高温干旱下缩节胺通过调节碳和氨基酸代谢提高Bt棉杀虫蛋白含量的生理机制

doi:10.3864/j.issn.0578-1752.2023.08.004

摘要/Abstract

摘要：

【目的】探讨高温干旱胁迫下缩节胺（mepiquat chloride，DPC）调控Bt（Bacillus thuringiensis）棉杀虫蛋白含量的生理机制，为高抗虫性Bt棉品种选育及高产高效栽培提供理论依据。【方法】2020—2021年以转Bt抗虫基因抗虫棉品种泗抗3号为材料，采用盆栽法，在人工气候室进行高温干旱胁迫，胁迫开始后立即用20 mg·L^-1 DPC和清水（对照）喷施。7 d后测定铃壳杀虫蛋白含量、α-酮戊二酸含量、丙酮酸含量以及谷氨酸合酶活性、谷氨酸草酰乙酸转氨酶活性、可溶性蛋白含量、游离氨基酸含量。并进行转录组测序，利用DESeq进行差异基因分析，通过GO富集和KEGG Pathway数据库注释参与DPC调节杀虫蛋白含量的差异表达基因。【结果】与清水对照相比，DPC可显著提高高温干旱条件下Bt棉铃壳中杀虫蛋白含量，提高幅度达4.7%—11.9%。在碳代谢方面，α-酮戊二酸含量、丙酮酸含量提高46%—57%和25%—29%；在氨基酸代谢方面，谷氨酸合酶活性、谷氨酸草酰乙酸转氨酶活性、可溶性蛋白含量、游离氨基酸含量分别提高32%—44%、30%—40%、28%和22%—27%。转录组分析结果表明，DPC处理后上调基因7 542条，下调基因10 449条。GO和KEGG结果显示，差异基因主要涉及氨基酸代谢、碳代谢等生物过程。其中编码6-磷酸果糖激酶和丙酮酸激酶、谷氨酸丙酮酸转氨酶、丙酮酸脱氢酶、柠檬酸合酶、异柠檬酸脱氢酶和α-酮戊二酸脱氢酶、谷氨酸合酶、吡咯啉-5-羧酸脱氢酶、谷氨酸草酰乙酸转氨酶、N-乙酰谷氨酸合成酶、乙酰鸟氨酸脱乙酰酶基因表达显著上调。【结论】高温干旱下，DPC通过调节碳、氨基酸代谢增加丙氨酸、α-酮戊二酸含量，提高天冬氨酸、谷氨酸、丙酮酸和精氨酸合成能力，进而提高Bt棉杀虫蛋白含量。

关键词: Bt棉, 缩节胺, 杀虫蛋白, 氨基酸代谢, 碳代谢

Abstract:

【Objective】The effects of mepiquat chloride (DPC) on the insecticidal protein contents in Bt (Bacillus thuringiensis) cotton shell under high temperature and drought stress were investigated to provide a theoretical reference for the Bt cotton breeding as well as high-yield and high-efficiency cotton cultivation.【Method】The study was undertaken on the Bt cotton cultivar Sikang 3 during the 2020 and 2021 growing seasons at the Yangzhou University Farm, Yangzhou, China. The potted cotton plants were exposed to high temperature and drought stress, and 20 mg·L^-1DPC and water (CK) were sprayed to cotton plants. Seven days after treatment, the insecticidal protein content, α-ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, free amino acid content in boll shell were analyzed, and the transcriptome sequencing was performed. DESeq was used for differential gene analysis. The GO and KEGG pathway databases were used to analyze the differentially expressed genes involved in regulating the insecticidal protein content through DPC.【Result】Compared with the water treatment (CK), the insecticidal protein contents under DPC treatment increased by 4.7%-11.9%. In terms of carbon metabolism, the contents of α-ketoglutarate and pyruvic acid were increased by 46%-57% and 25%-29%, respectively. In terms of amino acid metabolism, the activities of glutamate synthase and glutamic oxaloacetic transaminase, and the contents of soluble protein and free amino acid were increased by 32%-44%, 30%-40%, and 28%, 22%-27%, respectively. The transcriptome analysis revealed that there were 7 542 upregulation genes and 10 449 downregulation genes for DPC vs water. The GO and KEGG analysis showed that the differentially expressed genes were mainly involved in biological process such as amino acid metabolism and carbon metabolism. The genes coding 6-phosphofructokinase, pyruvate kinase, glutamic pyruvate transaminase, pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, glutamate synthase, 1-pyrroline-5-carboxylate dehydrogenase, glutamic oxaloacetic transaminase, amino-acid N-acetyltransferase, and acetylornithine deacetylase were all significantly up-regulated. 【Conclusion】 Under the stress of high temperature and drought, the DPC treatment increased the contents of α-ketoglutarate and pyruvic acid, and improved the synthesis ability of aspartic acid, glutamic acid, pyruvate and arginine, then enhanced the insecticidal protein contents in boll shell by regulating the carbon and amino acid metabolism.

Key words: Bt cotton, mepiquat chloride, insecticidal protein, amino acid metabolism, carbon metabolism

邢羽桐, 滕永康, 吴天凡, 刘媛媛, 陈源, 陈媛, 陈德华, 张祥. 高温干旱下缩节胺通过调节碳和氨基酸代谢提高Bt棉杀虫蛋白含量的生理机制[J]. 中国农业科学, 2023, 56(8): 1471-1483.

XING YuTong, TENG YongKang, WU TianFan, LIU YuanYuan, CHEN Yuan, CHEN Yuan, CHEN DeHua, ZHANG Xiang. Mepiquat Chloride Increases the Cry1Ac Protein Content Through Regulating Carbon and Amino Acid Metabolism of Bt Cotton Under High Temperature and Drought Stress[J]. Scientia Agricultura Sinica, 2023, 56(8): 1471-1483.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.08.004

https://www.chinaagrisci.com/CN/Y2023/V56/I8/1471

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高温干旱下DPC处理 vs 清水处理铃壳中差异表达基因的GO富集"

GO分类 GO classification	上调词条 Up term	差异基因数 Number of differential genes	下调词条 Down term	差异基因数 Number of differential genes
生物过程 Biological process	ATP水解耦合质子传输 ATP hydrolysis coupled proton transport	36	对几丁质的反应 Response to chitin	188
	三羧酸循环 Tricarboxylic acid cycle	41	植物型次生细胞壁生物合成 Plant-type secondary cell wall biogenesis	85
	对乙烯的反应Response to ethylene	97	防御反应的调控Regulation of defense response	59
	乙烯激活信号通路的负调控 Negative regulation of ethylene-activated signaling pathway	27	光合作用 Photosynthesis	79
	亮氨酸分解代谢过程Leucine catabolic process	11	对受伤的反应Response to wounding	139
	脂肪酸β氧化 Fatty acid beta-oxidation	35	诱导的系统抗性，茉莉酸介导的信号通路 Induced systemic resistance, jasmonic acid mediated signaling pathway	22
	线粒体中电子传递（细胞色素c到氧） Mitochondrial electron transport, cytochrome c to oxygen	9	对寒冷的反应 Response to cold	191
	盐胁迫反应 Response to salt stress	229	油菜素类固醇介导的信号通路调控 Regulation of brassinosteroid mediated signaling pathway	12
	糖酵解过程Glycolytic process	46	对缺水的反应Response to water deprivation	201
	氨基酸跨膜转运 Amino acid transmembrane transport	38	茉莉酸介导的信号通路调控 Regulation of jasmonic acid mediated signaling pathway	39
细胞组成 Cellular component	膜组成部分Integral component of membrane	1314	叶绿体类囊体膜Chloroplast thylakoid membrane	209
	高尔基体 Golgi apparatus	380	质膜锚定成分 Anchored component of plasma membrane	127
	内质网膜Endoplasmic reticulum membrane	282	叶绿体Chloroplast	805
	高尔基膜Golgi membrane	216	光系统II photosystem II	44
	内质网高尔基中间室 Endoplasmic reticulum-Golgi intermediate compartment	25	膜的锚固组件 Anchored component of membrane	120
	液泡膜Vacuolar membrane	216	质膜Plasma membrane	1154
	线粒体基质Mitochondrial matrix	56	叶绿体类囊体Chloroplast thylakoid	80
	内质网Endoplasmic reticulum	320	纤维素合酶复合物Cellulose synthase complex	12
	线粒体呼吸链复合体IV Mitochondrial respiratory chain complex IV	9	光系统I Photosystem I	24
	液泡Vacuole	228	叶绿体基质Chloroplast stroma	254
分子功能 Molecular function	谷胱甘肽转移酶活性 Glutathione transferase activity	42	DNA结合转录因子活性 DNA-binding transcription factor activity	934
	质子转运ATP酶活性，旋转机制 Proton-transporting ATPase activity, rotational mechanism	24	序列特异性DNA结合 Sequence-specific DNA binding	539
	细胞色素C氧化酶活性Cytochrome-C oxidase activity	9	木聚糖o-乙酰转移酶活性Xylan o-acetyltransferase activity	21
	氨基酸跨膜转运蛋白活性 Amino acid transmembrane transporter activity	39	叶绿素结合 Chlorophyll binding	37
	脂肪酰辅酶A结合Fatty-acyl-coA binding	15	微管结合Microtubule binding	111
	钴离子结合Cobalt ion binding	33	水道活动Water channel activity	26
	乙酰辅酶A C-酰基转移酶活性 Acetyl-CoA C-acyltransferase activity	8	亚油酸13S脂氧合酶活性 Linoleate 13S-lipoxygenase activity	10
	泛醌细胞色素C还原酶活性 Ubiquinol-cytochrome-C reductase activity	12	苯丙氨酸解氨酶活性 Phenylalanine ammonia-lyase activity	9
	烯醇辅酶A水合酶活性Enoyl-CoA hydratase activity	16	蓝光感光活性Blue light photoreceptor activity	10
	蔗糖跨膜转运蛋白活性 Sucrose transmembrane transporter activity	14	DNA结合 DNA binding	721

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年份 Year	处理 Treatment	α-酮戊二酸含量 α-ketoglutarat content (mg·g^-1 FW)	丙酮酸含量 Pyruvic acid content (mg·g^-1 FW)	谷氨酸合酶活性 Glutamate synthase activity (µmol·g^-1·min^-1)	谷氨酸草酰乙酸转氨酶活性 Glutamic oxaloacetic transaminase activity (µmol·g^-1 FW·h^-1)	可溶性蛋白含量 Soluble protein content (mg·g^-1 FW)	游离氨基酸含量 Free amino acid content (µmol·g^-1 FW)
2020	DPC	0.33±0.01a	1.5±0.6a	4.9±0.4a	26.8±1.1a	14.5±0.5a	52.5±3.1a
2020	清水Water	0.21±0.03b	1.2±0.7b	3.7±0.2b	19.2±0.5b	11.3±0.6b	41.2±5.4b
2021	DPC	0.41±0.05a	1.8±0.4a	4.6±0.3a	24.6±0.9a	15.6±0.4a	49.6±4.8a
2021	清水Water	0.28±0.04b	1.4±0.9b	3.2±0.1b	18.9±0.6b	12.2±0.1b	40.7±3.7b

处理 Treatment	重复 Repeat	原始数据 Raw reads	有效数据 Clean reads	总映射率 Total mapped rate (%)	唯一映射率 Uniquely mapped rate (%)	Q30 (%)	GC含量 GC content (%)
清水 Water	I	50384077	49660559	97.74	90.26	94.72	43.69
	II	54022636	53204746	97.78	89.89	95.10	43.73
	III	49503273	48769270	97.84	89.92	95.03	43.70
DPC	I	50205819	49387930	97.61	89.70	95.16	43.75
	II	50289705	49524244	97.67	89.85	95.17	43.75
	III	53487862	52617544	97.68	89.91	95.25	43.73