黑果枸杞不同发育时期果实花色苷合成的转录组分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0247

摘要/Abstract

摘要：

【目的】探究黑果枸杞果实发育过程中花色苷合成的分子机制，对深入理解黑果枸杞花色苷合成调控网络具有重要意义。【方法】选取青果期、转色初期、转色后期、成熟期和完全成熟期的黑果枸杞果实进行转录组测序，挖掘与黑果枸杞果实花色苷合成相关的候选基因。【结果】随黑果枸杞果实发育，花色苷含量逐渐升高，成熟期及完全成熟期果实中的花色苷含量显著高于青果期、转色初期及转色后期。5个发育时期共筛选出13 540个差异表达基因（differentially expressed genes, DEGs），以青果期为对照，随果实发育，各阶段DEGs数量逐渐增多。GO分析发现，DEGs共同富集的子集有苯丙烷代谢过程、多糖分解代谢过程、苯丙烷生物合成过程、类黄酮生物合成过程、细胞壁大分子代谢过程、膜锚定成分和营养库活性通路。KEGG分析表明，DEGs显著富集于类黄酮生物合成、苯丙烷生物合成、植物激素信号转导、二苯乙烯类、二芳基庚烷类和姜辣素生物合成及角质和木栓质和蜡质的生物合成通路。分析DEGs表达量与花色苷含量相关性，筛选出参与黑果枸杞果实发育过程花色苷合成的候选基因36个，主要包括10个花色苷合成通路结构基因，4个转录因子，9个ABA、8个GA及5个JA信号转导通路基因。RT-qPCR验证其中10个基因的表达趋势，与转录组数据一致。【结论】黑果枸杞果实发育过程中，花色苷合成途径的结构基因、转录因子及ABA、GA和JA信号转导通路中基因的表达调控果实着色。

关键词: 黑果枸杞, 果实, 转录组, 差异表达基因, 植物激素, 花色苷生物合成调控

Abstract:

【Objective】To explore the molecular mechanism involved in anthocyanin biosynthesis during the fruit development of Lycium ruthenicum Murr. is important for deeply understanding the regulatory network of anthocyanin biosynthesis.【Method】Transcriptome sequencing was performed on the samples at the five stages of fruit development: green fruit, early color-changing, late color-changing, ripeness, and complete ripeness. The candidate genes related to anthocyanin biosynthesis were mined.【Result】With the development of L. ruthenicum Murr. fruit, the anthocyanin content gradually increased. The anthocyanin content in the fruits at the ripeness and complete ripeness stages were significantly higher than that at the green fruit, early color-changing, and late color-changing stages. A total of 13 540 differentially expressed genes（DEGs）were identified from the five stages of fruit development. Using the green fruit stage as a control, the number of DEGs gradually increased with the development of the fruit. GO analysis showed that DEGs were co-enriched GO terms including phenylpropanoid metabolic process, polysaccharide catabolic process, phenylpropanoid biosynthetic process, flavonoid biosynthetic process, cell wall macromolecule metabolic process, anchored component of membrane, and nutrient reservoir activity. KEGG analysis indicated the significant enrichment of DEGs in flavonoid biosynthesis, phenylpropanoid biosynthesis, plant hormone signal transduction, stilbenoid, diarylheptanoid and gingerol biosynthesis, as well as cutin, suberine, and wax biosynthesis. The correlation between DEGs expression and anthocyanin content was performed and 36 candidate genes involved in anthocyanin biosynthesis during the L. ruthenicum fruit development were screened, including ten structural genes of anthocyanin biosynthesis pathway, four genes of transcription factors, nine, eight, and five genes of ABA, GA, and JA signal transduction pathway. Ten DEGs were selected for RT-qPCR analysis, and the results were consistent with RNA-seq data.【Conclusion】DEGs related to structural genes and transcription factors involved in anthocyanin biosynthesis, as well as genes involved in the ABA, GA, and JA signal transduction pathways, affect fruit coloration during the development of L. ruthenicum Murr. fruit.

Key words: Lycium ruthenicum Murr., fruit, transcriptome, differentially expressed gene, plant hormone, regulation of anthocyanin biosynthesis

聂祝欣, 郭瑾, 乔子洋, 李微薇, 张学燕, 刘春阳, 王静. 黑果枸杞不同发育时期果实花色苷合成的转录组分析[J]. 生物技术通报, 2024, 40(8): 106-117.

NIE Zhu-xin, GUO Jin, QIAO Zi-yang, LI Wei-wei, ZHANG Xue-yan, LIU Chun-yang, WANG Jing. Transcriptome Analysis of the Anthocyanin Biosynthesis in the Fruit Development Processes of Lycium ruthenicum Murr.[J]. Biotechnology Bulletin, 2024, 40(8): 106-117.

图/表 9

表1 本研究所用引物序列

Table 1 Primer sequences used in this study

基因ID Gene ID	GenBank登录号GenBank number	引物序列Primer sequence（5'-3'）	扩增长度Amplification length/bp
Cluster-53530	—	F: ACCGATGAAGAACTCGTCGTCCA	228
Cluster-53530	—	R: GGTAGCCTTCCAATAACCCGATGT	228
Cluster-54040	KY287796	F: CTAAATGCCCCCAACCAGAACT	124
Cluster-54040	KY287796	R: GTTACCCACTTCCCTTCATAGA	124
Cluster-55329	OQ414189	F: TTTTGGACTGCTGGTTCTTGTT	260
Cluster-55329	OQ414189	R: TTGAGGTCTTTGTTGATTATCGG	260
Cluster-57771	—	F: CAAGGAGGAAATGCAAAGAGGAGC	254
Cluster-57771	—	R: CTATTAGGGACCATCTGTTGCCAAG	254
Cluster-59360	—	F: CCGATGTAACGGATGGTGTCTA	182
Cluster-59360	—	R: GGGGCTGATTTGTCTCTATTGC	182
Cluster-67674	KY287799	F: AGAATGGGCACTGGCAGAAATGAT	210
Cluster-67674	KY287799	R: ACTACACACGGCTCGTTTGATACC	210
Cluster-67748	KF031378	F: TCCCTTTTCCTTCCGAGTTCAT	164
Cluster-67748	KF031378	R: TGTTTTGCCCTTCCACTGCTGC	164
Cluster-73437	KY287797	F: CACGGTAAGGAGACTGGTTTTCA	167
Cluster-73437	KY287797	R: CAGCCTTCTCTGCCAGTATCTTG	167
Cluster-74326	KY287798	F: CAGTGGTGAACTCGGATAGCAGCA	117
Cluster-74326	KY287798	R: GCTCCGCCATTACTGCTTTCTCTC	117
Cluster-76366	—	F: GCAATCTCTTGTTCCTGGTTGTGAC	288
Cluster-76366	—	R: GGATACCCGCTGTTAGTGTTAGGAA	288
LrH2B1	—	F: AGTGCTTCCTGGTGAATTGG	163
LrH2B1	—	R: TGGATAATACCTAGCCCTAGTTTCC	163

图1 黑果枸杞不同发育时期果实的表型（A）和花色苷含量（B） BS1：青果期；BS2：转色初期；BS3：转色后期；BS4：成熟期；BS5：完全成熟期；以BS1为对照，** P < 0.01，*** P < 0.001。下同

Fig. 1 Phenotypes（A）and anthocyanin contents（B）of fruit at different developmental stages of L. rutheni-cum Murr. BS1: Green fruit stage. BS2: Early color-changing stage. BS3: Late color-changing stage. BS4: Ripeness stage. BS5: Complete ripeness stage. Using BS1 as a control, ** P < 0.01, *** P < 0.001. The same below

图2 黑果枸杞不同发育时期果实中差异表达基因

Fig. 2 DEGs statistics in fruits at different stages of L. ru-thenicum Murr.

图3 黑果枸杞不同发育时期果实中差异表达基因GO富集分析绿色: 生物学过程；灰色: 细胞组分；紫色: 分子功能

Fig. 3 Gene ontology annotation of DEGs in the fruits at different stages of L. ruthenicum Murr. Green: Biological process. Gray: Cell component. Purple: Molecular function

图4 黑果枸杞不同发育时期果实中差异表达基因KEGG富集分析

Fig. 4 KEGG classification of DEGs in the fruits at different stages of L. ruthenicum Murr.

图5 KEGG通路中差异表达基因数量

Fig. 5 DEGs statistics in KEGG pathways

图6 KEGG富集通路上参与花色苷生物合成的候选基因及与花色苷的相关性 R2：基因表达量（FPKM）与花色苷含量的相关性，下同

Fig. 6 Candidate genes related to anthocyanin biosynthesis in KEGG pathway and their correlation with anthocyanins R2: Correlation between gene expression（FPKM）and anthocyanin content. The same below

图7 植物激素信号转导通路关键基因表达热图 A：ABA信号转导通路；B：GA信号转导通路；C：JA信号转导通路

Fig. 7 Heat map of expressions of key genes in plant hormone signal transduction pathway A: ABA signal transduction pathway. B: GA signal transduction pathway. C: JA signal transduction pathway

图8 转录组数据验证以BS1为对照，* P < 0.05，** P < 0.01和*** P < 0.001；R2：转录组数据（RNA-seq）与RT-qPCR结果（RT-qPCR）的相关性

Fig. 8 Validation of transcriptome data Using BS1 as a control, * P < 0.05, ** P < 0.01, and *** P < 0.001. R2: Correlation between transcriptome data（RNA-seq）and RT-qPCR result（RT-qPCR）

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