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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (10): 1485-1495.doi: 10.3724/SP.J.1006.2020.01013

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2020-10-12 Published:2020-06-12
  • Contact: Xian-Chun XIA,Jian-Jun LIU E-mail:zsn19870322@163.com;xiaxianchun@caas.cn;ljjsaas@163.com
  • 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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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

Table 1

Primers for screening Lcye mutations by TILLING"

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

Table 2

Primers of Lcye gene for qRT-PCR analysis"

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

Supplementary table 1

Table Supplementary table 1 Information of cDNA sequences of Lcye genes in 27 plant species"

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

Table 3

Summary of mutations in Lcye identified by TILLING"

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

Fig. 1

Distribution of mutation sites in Lcye Exons are represented by yellow arrows and introns by connecting lines."

Table 4

The severity of mutations affecting protein function by the PARSESNP"

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

Fig. 2

The relative expression analysis of Lcye and its homoeologs in grains of three genotypes in F2 population derived from the homozygous M090815 mutant crossed with the wild-type plant during grain development at 7, 14, 21, and 28 days post anthesis * P < 0.05, ** P < 0.01. Hom: homozygous mutants; Het: heterozygous mutants; WT: wild-type genotypes."

Fig. 3

Yellow pigment content of different genotypes in F2 populations derived from homozygous M090815 mutant crossed with wild-type plant * P < 0.05. Hom: homozygous mutants; Het: heterozygous mutants; WT: wild-type genotypes."

Fig. 4

Functional domains predication of Lcye by MEME"

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