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| Cloning and Functional Analysis of Cysteine Synthase Genes in Grass Pea (Lathyrus sativus) |
| SHEN Xiao1,*, SONG Yao-Yao1,*, CHEN Hong1, QU Rui-Hong1, LI Yi-Fan1, JIAO Cheng-Jin2, CHEN Peng1, XU Quan-Le1,** |
1 College of Life Sciences, Northwest A&F University, Yangling 712100, China;
2 College of Bioengineering and Biotechnology, Tianshui Normal University, Tianshui 741000, China |
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Abstract β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) is a neuro-excitatory amino acid found in Lathyrus sativus and also named as dencichine in Panaxnoto ginseng known for its hemorrhage stopping property. The biosynthesis of β-ODAP was proven to be related to several basic metabolisms especially sulfur and cysteine (Cys) metabolism. In this report, cysteine synthase (CS) genes were cloned from L. sativus based on the previous transcriptomics data. Firstly, multiple sequence alignment and phylogenetic tree analysis were conducted to detect the sequence characteristic and classification of LsCS genes. To confirm the property of LsCS gene expression product, functional complementation to Escherichia coli Cys-auxotrophic strain NK3, bacterial expression, recombinant protein purification and enzymatic activity assay were conducted. And then the key site for CS activity was detected via point mutants of LsCS. At last, gene expression level of LsCS in developing seeds was analyzed via qRT-PCR. The results suggested eight LsCS genes including LsCS1~LsCS7 and LsCAS separately in L. sativus. The LsCS genes were found homologous to those in soybean and other species and belong to β-substituted alanine synthase gene family. Expression of LsCS genes in E. coli Cys-auxotrophic strain NK3 resulted in bacterial growth and enzymatic activity assay suggesting a high cysteine synthase activity of LsCS, which were verified to be PLP-dependent. Among LsCS genes, LsCS1、LsCS2、LsCS4、LsCS6 and LsCS7 expressed with high level during seed development. These results will help to reveal gene functions of LsCS in β-ODAP biosynthesis of L. sativus.
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Received: 18 December 2020
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Corresponding Authors:
** xuql03@163.com
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| About author:: * These authors contributed equally to this work |
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[1] 董晓宁, 王小芳, 杨玲娟, 等. 2013.植物中一组杂环取代的丙氨酸衍生物研究进展[J]. 天然产物研究与开发, 25: 1759-1768.
(Dong X N, Wang X F, Yang L J, et al.2013. Study on the group of heterocyclic β-substituted α-alanines in plants[J]. Natural Product Research and Development, 25: 1759-1768.)
[2] 焦成瑾, 赵菲佚, 谢尚强, 等. 2014. 半胱氨酸合成酶与β-氰基丙氨酸合成酶活性检测[J]. 氨基酸与生物资源, 36(4): 66-72.
(Jiao C J, Zhao F Y, Xie S Q, et al.2014. Assay for activities of cysteine synthase and β-cyanoalanine synthase[J]. Amino Acid＆Biotic Resources, 36(4): 66-72.)
[3] 陶英杰, 刘凤娟, 毕春晓, 等. 2018. 山黧豆CASase基因DNA全长序列的扩增及CRISPR/Cas9敲除载体的构建[J]. 西北农林科技大学学报(自然科学版), 46(8): 23-28.
(Tao Y J, Liu F J, Bi C X, et al.2018. Amplification of CASase gene from Lathyrus sativus and construction of its knock-out vector via CRISPR/Cas9 system[J]. Journal of Northwest A＆F University (Natural Science), 46(8): 23-28.)
[4] 张明科, 刘凤娟, 陶英杰, 等. 2016. 山黧豆CASase基因的克隆及RNAi载体的构建[J]. 草地学报, 24(5): 1146-1149.
(Zhang M K, Liu F J, Tao Y J, et al.2016. Cloning of CASase gene from Lathyrus sativus and construction of its RNAi vector[J]. Acta Agrestia Sinica, 24(5): 1146-1149.)
[5] 赵钢. 2014. 三七素在制备治疗神经变性病药物中的应用的制作方法[P]. 中国, CN201210008092.9.
(Zhao G.2014. Notoginseng factor in preparing medicine for treating neurodegenerative diseases[P]. China, CN201210008092.9.)
[6] Alvarez C, Calo L, Romero LC, et al.2010. An O-acetylserine(thiol)lyase homolog with L-cysteine desulfhydrase activity regulates cysteine homeostasis in Arabidopsis[J]. Plant Physiology, 152(2): 656-669.
[7] Bermúdez M, Páez-Ochoa M, Gotor C, et al.2010. Arabidopsis S-sulfocysteine synthase activity is essential for chloroplast function and long-day light-dependent redox control[J]. Plant Cell, 22(2): 403-416.
[8] Chakraborty S, Mitra J, Samanta MK, et al.2018. Tissue specifific expression and in-silico characterization of a putative cysteine synthase gene from Lathyrus sativus L.[J]. Gene Expression Patterns, 27:128-134.
[9] Eric RB, Rebecca EC, Sarah MK, et al.2005. Molecular basis of cysteine biosynthesis in plants: Structural and functional analysis of O-acetylserinesulfhydrylase from Arabidopsis thaliana[J]. Journal of Biological Chemistry, 280(46): 38803-38813.
[10] Ikegami F, Murakoshi I.1994. Enzymic synthesis of nonprotein β-substituted alanines and some higher homologues in plants[J]. Phytochemistry, 35: 1089-1104.
[11] Ikegami F, Ongena G, Sakai R, et al.1993. Biosynthesis of β-(isoxazolin-5-on-2-yl)-alanine by cysteine synthase in Lathyrus sativus[J]. Phytochemistry, 33(1): 93-98.
[12] Jiao CJ, Xu QL, Wang CY, et al.2006. Accumulation pattern of toxin β-ODAP during lifespan and effect of nutrient elements on β-ODAP content in Lathyrus sativus seedlings[J]. Journal of Agricultural Science, 144(4): 369-375.
[13] Koh HL, Lau AJ, Chan CY.2005. Hydrophilic interaction liquid chromatography with tandem mass spectrometry for the determination of underivatized dencichine (β-N-oxalyl-L-α,β-diaminopropionic acid) in Panax medicinal plant species[J]. Rapid Communications in Mass Spectrometry, 19: 1237-1244.
[14] Kuo YH, Ikegami F, Lambein F.1998. Metabolic routes of beta-(isoxazolin-5-on-2-yl)-L-alanine (BIA), the precursor of the neurotoxin ODAP (beta-N-oxalyl-L-alpha, beta-diaminopropionic acid), in different legume seedlings[J]. Phytochemistry, 49(l): 43-48.
[15] Lambein F, Ongena G, Kuo Y H.1990. β-Isoxazolinone-alanine is involved in the biosynthesis of the neurotoxin β-N-oxalyl-L-α,β-diaminopropionic acid[J]. Phytochemistry, 29(12): 3793-3796.
[16] Lambein F, Travella S, Kuo Y H, et al.2019. Grass pea (Lathyrus sativus L.): Orphan crop, nutraceutical or just plain food[J]? Planta, 250(3): 821-838.
[17] Lan G, Chen P, Sun Q, et al.2013. Methods for treating hemorrhagic conditions[P]. United States Patent, No. US 8,362,081 B2.
[18] Liu FJ, Jiao CJ, Bi CX, et al.2017. Metabolomics approach to understand mechanisms of β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) biosynthesis in grass pea (Lathyrus sativus L.)[J]. Journal of Agricultural and Food Chemistry, 65(47): 10206-10213.
[19] Marrero-Degro J, Marcano-Velázquez J, Siritunga D.2011. Isolation and characterization of novel β-cyanoalanine synthase and cysteine synthase genes from cassava[J]. Plant Molecular Biology Reporter, 29: 514-524.
[20] Rao SLN.2011. A look at the brighter facets of β-N-oxalyl-L-α,β-diaminopropionic acid, homoarginine and the grass pea[J]. Food and Chemistry Toxicology, 49(3): 620-622.
[21] Rashid MU, Iwasaki H, Oogai S, et al.2018. Molecular characterization of cytosolic cysteine synthase in Mimosa pudica[J]. Journal of Plant Research, 131: 319-329.
[22] Song YY, Wang L, Liu FJ, et al.2021. β-cyanoalanine synthase regulates the accumulation of β-ODAP via interaction with serine acetyltransferase in Lathyrus sativus[J]. Journal of Agricultural and Food Chemistry, 69(6): 1953-1962.
[23] Wang Y, Zhong P, Zhang X, et al.2019. GRA78 encoding a putative S-sulfocysteine synthase is involved in chloroplast development at the early seedling stage of rice[J]. Plant Science, 280: 321-329.
[24] Warrilow AG, Hawkesford MJ.1998. Separation, subcellular location and influence of sulphur nutrition on isoforms of cysteine synthase in spinach[J]. Journal of Experimental Botany, 49: 1625-1636.
[25] Watanabe M, Kusano M, Oikawa A, et al.2008. Physiological roles of β-substituted alanine synthase gene family in Arabidopsis[J]. Plant Physiology, 146: 310-320.
[26] Xu Q L, Liu F J, Chen P, et al.2017. β-N-oxalyl-L-α,β- diaminopropionic acid (β-ODAP) content in Lathyrus sativus: the integration of nitrogen and sulfur metabolism through β-cyanoalanine synthase[J]. International Journal of Molecular Sciences, 18(3): 526.
[27] Xu Q L, Liu F J, Qu R H, et al.2018. Transcriptomic profiling of Lathyrus sativus L. metabolism of β‐ODAP, a toxin associated with neurodegenerative lower limb paralysis[J]. Plant Molecular Biology Reporter, 36(5-6): 832-843
[28] Yamaguchi Y, Nakamura T, Kusano T, et al.2000. Three Arabidopsis genes encoding proteins with differential activities for cysteine synthase and beta-cyanoalanine synthase[J]. Plant and Cell Physiology, l41: 465-476
[29] Yi H, Jez JM.2012. Assessing functional diversity in the soybean beta-substituted alanine synthase enzyme family[J]. Phytochemistry, 83: 15-24.
[30] Yi H, Juergens M, Jez JM.2012. Structure of soybean beta-cyanoalanine synthase and the molecular basis for cyanide detoxification in plants[J]. Plant Cell, 24(6): 2696-2706.
[31] Zhang C H, Meng Q C, Zhang M, et al.2008. Characterization of O-acetylserine(thiol)lyase encoding genes reveals their distinct but cooperative expression in cysteine synthesis of soybean (Glycine max (L) Merr)[J]. Plant Molecular Biology Reporter, 26(4): 277-291. |
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