甲哌鎓通过调节碳和氨基酸代谢提高高温和干旱下Bt棉Cry1Ac蛋白含量

doi:10.1016/j.jia.2023.11.013

摘要/Abstract

摘要：

本文研究了高温干旱胁迫下甲哌鎓（DPC）对Bt棉Cry1Ac蛋白含量的影响，以期为Bt棉育种和高产高效栽培提供理论参考。于2020年和2021年在扬州大学农牧场进行，以“泗抗3号”为材料，采用盆栽栽培。供试材料在人工气候室高温和干旱胁迫后，进行喷施20 mg L^-1 DPC和清水（CK）处理。处理7d后，测定Cry1Ac蛋白含量、α-酮戊二酸含量、丙酮酸含量、谷氨酸合成酶活性、谷氨酸草酰乙酸转氨酶活性、可溶性蛋白含量和氨基酸含量，并进行转录组测序。结果表明，与CK相比，DPC处理Cry1Ac蛋白含量增加了4.7%-11.9%，α-酮戊二酸含量、丙酮酸含量、谷氨酸合成酶活性、谷氨酸草酰乙酸转氨酶活性、可溶性蛋白含量和氨基酸含量均有所增加。转录组分析显示， DPC处理7542个基因上调，10449个基因下调。进一步分析表明，差异表达基因主要涉及氨基酸代谢和碳代谢等过程。其中编码6-磷酸果糖激酶、丙酮酸激酶、谷氨酸丙酮酸转氨酶、丙酮酸脱氢酶、柠檬酸合成酶、异柠檬酸脱氢酶、2-氧代戊二酸脱氢酶的基因显著上调，谷氨酸合成酶、1-吡咯啉-5-羧酸脱氢酶、谷氨酸草酰乙酸转氨酶、N-乙酰谷氨酸合成酶和乙酰鸟氨酸脱乙酰酶也显著上调。说明DPC提高糖酵解途径、柠檬酸循环的运行速率，增加了丙酮酸、α-酮戊二酸和草酰乙酸。DPC处理还显著上了调谷氨酸合成酶和吡咯烷-5-羧酸脱氢酶的编码基因，下调谷氨酰胺合成酶的编码基因，上调谷氨酸草酰乙酸转氨酶和N-乙酰谷氨酸合成酶的编码基因。综上，DPC处理通过调节高温干旱胁迫后Bt棉自身碳和氨基酸代谢能力，增强天门冬氨酸、谷氨酸、丙酮酸和精氨酸含量，进而为Cry1Ac蛋白合成提供充足原料，并提高其含量。

Abstract:

The effects of mepiquat chloride (DPC) on the Cry1Ac protein content in Bacillus thuringiensis (Bt) cotton boll shells under high temperature and drought stress were investigated to provide a theoretical reference for Bt cotton breeding and high-yield and -efficiency cotton cultivation. This study was conducted using Bt cotton cultivar ‘Sikang 3’ during the 2020 and 2021 growing seasons at Yangzhou University Farm, Yangzhou, Jiangsu Province, China. Potted cotton plants were exposed to high temperature and drought stress, and sprayed with either 20 mg L⁻¹ DPC or water (CK). Seven days after treatment, the Cry1Ac protein content, α-ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content were measured, and transcriptome sequencing was performed. DESeq was used for differential gene analysis. Under the DPC treatment, the Cry1Ac protein content increased by 4.7–11.9% compared to CK. The α-ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content all increased. Transcriptome analysis revealed 7,542 upregulated genes and 10,449 downregulated genes for DPC vs. CK. Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analyses showed that the differentially expressed genes were mainly involved in biological processes, such as carbon and amino acid metabolism. For example, genes encoding 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 upregulated. The DPC treatment increased pyruvate, α-ketoglutarate, and oxaloacetate by increasing the operational rate of the glycolytic pathway of the citric acid cycle. It also significantly upregulated the genes encoding glutamate synthase, pyrrolidine-5-carboxylic acid dehydrogenase, glutamate oxaloacetate transaminase, and N-acetylglutamate synthetase, while it downregulated the genes encoding glutamine synthetase. Therefore, the synthesis of aspartic acid, glutamic acid, pyruvate, and arginine increased after treatment with DPC, and the Cry1Ac protein content was increased by regulating carbon and amino acid metabolism.

Key words: amino acid metabolism , Bt cotton , carbon metabolism , Cry1Ac protein , mepiquat chloride

Dian Jin, Yuting Liu, Zhenyu Liu, Yuyang Dai, Jianing Du, Run He, Tianfan Wu, Yuan Chen, Dehua Chen, Xiang Zhang. 甲哌鎓通过调节碳和氨基酸代谢提高高温和干旱下Bt棉Cry1Ac蛋白含量[J]. Journal of Integrative Agriculture, 2024, 23(12): 4032-4045.

Dian Jin, Yuting Liu, Zhenyu Liu, Yuyang Dai, Jianing Du, Run He, Tianfan Wu, Yuan Chen, Dehua Chen, Xiang Zhang. Mepiquat chloride increases the Cry1Ac protein content of Bt cotton under high temperature and drought stress by regulating carbon and amino acid metabolism[J]. Journal of Integrative Agriculture, 2024, 23(12): 4032-4045.

参考文献

Anders S, Huber W. 2010. Differential expression analysis for sequence count data. Genome Biology, 11, R106.

Benedict J H, Sachs E S, Altman D W, Deaton W R, Kohel R J, Ring D R, Berberich S A. 1996. Field performance of cottons expressing transgenic Cry1A insecticidal proteins for resistance to Heliothis virescens and Helicoverpa zea (Lepidoptera, Noctuidae). Journal of Economic Entomology, 89, 230–238.

Celik I, Tuluce Y, Isik I. 2006. Influence of subacute treatment of some plant growth regulators on serum marker enzymes and erythrocyte and tissue antioxidant defense and lipid peroxidation in rats. Journal of Biochemical and Molecular Toxicology, 20, 174–182.

Chen D H, Ye G Y, Yang C Q, Chen Y, Wu Y K. 2005. The Effect of high temperature on the insecticidal properties of Bt cotton. Environmental and Experimental Botany, 53, 333–342.

Chen G, Xu J Z, Zhang W G. 2016. Progress in biosynthesis and metabolic engineering of L-arginine producer. Microbiology China, 43, 1379–1387. (in Chinese)

Chen S, Wu J Y, He X L, Huang J Q, Zhou B L, Zhang X X. 1997. Quantification using ELISA of Bacillus thuringiensis insecticidal protein expressed in the tissue of transgenic insect-resistant cotton. Jiangsu Journal of Agriculture Science, 13, 27–29. (in Chinese)

Chen Y, Wen Y J, Chen Y, Cothren J T, Zhang X, Wang Y, Payne W A, Chen D H. 2012. Effects of extreme air temperature and humidity on the insecticidal expression level of Bt cotton. Journal of Integrative Agriculture, 11, 1836–1844.

Chihiro K, Watanabe T H K T, Uchimiya H, Uesono Y, Terashima I, Noguchi K. 2010. Effects of AOX 1a deficiency on plant growth, gene expression of respiratory components and metabolic profile under low-nitrogen stress in Arabidopsis thaliana. Plant and Cell Physiology, 51, 810–822.

Dong Z Q, Shu W H, Zhai X J, Zhang B M, Liu F, Song G Q. 2005. The change and relationship of several endogenesis hormone in different organs of cotton plant. Acta Agriculturae Nucleatae Sinica, 19, 62–67. (in Chinese)

Gwathmey C O, Clement J D. 2010. Alteration of cotton source–sink relations with plant population density and mepiquat chloride. Field Crops Research, 116, 101–107.

Hernándezc A E, Sánchez E, Preciador P, Garcíab M L, Palomog A, Espinozab A. 2015. Nitrate reductase activity, biomass, yield, and quality in cotton in response to nitrogen fertilization. Phyton-International Journal of Experimental Botany, 84, 454–460.

Hodges M. 2002. Enzyme redundancy and the importance of 2-oxoglutarate in plant ammonium assimilation. Journal of Experimental Botany, 53, 905.

Kanehisa M, Araki M, Goto S. 2008. KEGG for linking genomes to life and the environment. Nucleic Acids Research, 36, 480–484.

Kim D, Langmead B, Salzberg S L. 2015. HISAT, a fast spliced aligner with low memory requirements. Nature Methods, 12, 357–360.

Lin M W, Watson J F, Baggett J R. 1995. Inheritance of soluble solids and pyruvic acid content of bulb onions. Journal of the American Society for Horticultural Science, 120, 119–122.

Liu Y Y. 2021. The effect of high temperature and drought on the content of insecticidal protein in Bt cotton and its physiology mechanism. MSc these, Yangzhou University, China. (in Chinese)

Liu Y T, Li H J, Chen Y, Tambel L I M, Liu Z Y, Wu S J, Sun S Q, Zhang X, Chen D H. 2024. Inhibition of protein degradation increases the Bt protein concentration in Bt cotton. Journal of Integrative Agriculture, 23, 1897–1909.

Liu Z Y, Wang G X, Zhang Z N, Zhang C X, Li H J, Wu T F, Zhang X, Chen D H. 2022. Recovery characteristics of Cry1Ac endotoxin expression and related physiological mechanisms in Bt transgenic cotton squares after high-temperature stress termination. Agronomy, 12, 668.

Martins C M, Beyene G, Hofs J L, Krüger K, Vyver C Vd, Schlüter U, Kunert K. 2008. Effect of water-deficit stress on cotton plants expressing the Bacillus thuringiensis toxin. Annals of Applied Biology, 152, 255–262.

Mustroph A, Albrecht G. 2003. Tolerance of crop plants to oxygen deficiency stress, fermentative activity and photosynthetic capacity of entire seedlings under hypoxia and anoxia. Physiologiaplantarum, 117, 508–520.

Rochester I J. 2006. Effect of genotype edaphic, environmental conditions, and agronomic practices on Cry1Ac protein expression in transgenic cotton. Journal of Cotton Science, 10, 252–262.

Sachs O, Katzman A, Abu-Johar E, Eidelman M. 2015. Treatment of adolescent blount disease using taylor spatial frame with and without fibular osteotomy, is there any difference? Journal of Pediatric Orthopedics, 35, 501.

Schnepf H E, Wong H C, Whiteley H R. 1985. The amino acid sequence of a crystal protein from Bacillus thuringiensis deduced from the DNA base sequence. The Journal of Biological Chemistry, 260, 6264–6272.

Sener G, Sehirli O, Tozan A, Velioğluo A, Gedik N, Omurtag G Z. 2007. Ginkgo biloba extract protects against mercury (II)-induced oxidative tissue damage in rats. Food and Chemical Toxicology, 45, 543–550.

Siebert J D, Stewart A M. 2006. Influence of plant density on cotton response to mepiquat chloride application. Agronomy Journal, 98, 1634–1639.

Tambel L I M. Zhou M Y, Chen Y, Zhang X, Chen D H. 2019. Amino acids application enhances flowers insecticidal protein content in Bt cotton. Journal of Cotton Research, 2, 38–43.

Wang J, Chen Y, Yao M H, Li Y, Wen Y J, Chen Y, Xiang Z, Chen D H. 2015. The effects of high temperature level on square Bt protein concentration of Bt cotton. Journal of Integrative Agriculture, 14, 1971–1979.

Xia L Q, Guo S D. 2004. The expression of Bt toxin gene under different thermal treatments. Scientia Agricultura Sinica, 37, 1733–1737. (in Chinese)

Xiang J L, Zhu W X, Han J Q, Li Z X, Ge H J, Lin D H. 2012. Analysis of organic acids in Chinese raisin tree (Hovenia dulcis) peduncles and their changes in liquid fermentation process. Food Science and Biotechnology, 21, 1119–1127.

Xu Y, Glansdorff N, Labedan B. 2006. Bioinformatic analysis of an unusual gene–enzyme relationship in the arginine biosynthetic pathway among marine gamma proteobacteria, implications concerning the formation of N-acetylated intermediates in prokaryotes. BMC Genomics, 7, 4.

Yang Z, You S S, Ji C D, Yin M Z, Yang W T, Shen J. 2016. Development of an amino acid-functionalized fluorescent nanocarrier to deliver a toxin to kill insect pests. Advanced Materials, 28, 1375–1380.

Yemm E, Cocking E, Ricketts R. 1955. The determination of amino acids with ninhydrin. The Analyst, 80, 209–214.

Zhang R, Wang N, Li S, Wang Y, Xiao S, Zhang Y, Egrinya E A, Zhang M, Wang B, Duan L, Li F, Tian X, Li Z. 2021. Gibberellin biosynthesis inhibitor mepiquat chloride enhances root K+ uptake in cotton by modulating plasma membrane H+-ATPase. Journal of Experimental Botany, 72, 6659–6671.

Zhang T, Zhao Q, Li G W. 2021. Progress in the effects of DPC on the development of cotton. Jiangsu Agricultural Science, 49, 14–18. (in Chinese)

Zhang X, Tian Q, Zhao Z, Dong Z, Chen Y, Chen D. 2021. Analysis of differentially expressed proteins affecting insecticidal protein content in Bt cotton under high-temperature and water deficit stress using label-free quantitation. Journal of Agronomy and Crop Science, 207, 1–11.

Zhang X, Tian Q F, Zhao C, Ma Y X, Wei C H, Chen Y, Chen D H. 2019. Exogenous hormones affect Bt protein content of two Bt cotton cultivars. Agronomy Journal, 3, 3076–3083.

Zhang X, Wang J, Peng S, Rui Q Z, Li L N, Chen Y, Chen Y, Chen D H. 2018. Relationship between temperature, soil moisture, and insecticidal protein content in Bt cotton boll shell and the mechanism of nitrogen metabolism. Scientia Agricultura Sinica, 51, 1261–1271. (in Chinese)

Zhang X Z. 1992. The Research Method of Crop Physiology. China Agriculture Press, China. pp. 139–142. (in Chinese)

Zhen Q S, Liu Y L. 2001. Effects of soaking seeds in DPC increasing the salinity tolerance in cotton (Gossypium hirsutum L.) seedlings and its mechanism. Cotton Science, 13, 278–282. (in Chinese)

Zhou H, Zhou Y, Zhang F, Guan W X, Su Y, Yuan X X, Xie Y J. 2021. Persulfidationof nitrate reductase 2 is involved in l-cysteine desulfhydrase-regulated rice drought tolerance. International Journal of Molecular Sciences, 22, 12119.

Zou Q. 2000. Experimental Instruct of Plant Physiology. China Agriculture Press, China. pp. 127–130. (in Chinese)