山黧豆半胱氨酸合成酶基因的克隆及功能分析  

Cloning and Functional Analysis of Cysteine Synthase Genes in Grass Pea(Lathyrus sativus)

作　　者：申肖 宋瑶瑶 陈红 曲瑞红 李轶凡 焦成瑾 陈鹏[1] 徐全乐[1] SHEN Xiao;SONG Yao-Yao;CHEN Hong;QU Rui-Hong;LI Yi-Fan;JIAO Cheng-Jin;CHEN Peng;XU Quan-Le(College of Life Sciences,Northwest A&F University,Yangling 712100,China;College of Bioengineering and Biotechnology,Tianshui Normal University,Tianshui 741000,China)

机构地区：[1]西北农林科技大学生命科学学院,杨凌712100 [2]天水师范学院生物工程与技术学院,天水741000

出　　处：《农业生物技术学报》2021年第8期1443-1452,共10页Journal of Agricultural Biotechnology

基　　金：陕西省自然科学基金(2020JM-167);国家自然科学基金(31660153);中国博士后科学基金特别资助(2019T120952);大学生创新创业训练计划项目(S202010712516)。

摘　　要：β-N-草酰-L-α,β-二氨基丙酸(β-N-oxalyl-L-α,β-diaminopropionic acid,β-ODAP)是山黧豆(Lathyrus sativus)中的重要神经激活性物质和三七(Panaxnoto ginseng)止血的活性成分(三七素),其生物合成途径与硫代谢尤其是半胱氨酸(cysteine,Cys)代谢密切相关。本研究基于山黧豆转录组数据克隆了山黧豆半胱氨酸合成酶(cysteine synthase,CS)家族基因。利用多序列比对、系统发育分析等解析基因序列特征并进行分类;利用Cys营养缺陷型大肠杆菌(Escherichia coli)突变体功能互补、原核表达及酶活检测等分析LsCS基因表达产物特征;利用氨基酸定点突变分析LsCS活性关键位点;利用qRT-PCR分析LsCS基因在种子发育期的表达特征。结果表明,山黧豆中至少存在8条LsCS基因,与大豆(Glycine max)等物种的CS基因同源性较高,均属于β-取代基丙氨酸合成酶(β-substituted alanine synthase,BSAS)基因家族;LsCS基因的表达产物均具有半胱氨酸合成酶活性,其活性发挥依赖于磷酸吡哆醛(pyridoxal phosphate,PLP);在山黧豆发育的种子中,LsCS1、LsCS2、LsCS4、LsCS6和LsCS7表达水平较高,可能是主要的半胱氨酸合成酶来源。上述研究为进一步分析山黧豆LsCS家族基因在硫代谢途径,尤其是β-ODAP生物合成过程中的功能奠定了基础。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 ofβ-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 toβ-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 inβ-ODAP biosynthesis of L.sativus.

关 键 词：山黧豆 β-N-草酰-L-α β-二氨基丙酸(β-ODAP) 硫代谢 半胱氨酸合成酶 功能互补 

分 类 号：S529[农业科学—作物学]