Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (8): 1471-1483.doi: 10.3864/j.issn.0578-1752.2023.08.004

• TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY • Previous Articles     Next Articles

Mepiquat Chloride Increases the Cry1Ac Protein Content Through Regulating Carbon and Amino Acid Metabolism of Bt Cotton Under High Temperature and Drought Stress

XING YuTong(), TENG YongKang, WU TianFan, LIU YuanYuan, CHEN Yuan, CHEN Yuan, CHEN DeHua(), ZHANG Xiang()   

  1. Agricultural College, Yangzhou University/Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Yangzhou 225009, Jiangsu
  • Received:2022-08-08 Accepted:2022-12-05 Online:2023-04-16 Published:2023-04-23

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-1 DPC 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

Fig. 1

Effects of DPC on the boll shell insecticidal protein content under the stress of high temperature and drought Different small letters meant significant difference at P<0.05 level among treatments"

Table 1

Effects of DPC on carbon, amino acid metabolism and soluble protein content in the boll shell under the stress of high temperature and drought"

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
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
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

Table 2

Statistics of Bt cotton transcriptome data quality"

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

Fig. 2

Volcanic diagram of differentially expressed genes in boll shell between DPC and water treatments"

Table 3

GO enrichment of differentially expressed genes in the boll shell for DPC vs water under high temperature and drought"

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

Fig. 3

KEGG pathway of differentially expressed genes in the boll shell for DPC and water under high temperature and drought The color of bubble represents the P value, and the smaller the P value, the more significant the enrichment result. The size of bubble represents the number of differentially expressed genes, a large bubble represents a large number of differential genes, and a small bubble represents a small number of differential genes"

Fig. 4

Effect of DPC on glycolysis pathway (ko00010) under the stress of high temperature and drought Red squares represent differentially up-regulated genes; dark green squares represent differentially down-regulated genes; yellow squares represent there are both differentially up-regulated genes and down-regulated genes; light green or purple squares represent specific genes and non-differentially significant genes; white squares represent genes that are not present or not detected in this species. The same as below"

Fig. 5

Effect of DPC on citrate cycle (TCA cycle) pathway (ko00020) under the stress of high temperature and drought"

Fig. 6

Effect of DPC on alanine, aspartate and glutamate metabolism pathway (ko00250) under the stress of high temperature and drought"

Fig. 7

Effect of DPC on arginine biosynthesis pathway (ko00220) under the stress of high temperature and drought"

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