菊花脑管状花响应蔗糖饲喂的差异基因分析

doi:10.16420/j.issn.0513-353x.2023-0321

摘要/Abstract

摘要：

为探明蔗糖饲喂对菊花脑花苞总黄酮积累的影响及其机制，以中国特有的菊属野生二倍体物种菊花脑（Chrysanthemum nankingense）为试材，对其离体花苞进行蔗糖饲喂，测定饲喂后0、12、36和60 h管状花和舌状花总黄酮和总酚的动态变化，并对管状花进行转录组测序。结果发现：蔗糖饲喂24 h以内菊花脑管状花类黄酮和总酚的含量显著增加，而同类物质在舌状花中变化较为平缓。蔗糖饲喂60 h与同时间无蔗糖饲喂相比，转录组分析仅获得3 423个差异表达基因；而与0 h无蔗糖饲喂相比，可获得最多的差异表达基因（9 135个），说明随饲喂时间的增加，管状花在生化层面上对蔗糖的响应程度增加。经KEGG分析，类黄酮生物合成途径被显著富集，32个类黄酮生物合成结构基因具有差异表达。相关性分析表明转录因子MYB、WRKY和MADS-box与类黄酮生物合成结构基因表达具有较强的共表达关系。在上述差异基因中随机挑选9个进行qRT-PCR验证，结果表明转录组数据具有较高的准确性；并对其中2个转录因子的编码基因进行了时空表达分析和蛋白亚细胞定位，为从代谢工程的角度研究菊花脑次生代谢筛选了目标基因。

关键词: 菊花脑, 蔗糖响应, 类黄酮生物合成, 差异表达基因, 管状花

Abstract:

To explore effects of sucrose feeding on the accumulation of total flavonoids in Chrysanthemum nankingense capitula and the underlying mechanism，Chrysanthemum nankingense，a unique wild diploid species of chrysanthemum in China，was used as the test material，and its detached capitula were fed with sucrose. The dynamic changes of total flavonoids and total phenols in disc and ray florets were determined at 0，12，36 and 60 h after sucrose feeding，and transcriptome sequencing of disc florets was performed. The results confirmed that sucrose feeding could increase the content of flavonoids and total phenols in disc florets within 24 h. However，in ray florets，these compounds changed slightly. A total of 9 135 differentially expressed genes（DEGs），which was also the maximum number of DEGs identified between treatments，were obtained by transcriptome analysis of disc florets sucrose-fed for 60 h and 0 h，while only 3 423 differentially expressed genes were obtained by transcriptome analysis of disc florets sucrose-fed for 60 h and no sucrose-fed for 60 h. These data indicate that the response degree of disc florets to sucrose increased with the increase of feeding time on the biochemistry level. KEGG analysis indicated that the flavonoid biosynthesis pathway was significantly enrichedand 32 flavonoid biosynthetic structural genes were differentially expressed. Correlation analysis showed that transcription factors MYBs，WRKYs and MADS-boxes had a strong co-expression relationship with flavonoid biosynthetic structural gene expression in disc florets in response to sucrose feeding. Nine DEGs were randomly selected for qRT-PCR，indicating that the transcriptome data were accurate. Spatio-temporal expression and subcellular localization of the encoding genes of two transcription factors were further analyzed to confirm the findings. Genes screened out from this study can offer potential targets for further efforts of metabolic engineering aiming to probe secondary metabolism in Chrysanthemum nankingense.

Key words: Chrysanthemum nankingense, response to sucrose, flavonoid metabolism, differentially expressed genes, disc florets

王稳, 张鹏, 王志敏, 刘根忠, 包志龙, 马方放. 菊花脑管状花响应蔗糖饲喂的差异基因分析[J]. 园艺学报, 2024, 51(6): 1297-1310.

WANG Wen, ZHANG Peng, WANG Zhimin, LIU Genzhong, BAO Zhilong, MA Fangfang. Analysis of Differential Genes in Response to Sucrose Feeding in Disc Florets of Chrysanthemum nankingense[J]. Acta Horticulturae Sinica, 2024, 51(6): 1297-1310.

图/表 9

表1 本试验中所用的引物

Table 1 Primers used in this experiment

引物用途 Primer usage	基因ID Gene ID	引物序列（5′-3′） Primer sequence
qRT-PCR	CHR00071024	F：CGTCGAGTTGATGGATTGCG
		R：AGAACAGTGTCACCCGTTGC
	CHR00071025	F：CGAGTTGATGGATTGCGACG
		R：AAAACTTACCCGTTGCCGGT
	CHR00072791	F：ACCAGAAAGATGGCCCAACA
		R：TCTGCCATCACCCATTCGAC
	CHR00057220	F：CTTGGCTCCGAGTGCTAAGA
		R：TCTCTTTGATCGCGAGTAGCA
	CHR00023313	F：ACGCAGGACCTTTTAGTCGG
		R：TCGAGCTCTTCTGTTGCCTT
	CHR00023554	F：CCAAAGAGTGGAGGGCAGTT
		R：GCCTCTTTCTCAAGGCCCAT
	CHR00068386	F：TGCCAGCATTTCGACACTGA
		R：GACGGGTTTCCACGTAGTCC
	CHR00001486	F：AGATTTGCCACCCCAGTCTT
		R：ATGGAGCAGTGTTCGGTTGT
	CHR00075330	F：AGTCTTCGACAGTTTCGGGC
		R：CAAACCGTTGTTGCCGGATT
	CnUbi	F：CTAATGAATGCTTACTGTGACCGAC
		R：TAGCTCATCGGGAGTCTGCT
亚细胞定位 Subcellular localization	CHR00001486	F：GCCAGTGCCAAGCTTCTATGGATTCAGCTGTTAAGAATGTGC
		R：CATGATTACGAATTCTAAGTTCATGGCATACCCTGTGTC
	CHR00075330	F：GCCAGTGCCAAGCTTCTATGGACGATGATGAAAAGCC
		R：CATGATTACGAATTCGTTGCGCATTATTGAAGGCAC

图1 蔗糖饲喂下菊花脑离体花苞的形态变化红色箭头指向雄蕊。

Fig. 1 Morphological changes of isolated flower buds of Chrysanthemum nankingense after sucrose feeding The red arrow points to the stamen.

图2 蔗糖饲喂后不同时间点菊花脑管状花和舌状花中总黄酮和总酚的含量

Fig. 2 Contents of total flavonoids and total phenols in disc florets and ray florets of Chrysanthemum nankingense after sucrose feeding

图3 有/无蔗糖饲喂的管状花不同时间点表达基因主成分分析（PCA）（A）及差异表达基因数量的比较（B、C） Su：蔗糖饲喂；NS：无蔗糖饲喂。下同。

Fig. 3 The principal component analysis（PCA）（A）of the expression genes and the comparison（B，C）of the number of differentially expressed genes in different time points of disc florets feeding with or without sucrose Su：Sucrose feeding；NS：No sucrose. The same below.

图4 蔗糖饲喂60 h后与无蔗糖培养60 h/蔗糖饲喂0 h后的管状花差异基因表达分析（A、C）和KEGG富集分析（B、D） R1 ~ R3表示每个时间点样品的3个生物学重复。

Fig. 4 Differential gene expression analysis（A，C）and KEGG enrichment analysis（B，D）of disc florets after feeding with sucrose for 60 h and feeding without sucrose for 60 h/feeding with sucrose for 0 h R1-R3 indicate three biological replicates for each time-point sample in RNAseq analysis.

图5 蔗糖饲喂对管状花类黄酮生物合成途径相关酶基因（A）和关键转录因子（B）表达的影响

Fig. 5 Effects of sucrose feeding on the expression of genes（A）related to flavonoid biosynthesis pathway and key transcription factors（B）in disc florets

图6 类黄酮生物合成相关酶基因与重要转录因子的相关性分析

Fig. 6 Correlation analysis between flavonoid biosynthesis related genes and important transcription factors

图7 qRT-PCR 验证蔗糖饲喂60 h后关键差异基因的表达模式

Fig. 7 Expression analysis of key differential genes after 60 h of sucrose feeding via qRT-PCR * P < 0.05.

图8 CHR00001486和CHR00075330在不同组织中表达量（A）及其编码蛋白亚细胞定位（B）不同字母表示在不同组织中基因表达差异显著（P < 0.05）。

Fig. 8 The expression of CHR00001486 and CHR00075330 in different tissues of Chrysanthemum nankingense（A）and subcellular localization of their proteins（B） Different lowercase letters indicate significant differences of genes expression in different tissues（P < 0.05）.

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