摘要
【目的】分析黄瓜基因组中与叶酸合成代谢相关的基因数量、定位以及表达特征,对关键酶基因进行生物信息学分析与克隆,旨在为黄瓜叶酸合成调控研究奠定基础。【方法】根据已报道的拟南芥叶酸合成相关基因,利用黄瓜基因组数据库中9930_V3版本进行BLAST比对。利用MapChart绘制黄瓜染色体物理图谱并对基因定位。利用qRT-PCR分析这些基因在黄瓜果实发育不同时期和不同材料中的表达量。通过MEGA、WebLOGO、ExPASy等工具对关键酶基因进行生物信息学分析。通过PCR扩增对关键酶基因进行克隆,并测序分析基因的序列差异。【结果】同源比对获得19个黄瓜叶酸代谢相关基因,这些基因不均匀分布在黄瓜7条染色体上,且以Chr.4和Chr.5上分布最多。通过对其中11个调控叶酸合成的基因在测序黄瓜9930果实发育不同时期以及果实叶酸含量高低差异显著的2份材料的表达量分析,发现CsFPGS、CsHPPK/CsDHPS和CsDHNA 3个基因与果实叶酸含量变化趋势完全一致;CsADCS、CsADCL、CsDHNA、CsHPPK/CsDHFS、CsFPGS、CsDHFS等基因的表达量在2份材料中具有显著差异。通过对2个调控叶酸合成限速步骤的关键酶基因CsGCHI和CsADCS的蛋白序列及蛋白结构域分析,发现各物种中CsGCHI的同源基因均具有2个GTP_cyclohydroI结构域;CsADCS的同源基因均具有2个GATase结构域、1个Anth_synt_I_N结构域和1个Chorismate_bind结构域。它们在不同物种中高度保守,进化树分析亲缘关系近的物种聚类到一起。分别扩增黄瓜果实低叶酸含量自交系65G和高叶酸含量自交系02245中CsGCHI和CsADCS的同源基因,序列分析表明CsaV3_1G041250全长为3012 bp,CDS序列长度为1413 bp,3个SNP位点的突变导致了氨基酸序列的变异;CsaV3_7G026240全长为3047 bp,CDS长度1407 bp,序列无变异;CsaV3_5G036360全长7941 bp,CDS序列长度为2706 bp,序列无变异。【结论】鉴定出19个不均匀分布在7条染色体上的黄瓜叶酸代谢相关基因,基因CsFPGS、CsHPPK/CsDHPS、CsDHNA和CsADCS是影响黄瓜果实叶酸含量变化、导致叶酸含量高低显著差异的关键基因,调控叶酸合成限速步骤的关键酶基因GCHI及ADCS功能相对保守,CsGCHI在65G、02245中有3个SNP位点的突变导致了氨基酸序列的差异。
【Objective】This study analyzed the quantity,location and expression pattern of folic acid metabolization-related genes in cucumber,cloned and made bioinformatics analysis of the key enzyme genes,aiming to lay a foundation for the study on the regulation mode of folic acid synthesis in cucumber.【Method】The reported folic acid metabolism related genes in Arabidopsis thaliana was blasted in the cucumber genome database 9930_V3 to obtain the folic acid metabolism related genes in cucumber.These genes were mapped onto the cucumber chromosome by using Mapchart,and their expression pattern was examined in different materials and at different developmental stages.Bioinformatics analysis of key enzyme genes was conducted by MEGA,Web LOGO,and ExPASy.Key enzyme genes were cloned by PCR amplification,and sequence differences were analyzed.【Result】A total of 19 genes related to folate metabolism were blasted in cucumber,which were distributed non-uniformly on seven chromosomes,mostly on Chr.4 and Chr.5.The expression levels of 11 folic acid synthetic genes in fruits of sequenced material 9930,inbred line with low folic acid 65G,and inbred line with high folic acid 02245 at different developmental stages were analyzed.It was found that the expression pattern of CsFPGS,CsHPPK/CsDHPS,and CsDHNA were consistent with the changes of folic acid content,and there were significant differences in the expression levels of CsADCS,CsADCL,CsDHNA,CsHPPK/CsDHFS,CsFPGS and CsDHFS between 65G and 02245.CsGCHI and CsADCS were two key enzymes regulate folate synthesis in rate-limiting steps,and then their amino acid sequences and protein domains were analyzed.The result showed that it turned out that CsGCHI homologs all had two GTP_cyclohydroI domains,and CsADCS homologs all had two GATase domains,including one Anth_synt_I_N domain,and one Chorismate_bind domain.The domains were highly conserved in different species,evolutionary tree analysis clustered the proteins of closely related species together.The GCHI and ADCS gene were cloned from 65G and 02245,respectively.Sequence analysis showed that the full length of CsaV3_1G041250 was 3012 bp,the length of CDS sequence was 1413 bp,and the mutations in the three SNP sites led to the variation of amino acid sequence.The full length of CsaV3_7G026240 was 3047 bp,and the CDS length was 1407 bp with no sequence variation.The total length of CsaV3_5G036360 was 7941 bp,and the length of CDS sequence was 2706 bp.【Conclusion】It was identified that 19 genes were related to folate metabolism in cucumber.These genes were distributed unequally on seven chromosomes.CsFPGS,CsHPPK/CsDHPS,CsDHNA and CsADCS affected folic acid content and trend in cucumber fruit mostly,while CsGCHI and CsADCS were the Key enzyme genes regulating rate-limiting steps in folic acid synthesis,which was relatively conservative in function,and 3 SNP mutations led to variations in protein sequences in CsGCHI between 65G and 02245.
作者
周琪
刘小萍
薄凯亮
苗晗
董邵云
顾兴芳
张圣平
ZHOU Qi;LIU XiaoPing;BO KaiLiang;MIAO Han;DONG ShaoYun;GU XingFang;ZHANG ShengPing(Institute of Vegetable and Flowers,Chinese Academy of Agricultural Sciences/Key Laboratory of Horticultural Crop Biology and Germplasm Creation,Ministry of Agriculture and Rural Areas,Beijing 100081)
出处
《中国农业科学》
CAS
CSCD
北大核心
2020年第18期3764-3776,I0013-I0016,共17页
Scientia Agricultura Sinica
基金
中国农业科学院创新工程(CAAS-ASTIP-2017-IVF)
国家现代农业产业技术体系(CARS-23)。
关键词
黄瓜
叶酸
关键酶
同源基因
克隆
cucumber
folate
key enzyme
homology gene
cloning
