基于转录组和WGCNA的甘薯花青素合成相关基因共表达网络的构建及核心基因的挖掘<sup>*</sup>

doi:10.13523/j.cb.2106048

中国生物工程杂志

2021, Vol. 41

Issue (9): 27-36 DOI: 10.13523/j.cb.2106048

研究报告

基于转录组和WGCNA的甘薯花青素合成相关基因共表达网络的构建及核心基因的挖掘^*

贺立恒¹,张毅²,张洁¹,任豫超¹,解红娥³,唐锐敏¹,贾小云^2,^**(

),武宗信^3,^**(

)

1 山西农业大学农学院太谷 030801
2 山西农业大学生命科学学院太谷 0308012
3 山西农业大学棉花研究所运城 044000

Construction of Gene Co-expression Network and Identification of Hub Genes Related to Anthocyanin Biosynthesis Based on RNA-seq and WGCNA in Sweetpotato

HE Li-heng¹,ZHANG Yi²,ZHANG Jie¹,REN Yu-chao¹,XIE Hong-e³,TANG Rui-min¹,JIA Xiao-yun^2,^**(

),WU Zong-xin^3,^**(

)

1 College of Agriculture, Shanxi Agricultural University, Taigu 030801, China
2 College of Life Science, Shanxi Agricultural University, Taigu 030801, China
3 Institute of Cotton Research, Shanxi Agricultural University, Yuncheng 044000, China

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摘要：

加权基因共表达网络分析(weighted gene co-expression network analysis, WGCNA)可通过聚类鉴定共表达的基因模块来研究生物学数据与相应性状之间的关系。甘薯[Ipomoea batatas (L.)Lam.]是世界上营养丰富的块根作物之一,紫薯是甘薯的一种特殊品种,因含有大量的花青素而具有较高的营养价值。因此,培育花青素含量高的优质紫薯一直以来都是甘薯育种家所追求的目标。利用传统的育种方法已经培育了一些紫薯品种,但其周期长、工作量大、见效慢,所以急需通过分子设计育种手段来培育高产优质的紫薯新品种。花青素合成相关关键基因的挖掘对紫薯的分子育种具有重要意义。为了挖掘甘薯花青素合成相关基因,以紫薯品种‘徐紫薯3号'和白薯品种‘徐薯18号'的块根为材料进行了转录组测序(RNA-seq),并结合公共数据库中已公布的甘薯基因组信息以及43份紫薯和45份非紫薯块根的RNA-seq数据,通过分析在不同样本间表达量差异大的前50%的基因中选择了26 760个基因进行WGCNA分析。结果表明,利用WGCNA鉴定出28个共表达模块,其中4个为紫薯特异性模块(Grey60模块和Black模块与紫薯显著正相关,Brown模块和Blue模块与紫薯显著负相关)。利用GO功能富集分析发现紫薯特异性模块Grey60可以显著富集到类黄酮和花青素代谢过程。通过计算模块内基因的连通性,分析挖掘到Grey60模块中有47个核心基因,其中包括已报道的8个花青素合成相关基因MYB113、CHS、3个CHI、F3H、GST和LDOX。利用qRT-PCR验证了其中7个核心基因的表达模式。通过构建核心基因的互作网络发现:MYB不仅与已知的花青素合成相关基因bHLH、CHI、GST、F3'H、CHS等存在互作,同时也与DUF914、ABCC4等转运蛋白基因互作;WRKY3与多个核心基因存在互作,如LDOX、GST、CHS等。为高花青素含量紫薯新品种的培育和紫薯花青素生物合成机制的解析提供了理论基础和新思路。

关键词： 紫薯; 花青素; 加权基因共表达网络分析; 转录组; 核心基因

Abstract:

Weighted gene co-expression network analysis (WGCNA) is a systematic biological method. It can be used to explore the relationship between genes and related traits by the identification of co-expressed modules and hub genes in the network. Sweetpotato [Ipomoea batatas (L.)Lam.] is one of the nutritious tuberous crops in the world. Purple-fleshed sweetpotato (PFSP) is a special variety of sweetpotato. In addition to the nutritional components of common sweetpotato, PFSP is also enriched in anthocyanin with antioxidant function that is beneficial to health. Therefore, the cultivation of PFSP with high anthocyanin content has always been the goal of scientists. Although some PFSP varieties have been bred by traditional hybridization breeding techniques, which has some disadvantages, such as long breeding cycle, labor consuming and low efficiency. Therefore, it is urgent to develop PFSP with high yield and quality by molecular design breeding. Studying on key genes related to anthocyanin biosynthesis of sweetpotato can shed light on molecular breeding of PFSP. In order to explore genes related to anthocyanin biosynthesis in sweetpotato, white-fleshed sweetpotato‘Xushu-18'and PFSP‘Xuzishu-3' were used for transcriptome sequencing in this study. By integrated analysis of the published genomic information of sweetpotato and the transcriptome sequencing data of 43 purple-fleshed and 45 non purple-fleshed tuberous roots in public database, the top 50% differentially expressed genes (26 760) with large variation in different samples were identified and selected for WGCNA. A total of 28 gene co-expression modules were identified, in which four modules were specific in purple-fleshed sweetpotato (Grey60 module and Black module are significantly positively correlated to PFSP, while Brown modules and Blue modules are significantly negatively correlated to PFSP). GO analysis showed that the Grey60 module was significantly enriched in the metabolic process of flavonoids and anthocyanin. A total of 47 core hub genes related to anthocyanin biosynthesis were screened in Grey60 module by calculating gene connectivity in the corresponding networks. Of them, MYB113, CHS, three CHI and F3H, GST and LDOX have been previously reported to be related to anthocyanins biosynthesis. The expression patterns of seven of the 47 hub genes were verified by qRT-PCR, which were consistent with the results by transcriptome sequencing. Analysis of the interaction network of hub genes indicated that MYB interacts not only with the known genes related to anthocyanin biosynthesis, such as bHLH, CHI, GST, F3'H and CHS, but also with the transporter genes DUF914 and ABCC4, etc. WRKY3 interacts with many core genes, such as LODX, GST, and CHS. In summary, this study will provide new ideas for the cultivation of high anthocyanin content purple-fleshed sweetpotato varieties and theoretical foundation for dissection of anthocyanin biosynthesis mechanism.

Key words: Purple-fleshed sweetpotato Anthocyanin WGCNA Transcriptome sequencing Hub genes

收稿日期: 2021-06-27 出版日期: 2021-09-30

ZTFLH:

Q812

基金资助: * 国家重点研发专项(2018YFD1000705);国家重点研发专项(2018YFD1000700);山西省研究生教育创新项目(2020BY057);山西省农业科学院应用基础研究计划(YGC2019FZ4);山西省新兴产业领军人才项目(2019061)

通讯作者: 贾小云,武宗信 E-mail: jiaxiaoyun@sxau.edu.cn;mhswzx@126.com

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引用本文:

贺立恒,张毅,张洁,任豫超,解红娥,唐锐敏,贾小云,武宗信. 基于转录组和WGCNA的甘薯花青素合成相关基因共表达网络的构建及核心基因的挖掘^*[J]. 中国生物工程杂志, 2021, 41(9): 27-36.

HE Li-heng,ZHANG Yi,ZHANG Jie,REN Yu-chao,XIE Hong-e,TANG Rui-min,JIA Xiao-yun,WU Zong-xin. Construction of Gene Co-expression Network and Identification of Hub Genes Related to Anthocyanin Biosynthesis Based on RNA-seq and WGCNA in Sweetpotato. China Biotechnology, 2021, 41(9): 27-36.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2106048 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I9/27

图1 软阈值的筛选

图2 基因聚类树状图及模块身份

图3 各模块中的基因数目

图4 模块中转录因子分布图

图5 共表达基因模块与紫薯性状的相关性热图

图6 紫薯特异性模块Grey60中基因的GO富集分析气泡图

表1 Grey60模块中花青素合成相关的核心基因及功能注释

图7 Grey60模块中花青素合成相关核心基因的互作网络

图8 7个核心基因的表达分析

[1]	黄钢, 刘永红, 何文铸, 等. 甘薯在抗震救灾中的重要作用及技术对策. 四川农业科技, 2008(6):12-13.
	Huang G, Liu Y H, He W Z, et al. The important role and technical countermeasures of sweetpotato in earthquake relief. Science and Technology of Sichuan Agriculture, 2008(6):12-13.
[2]	Kwak S S. Biotechnology of the sweetpotato: ensuring global food and nutrition security in the face of climate change. Plant Cell Reports, 2019, 38(11):1361-1363. doi: 10.1007/s00299-019-02468-0
[3]	高路. 薯中奇葩:紫甘薯. 沈阳: 白山出版社, 2009.
	Gao L. Special varieties of sweetpotato:Purple sweetpotato. Shenyang: Baishan Publishing House, 2009.
[4]	He W, Zeng M M, Chen J, et al. Identification and quantitation of anthocyanins in purple-fleshed sweet potatoes cultivated in China by UPLC-PDA and UPLC-QTOF-MS/MS. Journal of Agricultural and Food Chemistry, 2016, 64(1):171-177. doi: 10.1021/acs.jafc.5b04878
[5]	Gras C C, Nemetz N, Carle R, et al. Anthocyanins from purple sweet potato[Ipomoea batatas (L.) Lam.] and their color modulation by the addition of phenolic acids and food-grade phenolic plant extracts. Food Chemistry, 2017, 235:265-274. doi: 10.1016/j.foodchem.2017.04.169
[6]	裴苓荃. 甘薯bHLH基因家族的鉴定与初步分析. 徐州: 江苏师范大学, 2017.
	Pei L Q. Identification and preliminary analysis of bHLH gene family in sweet potato. Xuzhou: Jiangsu Normal University, 2017.
[7]	吴鑫宝. 紫甘薯花青素对大鼠放射性肠损伤作用的实验研究. 衡阳: 南华大学, 2015.
	Wu X B. Experimental studies in rats purple sweet potato anthocyanins intestinal radiation injury. Hengyang: University of South China, 2015.
[8]	Lim S, Xu J T, Kim J, et al. Role of anthocyanin-enriched purple-fleshed sweet potato p40 in colorectal cancer prevention. Molecular Nutrition & Food Research, 2013, 57(11):1908-1917.
[9]	王艳丽. 紫甘薯酰化花色苷与肠道菌群的相互作用研究. 天津: 天津科技大学, 2018.
	Wang Y L. Study on the interaction between acylated purple sweet potato anthocyanins and intestinal bacteria. Tianjin: Tianjin University of Science & Technology, 2018.
[10]	Zhao W, Langfelder P, Fuller T, et al. Weighted gene coexpression network analysis: state of the art. Journal of Biopharmaceutical Statistics, 2010, 20(2):281-300. doi: 10.1080/10543400903572753 pmid: 20309759
[11]	Weirauch M T. Gene coexpression networks for the analysis of DNA microarray data. Applied Statistics for Network Biology. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2011: 215-250.
[12]	宋长新, 雷萍, 王婷. 基于WGCNA算法的基因共表达网络构建理论及其R软件实现. 基因组学与应用生物学, 2013, 32(1):135-141.
	Song C X, Lei P, Wang T. Gene co-expression network analysis based on WGCNA algorithm-theory and implementation in R software. Genomics and Applied Biology, 2013, 32(1):135-141.
[13]	王跃, 毛开云, 王恒哲, 等. 转录组学测序技术应用与市场分析. 生物产业技术, 2017(5):11-17.
	Wang Y, Mao K Y, Wang H Z, et al. Application and market analysis of transcriptomic sequencing technologies. Biotechnology & Business, 2017(5):11-17.
[14]	熊强强, 魏雪娇, 施翔, 等. 多层组学在植物逆境及育种中的研究进展. 江西农业大学学报, 2018, 40(6):1197-1206.
	Xiong Q Q, Wei X J, Shi X, et al. Progress in research on multi-omics in plant stress and breeding. Acta Agriculturae Universitatis Jiangxiensis, 2018, 40(6):1197-1206.
[15]	Xiao D, Li X, Zhou Y Y, et al. Transcriptome analysis reveals significant difference in gene expression and pathways between two peanut cultivars under Al stress. Gene, 2021, 781:145535. doi: 10.1016/j.gene.2021.145535
[16]	Leng F, Ye Y L, Zhu X H, et al. Comparative transcriptomic analysis between ‘Summer Black' and its bud sport ‘Nantaihutezao' during developmental stages. Planta, 2021, 253(1):1-12. doi: 10.1007/s00425-020-03501-3
[17]	El-Sharkawy I, Liang D, Xu K N. Transcriptome analysis of an apple (Malus×domestica) yellow fruit somatic mutation identifies a gene network module highly associated with anthocyanin and epigenetic regulation. Journal of Experimental Botany, 2015, 66(22):7359-7376. doi: 10.1093/jxb/erv433 pmid: 26417021
[18]	Kroll K W, Mokaram N E, Pelletier A R, et al. Quality control for RNA-seq (QuaCRS): an integrated quality control pipeline. Cancer Informatics, 2014, 13(Suppl 3):7-14. doi: 10.4137/CIN.S14022 pmid: 25368506
[19]	Chen S F, Zhou Y Q, Chen Y R, et al. Fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 2018, 34(17):i884-i890. doi: 10.1093/bioinformatics/bty560
[20]	Kim D, Langmead B, Salzberg S L. HISAT: a fast spliced aligner with low memory requirements. Nature Methods, 2015, 12(4):357-360. doi: 10.1038/NMETH.3317
[21]	Liao Y, Smyth G K, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 2014, 30(7):923-930. doi: 10.1093/bioinformatics/btt656
[22]	Yang J, Moeinzadeh M H, Kuhl H, et al. Haplotype-resolved sweet potato genome traces back its hexaploidization history. Nature Plants, 2017, 3(9):696-703. doi: 10.1038/s41477-017-0002-z pmid: 28827752
[23]	Love M I, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 2014, 15(12):550. doi: 10.1186/s13059-014-0550-8
[24]	张毅, 吴万亿, 刘霞宇, 等. 利用WGCNA鉴定甘薯耐盐相关共表达网络及核心基因. 河南农业科学, 2021, 50(6):16-27.
	Zhang Y, Wu W Y, Liu X Y, et al. Identification of salt tolerance co-expression modules and hub genes in Ipomoea batatas by WGCNA. Journal of Henan Agricultural Sciences, 2021, 50(6):16-27.
[25]	Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics, 2008, 9:559. doi: 10.1186/1471-2105-9-559 pmid: 19114008
[26]	Mano H, Ogasawara F, Sato K, et al. Isolation of a regulatory gene of anthocyanin biosynthesis in tuberous roots of purple-fleshed sweet potato. Plant Physiology, 2007, 143(3):1252-1268. doi: 10.1104/pp.106.094425
[27]	Kim C Y, Ahn Y O, Kim S H, et al. The sweet potato IbMYB1 gene as a potential visible marker for sweet potato intragenic vector system. Physiologia Plantarum, 2010, 139(3):229-240.
[28]	Zuker A, Tzfira T, Ben-Meir H, et al. Modification of flower color and fragrance by antisense suppression of the flavanone3-hydroxylasegene. Molecular Breeding, 2002, 9(1):33-41. doi: 10.1023/A:1019204531262
[29]	Feng F J, Li M J, Ma F W, et al. Phenylpropanoid metabolites and expression of key genes involved in anthocyanin biosynthesis in the shaded peel of apple fruit in response to Sun exposure. Plant Physiology and Biochemistry, 2013, 69:54-61. doi: 10.1016/j.plaphy.2013.04.020
[30]	Yang Y N, Yao G F, Zheng D M, et al. Expression differences of anthocyanin biosynthesis genes reveal regulation patterns for red pear coloration. Plant Cell Reports, 2015, 34(2):189-198. doi: 10.1007/s00299-014-1698-0 pmid: 25323492
[31]	Xu W P, Peng H, Yang T B, et al. Effect of calcium on strawberry fruit flavonoid pathway gene expression and anthocyanin accumulation. Plant Physiology and Biochemistry, 2014, 82:289-298. doi: 10.1016/j.plaphy.2014.06.015
[32]	Olsen K M, Lea U S, Slimestad R, et al. Differential expression of four Arabidopsis PAL genes; PAL1 and PAL2 have functional specialization in abiotic environmental-triggered flavonoid synthesis. Journal of Plant Physiology, 2008, 165(14):1491-1499. doi: 10.1016/j.jplph.2007.11.005
[33]	Amato A, Cavallini E, Zenoni S, et al. A grapevine TTG2-like WRKY transcription factor is involved in regulating vacuolar transport and flavonoid biosynthesis. Frontiers in Plant Science, 2016, 7:1979.

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