Differential Expression of Grain Pigment-Related Genes of Guizimai No. 1

XU Xi1 REN MingJian1 LI LuHua1 YANG XiCui1 XU RuHong1

(1.College of Agriculture, Guizhou University/Guizhou Sub-Center of National Wheat Improvement Center, Guiyang 550025)

【Abstract】[Objective] The objective of this study is to investigate the transcriptome difference after and before purple-changing periods of grain at filling stage of Guizimai No. 1, explore the key genes and enzymes that contribute to the biogenesis of anthocyanin, then enrich the transcriptome data of grain pigment in wheat, and provide references for the cloning and expression of the transcription factor. [Method] RNA-seq, library construction and quality assessment were carried out for two periods before and after purple-changing of Guizimai No. 1 by using the Illumina Hiseq 2000TM sequencing platform, and the sequencing result was analyzed by bioinformatics. TTM was used to standardize the read count data and then DEGseq was used to analyze the difference. The q-value < 0.005 and |log2 (fold change)| > 1 were set as the threshold. The differential expression genes (DEGs) were obtained through selecting, in accordance with the transcriptome sequencing. Then, these DEGs were analyzed by BLAST search, NR annotation, GO functional enrichment and KEGG pathway method to find out the key genes and enzymes associated with anthocyanins, and qRT-PCR was combined to verify the expression level of the key genes and key enzymes in different periods. Finally, the information of these key genes was mastered. [Result] The RNA-seq results showed that 13.36 G and 12.69 G clean bases were obtained and 106 906 108 and 101 547 534 clean reads accounted for 93.73% and 94.90% of the raw reads after and before purple-changing of Guizimai No. 1, respectively. Clean reads were spliced by Trinity and totally 170 396 transcripts were obtained with the length of 119 020 625. There were 119 572 Unigenes after splicing clean reads. In the BLAST search, 86 004 (71.92%) Unigenes out of 119 572 high-quality unique sequences had at least one significant match to the existing gene models. According to Unigenes’Nr database alignment, at least 5 Unigenes with similar gene identities and known sequence homologies to Aegilops tauschii, Triticum urartu, Brachypodium distachyon, Hordeum vulgare, Triticum aestivum, and so on were identified. The results of KOG database alignment showed that the annotated genes were classified according to 26 groups in KOG, and the greater percentages of generally functional genes, posttranslational modification and transport, molecular chaperones and translation, ribosomal structure and biosynthesis were 15.79%, 14.51% and 10.54%, respectively. A total of 643 DEGs were found, 236 DEGs were up-regulated and 407 DEGs were down-regulated. The GO annotation indicated that there were 44 terms in accordance with biological process, cellular component, molecular function of the next level of classification, the differential genes significantly enriched in the carbohydrate metabolism process (GO: 0005975, 16.03%), stress response (GO: 0006950, 10.83%) and hydrolase activity (GO: 0016787, 34.84%) and other categories. The KEGG pathway enrichment analysis showed that the 353 different genes were enriched in 153 related pathways, among which, the pathways of starch and sucrose metabolism, phenylpropanoid biosynthesis and flavonoid biosynthesis were significantly enriched. There were 66 genes related to flavonoid biosynthesis, and two up-regulated Unigenes, involving two key enzyme genes of CHS, ANS. The log2 (fold change) values were 3.416 4 and 2.125 8, respectively. The qRT-PCR results showed that the expression of CHS and ANS after purple-changing was significantly up-regulated, which was consistent with the results of RNA-Seq analysis, and the RNA-seq results were reliable. [Conclusion] Compared the RNA-seq after and before purple-changing periods of Guizimai No. 1 grain, a large number of Unigenes and DEGs were obtained. It is identified that the two key enzyme genes (CHS and ANS) in flavonoid metabolism pathway play a significant role in the regulation of anthocyanin synthesis in Guizimai No. 1.

【Keywords】 Guizimai No. 1; grain; filling stage; anthocyanin; transcriptome; Illumina sequencing;


【Funds】 National Natural Science Foundation of China (31660390) Agricultural Achievement Transformation Plan in Guizhou Province (QianKeHeChengGuo (2016) 4022) National Key Research and Development Program for “Breeding of Seven Major Crops” (2017YFD0100900) Key Provincial Disciplilne Construction Plan for Crop Science in Guizhou Province (QianXueWeiHeZi ZDXK[2014]8) Key Laboratory Project for Genetic Improvement and Eco-Physiological Characteristics of Grain and Oil Crops at Universities in Guizhou Province (QianJiaoheKYZi[2015]333)

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(Translated by CHEN T)


    [1] LIU X F, LI F, YIN X R, XU C J, CHEN K S. Recent advances in the transcriptional regulation of anthocyanin biosynthesis. Acta Horticulturae Sinica, 2013, 40 (11): 2295–2306 (in Chinese).

    [2] LI N N. Genetic analysis and preliminary gene mapping of characteristics with purple in wheat (Triticum aestivum L.) [D]. Taian: Shandong Agricultural University, 2013 (in Chinese).

    [3] LI N N, ZHANG W D, GAO Q R, ZHANG B L, ZHANG Y Y, GAO J H, WANG H N. Genetic analysis of characteristics with purple in wheat SN066559 (Triticum aestivum L.). Journal of Triticeae Crops, 2013, 33 (1): 18–22 (in Chinese).

    [4] XU B Y, LI X P, LAN S Q, BAI F. Location of pigment gene for wheat with purple grains using microsatellite marker. Journal of Hebei Normal University (Natural Science Edition), 2007, 31 (5): 658 (in Chinese).

    [5] DOBROVOLSKAYA O, ARBUZOVA V S, LOHWASSER U, RÖDER M S, BÖRNER A. Microsatellite mapping of complementary genes for purple grain colour in bread wheat (Triticum aestivum L.). Euphytica, 2006, 150 (3): 355–364.

    [6] HUANG B G. Genetic analysis of purple and waxy grain in wheat. Scientia Agricultura Sinica, 2011, 44 (17): 3501–3507 (in Chinese).

    [7] ZONG X F, ZHANG J K, LI B X, YU G D, SHI Y M, WANG S G. Relationship between antioxidation and grain colors of wheat (Triticum aestivum L.). Acta Agronomica Sinica, 2006, 32 (2): 237–242 (in Chinese).

    [8] LI X P, LAN S Q, LIU Y P. Studies on pigment and its related physio-biochemical properties of blue or purple grain wheat. Acta Agronomica Sinica, 2003, 29 (1): 157–158 (in Chinese).

    [9] SHAKED-SACHRAY L, WEISS D, REUVENI M, NISSIM-LEVI A, OREN-SHAMIR M. Increased anthocyanin accumulation in aster flowers at elevated temperatures due to magnesium treatment. Physiologia Plantarum, 2002, 114 (4): 559–565.

    [10] STILES E A, CECH N B, DEE S M, LACEY E P. Temperaturesensitive anthocyanin production in flowers of Plantago lanceolata. Physiologia Plantarum, 2007, 129 (4): 756–765.

    [11] MORI K, GOTO-YAMAMOTO N, KITAYAMA M, HASHIZUME K. Loss of anthocyanins in red-wine grape under high temperature. Journal of Experimental Botany, 2007, 58 (8): 1935–1945.

    [12] KE Y, GAO F, JIN T, ZHENG L. Review of temperature effect on anthocyanin synthesis. Chinese Agricultural Science Bulletin, 2015, 31 (19): 101–105 (in Chinese).

    [13] GRIESBACH R J. Correlation of p H and light intensity on flower color in potted Eustoma grandiflorum Grise. Hort Science, 1992, 27 (7): 817–818.

    [14] HU K, HAN K T, DAI S L. Regulation of plant anthocyanin synthesis and pigmentation by environmental factors. Chinese Bulletin of Botany, 2010, 45 (3): 307–317 (in Chinese).

    [15] MA Y P, DAI S L. Research progress in the molecular mechanisms of flowering in higher plants. Molecular Plant Breeding, 2007, 5 (6S): 21–28 (in Chinese).

    [16] QI T, SONG S, REN Q, WU D, HUANG H, CHEN Y, FAN M, PENG W, REN C, XIE D X. The jasmonate-ZIM-domain proteins interact with the WD-repeat/bHLH/MYB complexes to regulate jasmonatemediated anthocyanin accumulation and trichome initiation in Arabidopsis thaliana. The Plant Cell, 2011, 23 (5): 1795–1814.

    [17] ZHAO J T. Research progresses on molecular mechanism of hormone regulation of plant anthocyanin biosynthesis. Molecular Plant Breeding, 2016, 14 (7): 1884–1891 (in Chinese).

    [18] LORETI E, POVERO G, NOVI G, SOLFANELLI C, ALPI A, PERATA P. Gibberellins, jasmonate and abscisic acid modulate the sucrose-induced expression of anthocyanin biosynthetic genes in Arabidopsis. New Phytologist, 2008, 179 (4): 1004–1016.

    [19] ZHOU Y. Whole genome wide association study of tocopherol and genetic analysis of anthocyanin biosynthesis pathway in rice [D]. Wuhan: Huazhong Agricultural University, 2014 (in Chinese).

    [20] BOVY A, DE V R, KEMPER M, SCHIJLEN E, ALMENAR P M, MUIR S, ROBINSON S, VERHOEYEN M, HUQHES S, SANTOS B C, VAN TUNEN A. High-flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes LC and C1. The Plant Cell, 2002, 14 (10): 2509–2526.

    [21] LI Y, ZHANG T, SHEN Z W, XU Y, LI J Y. Overexpression of maize anthocyanin regulatory gene Lc affects rice fertility. Biotechnology Letters, 2013, 35 (1): 115–119.

    [22] ZHANG C Q, DING Y L. Analysis of antioxidants metabolic pathway and expression of anthocyanin biosynthetic genes in blueberry flower and fruit. Journal of Jingling Institute of Technology, 2016, 32 (3): 63–66 (in Chinese).

    [23] COCK P J, FIELDS C J, GOTO N, HEUER M L, RICE P M. The Sanger FASTQ file format for sequences with quality scores, and the Solexa/Illumina FASTQ variants. Nucleic Acids Research, 2010, 38 (6): 1767–1771.

    [24] GRABHERR M G, HAAS B J, YASSOUR M, LEVIN J Z, THOMPSON D A, AMIT I, ADICONIS X, FAN L, RAYCHOWDHURY R, ZENG Q, CHEN Z, MAUCEI E, HACOHEN N, GNIRKE A, RHIND N, PALMA D F, BIRREN B W, NUSBAUM C, LINDBLADTOH K, FRIEDMAN N, REGEV A. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology, 2011, 29 (7): 644–652.

    [25] WANG L, FENG Z, WANG X, WANG X, ZHANG X. DEGseq: an R package for identifying differential expression genes from RNA-seq data. Bioinformatics, 2010, 26 (1): 136–138.

    [26] YOUNG M D, WAKEFIELD M J, SMYTH G K, OSHLACK A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biology, 2010, 11 (2): R14.

    [27] KANEHISA M, ARAKI M, GOTO S, HATTORI M, HIRAWA M, ITOH M, KATAYAMA T, KAWASHIWA S, OKUDA S, TOKIMATSU T, YAMANISHI Y. KEGG for linking genomes to life and the environment. Nucleic Acids Research, 2008, 36 (Database issue): D480–D484.

    [28] WU J. Transcriptome study on rice (Oryza sativa L.) in response to high night temperature stress at early milky stage [D]. Nanchang: Jiangxi Agricultural University, 2016 (in Chinese).

    [29] KE J. Effect of nitrogen management on rice population quality and transcriptomic analysis of ear organs [D]. Hefei: Anhui Agricultural University, 2014 (in Chinese).

    [30] WANG J P, SUN G Z, WANG H B. Transcriptome analysis of promotive effects of active carbon on growth and development of maize seedlings from in vitro cultured immature embryos. Acta Agronomica Sinica, 2017, 43 (10): 1489–1498 (in Chinese).

    [31] LI H Z. Transcriptomics researches on wheat (Triticum aestivum L.) grain development morphogenesis [D]. Yangling: Northwest A&F University, 2014 (in Chinese).

    [32] ZHAO S C, LIU B, ZHAO L J, GUO D L, MAO J S, GUO C Y, REN F S, WANG X Z, TIAN J C. Research of anthocyanin composition in blue and purple wheat grains. Scientia Agricultura Sinica, 2010, 43 (19): 4072–4080 (in Chinese).

    [33] KIM S H, LEE J R, HONG S T, YOO Y K, AN G, KIM S R. Molecular cloning and analysis of anthocyanin biosynthesis genes preferentially expressed in apple skin. Plant Science, 2003, 165 (2): 403–413.

    [34] REDDY A M, REDDY V S, SCHEFFLER B E, WIENAND U, REDDY A R. Novel transgenic rice overexpressing anthocyanidin synthase accumulates a mixture of flavonoids leading to an increased antioxidant potential. Metabolic Engineering, 2007, 9 (1): 95–111.

    [35] LI X L, ZHANG M S, LÜX. The research progress on plant anthocyanin synthetase ANS gene. Plant Physiology Journal, 2016, 52 (6): 817–827 (in Chinese).

    [36] FU S L. Identification and expression analysis of b HLH transcription factor genes in wheat [D]. Beijing: Chinese Academy of Agricultural Sciences, 2014 (in Chinese).

    [37] NESI N, DEBEAUJON I, JOND C, PELLETIER G, CABOCHE M, LEPINIEC L. The TT8 gene encodes a basic helix-loop-helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques. The Plant Cell, 2000, 12 (10): 1863–1878.

    [38] XIE J, XIA J, ZHANG P F, ZHANG P. Cloning and expression of PKR gene and affinity purification of PKR interacting proteins. Chinese Journal of Cellular and Molecular Immunology, 2012, 28 (6): 660–662 (in Chinese).

    [39] YIN X J. Construction of PKR gene expression vector and Agrobacterium tumefaciens mediated transformation of shoot apex of maize [D]. Lanzhou: Gansu Agricultural University, 2011 (in Chinese).

This Article


CN: 11-1328/S

Vol 51, No. 02, Pages 203-216

January 2018


Article Outline


  • 0 Introduction
  • 1 Materials and methods
  • 2 Results
  • 3 Discussion
  • 4 Conclusions
  • References