63 (Oryza sativa). Apparent contribution of transferred NSC to grain yield (AC(NSC) ) ranged from approximately 1 to 28% under LN and from 1 to 15% under NN. Concentration and total mass of NSC in stem (TM(NSC) ) at heading, apparent transferred mass of NSC (ATM(NSC) ) and AC(NSC) were larger under LN compared with NN. However, there was no significant difference in the apparent ratio of transferred NSC from stems to grain (AR(NSC) ). ATM(NSC) was positively correlated with grain yield, 1000-grain weight and AC(NSC) under both nitrogen levels, whereas AR(NSC) was highly correlated with harvest index and AC(NSC) . Leaf area contributed more strongly to grain yield compared with ATM(NSC) under both LN and NN. ATM(NSC) showed larger direct effects on grain yield under LN compared with NN. TM(NSC) at heading, small vascular bundles (SVBs) and spikelets per m(2) under LN had positive direct effects on ATM(NSC) . SVB and spikelets per m(2) under LN had larger and positive direct effects, and large vascular bundles had negative direct effects on AR(NSC) . TM(NSC) at heading and SVB under LN had positive direct effects on AC(NSC) . In brief, LN supply increased stem NSC accumulation and translocation to developing grain. Components of the source-sink-flow system showed different effects on NSC translocation and contribution to yield formation, depending on genotype and nitrogen level.

[30] 潘俊峰, 李国辉, 崔克辉. 水稻茎鞘非结构性碳水化合物再分配及其在稳产和抗逆中的作用
中国水稻科学, 2014,28(4):335-342.
[本文引用: 1]

PAN J F, LI G H, CUI K H. Re-partitioning of non-structural carbohydrates in rice stems and their roles in yield stability and stress tolerance
Chinese Journal of Rice Science, 2014,28(4):335-342. (in Chinese)
[本文引用: 1]

[31] LINGLE S E. Sugar metabolism during growth and development in sugarcane internode
Crop Science, 1999,39(2):480-486.
[本文引用: 1]

[32] 沈成国. 植物衰老生理与分子生物学. 北京: 中国农业出版社, 2001.
[本文引用: 1]

SHEN C G. Plant Senescence Physiology and Molecular Biology. Beijing: China Agriculture Press, 2001. (in Chinese)
[本文引用: 1]

[33] 张木, 唐拴虎, 张发宝, 黄旭, 黄巧义, 逄玉万, 易琼. 硒对水稻碳氮代谢及产量的影响
中国土壤与肥料, 2016(5):79-84.
[本文引用: 1]

ZHANG M, TANG S H, ZHANG F B, HUANG X, HUANG Q Y, PANG Y W, YI Q. Effects of selenium on carbon-nitrogen metabolism and yield of rice
.Soil and Fertilizer Sciences in China, 2016(5):79-84. (in Chinese)
[本文引用: 1]

[34] ZHANG P, FU J, HU L. Effects of alkali stress on growth, free amino acids and carbohydrates metabolism in Kentucky bluegrass (Poa pratensis)
Ecotoxicology, 2012,21:1911-1918.
DOI:10.1007/s10646-012-0924-1URL [本文引用: 1]

[35] YANG J C, ZHANG J H. Grain-filling problem in ‘super’ rice
Journal of Experimental Botany, 2010,61(1):1-5.
DOI:10.1093/jxb/erp348URLPMID:19959608 [本文引用: 1]
Modern rice (Oryza sativa L.) cultivars, especially the newly bred 'super' rice, have numerous spikelets on a panicle with a large yield capacity. However, these cultivars often fail to achieve their high yield potential due to poor grain-filling of later-flowering inferior spikelets (in contrast to the earlier-flowering superior spikelets). Conventional thinking to explain the poor grain-filling is the consequence of carbon limitation. Recent studies, however, have shown that carbohydrate supply should not be the major problem because they have adequate sucrose at their initial grain-filling stage. The low activities of key enzymes in carbon metabolism may contribute to the poor grain-filling. Proper field practices, such as moderate soil drying during mid- and late grain-filling stages, could solve some problems in poor grain-filling. Further studies are needed by molecular approaches to investigate the signal transport, the hormonal action, the gene expressions, and the biochemical processes in inferior spikelets.

[36] 胡钧铭, 江立庚. 籼型稻米整精米率影响因子研究进展
粮食食品科技, 2007,15(3):4-6.
[本文引用: 1]

HU J M, JIANG L G. The progress of analysis of the influence factors on the head rice rate of Indica rice
Science and Technology of Cereals, Oils and Foods, 2007,15(3):4-6. (in Chinese)
[本文引用: 1]

[37] 徐富贤, 熊洪, 朱永川, 谢戎, 王贵雄. 川东南高温伏旱区杂交中稻超稀栽培对稻米整精米率的影响与组合间库源结构的关系
植物生态学报, 2005,29(5):829-835.
[本文引用: 1]

XU F X, XIONG H, ZHU Y C, XIE R, WANG G X. The effects of cultivation density on the percent of head milled rice and source to sink ratios of mid-season hybrid rice in eastern and southern Sichuan Province
Acta Phytoecologica Sinica, 2005,29(5):829-835. (in Chinese)
[本文引用: 1]