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
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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