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作物学报 ›› 2020, Vol. 46 ›› Issue (10): 1485-1495.doi: 10.3724/SP.J.1006.2020.01013

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦类胡萝卜素合成途径关键基因Lcye功能分析

翟胜男1(), 郭军1, 刘成1, 李豪圣1, 宋健民1, 刘爱峰1, 曹新有1, 程敦公1, 李法计1, 何中虎2, 夏先春2,*(), 刘建军1,*()   

  1. 1 山东省农业科学院作物研究所, 山东济南 250100
    2 中国农业科学院作物科学研究所, 北京 100081
  • 收稿日期:2020-02-18 接受日期:2020-06-02 出版日期:2020-10-12 网络出版日期:2020-06-12
  • 通讯作者: 夏先春,刘建军
  • 作者简介:E-mail: zsn19870322@163.com, Tel: 0531-66659561
  • 基金资助:
    国家自然科学基金项目(31701420);山东省农业科学院农业科技创新工程(CXGC2018E01);中国科协青年人才托举工程(2017QNRC001);山东省农业良种工程项目(2019LGC001)

Functional analysis of Lcye gene involved in the carotenoid synthesis in common wheat

ZHAI Sheng-Nan1(), GUO Jun1, LIU Cheng1, LI Hao-Sheng1, SONG Jian-Min1, LIU Ai-Feng1, CAO Xin-You1, CHENG Dun-Gong1, LI Fa-Ji1, HE Zhong-Hu2, XIA Xian-Chun2,*(), LIU Jian-Jun1,*()   

  1. 1 Crop Research Institute, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China
    2 Institute of Crop Sciences, Chnese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2020-02-18 Accepted:2020-06-02 Published:2020-10-12 Published online:2020-06-12
  • Contact: Xian-Chun XIA,Jian-Jun LIU
  • Supported by:
    National Natural Science Foundation of China(31701420);Agricultural Scientific and Technological Innovation Project of Shandong Academy of Agricultural Sciences(CXGC2018E01);Young Elite Scientists Sponsorship Program by CAST(2017QNRC001);Agricultural Variety Improvement Project of Shandong Province(2019LGC001)

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摘要：

小麦籽粒黄色素是面粉及面制品黄度形成的主要原因, 其主要成分是类胡萝卜素。ε-番茄红素环化酶(LCYE)是小麦类胡萝卜素生物合成途径的关键酶, 前人对其研究多集中于QTL定位、基因克隆和分子标记开发, 而基因功能和遗传调控机制尚不明确。本研究利用TILLING技术筛选EMS诱变群体, 对Lcye功能及遗传调控机制进行研究, 以期深入认识小麦籽粒黄色素含量形成的分子机制。在2491份M2代EMS诱变群体中共检测到21个Lcye基因的点突变, 包含6个错义突变, 2个同义突变和13个内含子突变, Lcye基因在该诱变群体中的突变频率为1/266.1 kb。PARSENP软件预测分析显示, M090815 (C2202T)和M091648 (G3284A)两个错义突变可能严重影响蛋白质功能。MEME分析结果表明, M090815和M092230 (G2195A)突变位点位于Lcye基因保守结构域内。6个错义突变植株与野生型杂交构建的F2代群体中, M090815突变位点显著降低籽粒黄色素含量, 证实该位点对LCYE功能具有重要影响。qRT-PCR (quantitative real-time PCR)分析也显示, M090815突变位点显著降低Lcye基因表达水平, 且Lcye-B1和Lcye-D1基因表达降低趋势相似, 而Lcye-A1在花后14~28 d表现出补偿效应。本研究不仅验证Lcye基因功能, 也为面粉及其制品颜色性状改良提供了理论依据和种质资源。

关键词: EMS, TILLING, 面粉颜色, 黄色素含量, 遗传育种

Abstract:

Yellow pigment in wheat grains, mostly composed of carotenoids, is a main factor for the yellowness of flour and its end-used products. Lycopene epsilon cyclase (LCYE) is a key enzyme for the carotenoid biosynthesis pathway in wheat. Previous studies of Lcye gene mainly focused on QTL mapping, gene cloning, and molecular marker development, but its function and genetic regulatory mechanisms remained unclear. In the present study, in order to further understanding the molecular mechanism of yellow pigment formation in wheat grains, the function and genetic regulation of Lcye were studied by TILLING to screen the EMS-mutagenised population. A total of 21 Lcye mutations including six missense mutations, two synonymous mutations and 13 intron mutations were detected consisted from 2491 M2 EMS-mutagenised population. The mutation frequency of Lcye in the population was 1/266.1 kb. Two missense mutations (M090815 and M091648) were predicted to have severe effects on LCYE protein function based on PARSENP software. MEME analysis showed that the mutation sites of M090815 and M092230 were located on the conserved domain of Lcye gene. In F2 populations crossing by six missense mutants and the wild type, C2202T mutation in M090815 significantly reduced yellow pigment content in grains, indicating the mutation played an important effect on LCYE function. The quantitative real-time PCR (qRT-PCR) results also showed that the expression level of Lcye gene were significantly reduced, and the decrease trend of Lcye-B1 and Lcye-D1 expression level was similar during different seed developmental stages, while the expression level of Lcye-A1 exhibited a compensation effect at 14-28 days post anthesis. This study identified Lcye gene function, and provided germplasms and a theoretical basis for the improvement of flour color traits and end-used products.

Key words: EMS, TILLING, flour color, yellow pigment content, genetics and breeding

表1

利用TILLING技术筛选Lcye突变体的引物信息"

基因
Gene		 名称
Name		 上游引物序列
Forward primer (5′-3′)		 下游引物序列
Reverse primer (5′-3′)		 扩增长度
Length (bp)
Lcye-A1 A3F-A7R CCACAGTAGCAAAAATTAGTCA TGCTACATTTCACAGTGGTGAA 1450
Lcye-A1 A8F-A9R GGTTGAAAGATATCCGTACAAC TTTGGGTAACCGGAAAAAGGTT 978
Lcye-B1 B4F-B6R CACCAACCCTGCACAAAGTGCC GGAATATAAGACCACTCCTGAG 578
Lcye-D1 D2F-D5R GCTGAGAAGGTACATTCTATCA TTGAACTGGTGCACAAACAACA 437

表2

Lcye基因的qRT-PCR引物信息"

基因Gene 名称Name 序列Sequence (5'-3')
Lcye-all Lcye-all-F2 TGACCACYGAATATCCAGTTGC
Lcye-all-R6 AGTTTTCTTTGAGGAAACATGC
Lcye-A1 Lcye-A1-F7 GTTGCTGAGAAGATGCAACGAT
Lcye-A1-R7 CAAAGTATCTTGCGGTCCCTTT
Lcye-B1 Lcye-B1-F3 ATCTCCAGATGGACATCGAGTG
Lcye-B1-R3 TCCAACCTCATACTCTAGAAGT
Lcye-D1 Lcye-D1-F3 TTGGCCCTGATCTTCCATTC
Lcye-D1-R1 ATATACTACTCGATGTCCATCA
β-actin Actin-F CTGATCGCATGAGCAAAGAG
Actin-R CCACCGATCCAGACACTGTA

附表1

27个物种Lcye基因cDNA序列信息"

种名
Species name		 GenBank登录号
GenBank accession number		 种名
Species name		 GenBank登录号
GenBank accession number
拟南芥Arabidopsis thaliana NM_125085 小立碗藓Physcomitrella patens XM_001753846
二穗短柄草Brachypodium distachyon XM_003569209 碧桃Prunus persica XM_007203578
大白菜Brassica rapa XM_009133907 蓖麻Ricinus communis XM_002514090
荠菜Capsella rubella XM_006280236 谷子Setaria italica XM_004969360
莱茵衣藻Chlamydomonas reinhardtii XM_001696477 番茄Solanum lycopersicum EU533951
柑橘Citrus sinensis AY533827 马铃薯Solanum tuberosum XM_006353482
黄瓜Cucumis sativus XM_004157912 高粱Sorghum bicolor XM_002455793
草莓Fragaria vesca XM_004287534 可可Theobroma cacao XM_007012707
大豆Glycine max XM_003533727 普通小麦Triticum aestivum EU649785
大麦Hordeum vulgare AK371513 圆锥小麦Triticum turgidum GAKM01004311
亚麻Linum usitatissimum KC565894 乌拉尔图小麦Triticum urartu GAKL01018490
苹果Malus domestica XM_008389970 葡萄Vitis vinifera JQ319637
蒺藜苜蓿Medicago truncatula XM_003595195 玉米Zea mays EU924262
水稻Oryza sativa NM_001049945

表3

利用TILLING技术筛选获得Lcye突变体信息"

基因
Gene		 突变体编号
Number of M2 plant		 外显子/内含子
Exon/intron		 核苷酸改变
Nucleotide change		 密码子改变
Codon change		 氨基酸改变
Amino acid change		 基因型
Zygosity
Lcye-A1 M091034 Intron C1184T Hom
M091686 Intron C1243T Hom
M091772 Intron C1418T Hom
M092043 Intron C1478T Hom
M092852 Intron C3222T Hom
M090431 Intron C858T Het
M090631 Intron T1461C Het
M090897 Intron G1068A Hom
M091996 Intron G1575A Hom
M090147 Intron G3073A Hom
M091648 Exon G3284A GGA→GAA G392E Hom
M090201 Exon G3306A TTA→TTG L399= Hom
Lcye-B1 M091626 Intron G2406A Hom
Lcye-D1 M092404 Intron C2014T Het
M091884 Intron C2017T Het
M090945 Exon C2086T TAC→TAT Y214= Hom
M092089 Exon C2087T CTC→TTC L215F Hom
M091328 Exon C2121T CCT→CTT P226L Het
M090815 Exon C2202T TCT→TTT S253F Het
M092230 Exon G2195A GCA→ACA A251T Hom
M091075 Exon G2262A GGT→GAT G273D Hom

图1

Lcye突变位点分布图 黄色箭头代表外显子; 连线代表内含子。"

表4

突变位点对蛋白功能影响严重度预测"

基因
Gene		 突变体编号
Number of M2 plant		 核苷酸改变
Nucleotide change		 氨基酸改变
Amino acid change		 PSSM值
PSSM difference		 SIFT值
SIFT score
Lcye-D1 M090815 C2202T S253F 26.9 0
Lcye-A1 M091648 G3284A G392E 27.7 0

图2

突变体M090815的F2群体中不同基因型植株籽粒Lcye及其同源基因的相对表达量 * 和**分别代表0.05和0.01显著水平的差异。Hom: 纯合突变型; Het: 杂合突变型; WT: 野生型。"

图3

突变体M090815的F2群体中不同基因型植株籽粒黄色素含量 * 代表0.05显著水平的差异。Hom: 纯合突变型; Het: 杂合突变型; WT: 野生型。"

图4

Lcye功能结构域预测"

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