Transcriptome Analysis of the Anthocyanin Biosynthesis in the Fruit Development Processes of Lycium ruthenicum Murr.

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

Abstract

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

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.

Figures/Tables 9

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

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

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

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

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

Fig. 5 DEGs statistics in KEGG pathways

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

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

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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