Characteristics Analysis of Seed Microrhizome Gene Expression of Polygonatum cyrtonema

doi:10.13560/j.cnki.biotech.bull.1985.2023-0115

Abstract

Abstract:

The germination process of Polygonatum cyrtonema seeds have a special germination phenomenon of micro rhizomes formation. The purpose of explaining the changes of key genes in the germination process is to lay a foundation for related research on seed physiology, morphological structure development and so on. In this paper, based on high-throughput sequencing technology, the transcriptome sequencing and bioinformatics analysis were performed on the seeds of P. cyrtonema in different germination states. The results showed that 17 907 differentially expressed genes were identified in microrhizome formation, compared to radicle breakthrough seed coat, these genes were in significantly different expressions in terms of metabolic process, catalytic activity, protein phosphorylation, etc. Pathway significance enrichment indicated that the differential genes were mainly enriched in plant hormone signal transduction, starch and sucrose metabolism, flavonoid biosynthesis, and other pathways. Genes enriched were abundantly expressed in plant hormone signal transduction pathways. Especially, the expression genes were up-regulated in brassinosteroid biosynthesis pathway. There were 26 833 significant differences Unigenes before and after the microrhizomes turned green, mainly enriched in metabolic process, peptide biosynthesis process, catalytic activity and other pathways. The differential genes were mainly enriched in, starch and sucrose metabolism, photosynthesis and other pathways by KEGG pathway enrichment analysis. The germination process of P. cyrtonema seeds is a complex regulatory network of a series of genes, and genes related with in brassinosteroid biosynthesis pathway may have an important role in the formation of microhizome. The results also provides a reference for in-depth research on the micro-rhizome formation physiology, micro-rhizome rapid expansion in production, and microhizom exploitation.

Key words: Polygonatum cyrtonema, seeds, microrhizome, plant hormone, starch and sucrose, photosynthesis, brassinosteroid

LIU Bao-cai, CHEN Jing-ying, ZHANG Wu-jun, HUANG Ying-zhen, ZHAO Yun-qing, LIU Jian-chao, WEI Zhi-cheng. Characteristics Analysis of Seed Microrhizome Gene Expression of Polygonatum cyrtonema[J]. Biotechnology Bulletin, 2023, 39(8): 220-233.

Figures/Tables 14

Fig. 1 Germination process of P. cyrtonema seeds A: Radicle breaks through seed coat; B: radicle elongation; C: hypocotyl enlargement; D: microrhizomes; E: green microrhizomes; F: seedlings. A, D and E in germination stage were used as experimental materials in this experiment

Table 1 Gene-specific primers used in RT-qPCR

基因 Gene	引物 Primer	序列 Sequence（5'-3'）
AUX1	Cluster-68615.91119-F	CCTCTCCTTCTTGGTCCTGTA
AUX1	Cluster-68615.91119-R	GGTGGCTCAACTTATGATGCT
AUX2	Cluster-68615.87861-F	TGCTCATCCATCAGTTCATAACC
AUX2	Cluster-68615.87861-R	GAATACGATTGCGAGGAACCATA
CH3	Cluster-58608.0-F	AAGAAGACCTCCAGAAGAGCATA
CH3	Cluster-58608.0-R	GACAATCTCCCAGAACAACACAT
TF	Cluster-68615.45537-F	CTTCCTGCTGCTTTCTCTTAGTG
TF	Cluster-68615.45537-R	TGCGGCTGCTCAGATTATTG
SnPK2	Cluster-68615.69985-F	ATGCTGATGACTCTGATTCTGATG
SnPK2	Cluster-68615.69985-R	GCCTACAACCGATACTTCTGATAC
MYC2	Cluster-68615.81195-F	AGATCAGCTCAGCTTCCATCA
MYC2	Cluster-68615.81195-R	CATTCCAGTTCCTTCGCCATT
β-glucosidase	Cluster-68615.43996-F	ACAAGGGTTAATGGGACTTCTCT
β-glucosidase	Cluster-68615.43996-R	CTGAAGAATCGCCTCCAAGTG
Actin	Actin_Cluster-F	CACCGATTGACACAAGGAGAG
Actin	Actin_Cluster-R	AGGATGGCTTACTACATTGACTTC

Table 2 Mapped results of sample reads and assembly transcripts

样品 Sample	总读长 Total reads/bp	总匹配数 Total mapped ones/bp
A1	40 179 174	29 824 074（74.23%）
A2	45 826 078	32 824 940（71.63%）
A3	44 560 782	32 498 548（72.93%）
D1	45 873 682	34 073 516（74.28%）
D2	41 999 498	31 537 406（75.09%）
D3	45 760 904	33 679 092（73.60%）
E1	41 290 430	30 888 404（74.81%）
E2	44 373 676	33 739 212（76.03%）
E3	44 063 488	33 299 498（75.57%）

Table 3 FPKM interval statistics of sample expressed levels

样品 Sample	FPKM 区间FPKM Interval
样品 Sample	0≤FPKM≤0.1	0.1<FPKM≤0.3	0.3<FPKM≤3.57	3.57<FPKM≤15	15<FPKM≤60	FPKM>60
A1	45 729（25.64%）	8 928（5.01%）	84 666（47.48%）	26 418（14.82%）	9 731（5.46%）	2 847（1.60%）
A2	90 461（50.73%）	4 155（2.33%）	46 585（26.12%）	25 282（14.18%）	8 776（4.92%）	3 060（1.72%）
A3	92 713（51.99%）	2 373（1.33%）	42 307（23.73%）	28 240（15.84%）	9 676（5.43%）	3 010（1.69%）
D1	68 567（38.45%）	14 059（7.88%）	64 246（36.03%）	19 173（10.75%）	9 466（5.31%）	2 808（1.57%）
D2	68 767（38.56%）	13 366（7.50%）	64 688（36.28%）	19 321（10.84%）	9 411（5.28%）	2 766（1.55%）
D3	69 033（38.71%）	13 351（7.49%）	64 127（35.96%）	19 404（10.88%）	9 570（5.37%）	2 834（1.59%）
E1	63 577（35.65%）	11 564（6.49%）	70 391（39.47%）	20 459（11.47%）	9 569（5.37%）	2 759（1.55%）
E2	61 068（34.25%）	13 276（7.45%）	72 028（40.39%）	19 855（11.13%）	9 390（5.27%）	2 702（1.52%）
E3	59 900（33.59%）	13 120（7.36%）	73 014（40.95%）	20 113（11.28%）	9 453（5.30%）	2 719（1.52%）

Fig. 2 DvsA different expressed genes A01: Protein phosphorylation. A02: Phosphorylation. A03: Phosphate-containing compound metabolic process. A04: Phosphorus metabolic process. A05: Metabolic process. A06: Oxidation-reduction process. A07: Microtubule-based movement. A08: Cellular carbohydrate metabolic process. A09: Carbohydrate metabolic process. A10: Negative regulation of translation. A11: Negative regulation of cellular amide metabolic process. A12: Cellular polysaccharide metabolic process. A13: Cellular protein modification process. A14: Protein modification process. A15: Microtubule. A16: Tubulin complex. A17: Apoplast. A18: Cell wall. A19: Catalytic activity. A20: Hydrolase activity, acting on glycosyl bonds. A21:Transferase activity. A22: Tetrapyrrole binding, A23: Heme binding. A24: Protein kinase activity. A25: Hydrolase activity, hydrolyzing O-glycosyl compounds. A26: RNA glycosylase activity. A27: rRNA N-glycosylase activity. A28: Hydrolase activity. A29: Phosphotransferase activity, alcohol group as acceptor. A30: Oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen. A31: Microtubule binding. A32: Kinase activity. A33: Transferase activity, transferring acyl groups other than amino-acyl groups. A34: Oxidoreductase activity. A35: Microtubule motor activity. A36: Ion binding. A37: Iron ion binding. A38: Tubulin binding. A39: Xyloglucan: xyloglucosyl transferase activity. A40: Transferase activity, transferring phosphorus-containing groups

Fig. 3 KEGG pathway enrichment of DvsA differential gene

Fig. 4 KEGG enrichment results of TOP 20 differential genes

Fig. 5 DvsA differentially expressed genes involved in starch and sucrose metabolism The red border of the node contains up-regulated differential genes; the green border of the node contains the down-regulated differential genes; and the yellow border of the node contains the up-regulated and down-regulated differential genes. The same below

Fig. 6 DvsA differentially expressed genes involved in plant hormone signal transduction

Fig. 7 EvsD differently expressed genes B01: Ribosome biogenesis. B02: Translation. B03: Ribonucleoprotein complex biogenesis. B04: Peptide biosynthetic process. B05: Peptide metabolic process. B06: Cellular amide metabolic process. B07: Amide biosynthetic process. B08: Oxidation-reduction process. B09: Organonitrogen compound biosynthetic process. B10: Organonitrogen compound metabolic process. B11: Cellular component biogenesis. B12: Protein metabolic process. B13: Metabolic process. B14: Cellular protein metabolic process. B15: Biosynthetic process. B16: Organic substance biosynthetic process. B17: Cellular macromolecule biosynthetic process. B18: Cellular component organization or biogenesis. B19: Macromolecule biosynthetic process. B20: Cellular nitrogen compound biosynthetic process. B21: Cellular biosynthetic process. B22: Single-organism metabolic process. B23: Obsolete peroxidase reaction. B24: Ribosome. B25: Ribonucleoprotein complex. B26: Non-membrane-bounded organelle. B27: Intracellular non-membrane-bounded organelle. B28: Cytoplasmic part. B29: Cytoplasm. B30: Macromolecular complex. B31: Structural constituent of ribosome. B32: Oxidoreductase activity. B33: Structural molecule activity. B34: Catalytic activity. B35: Antioxidant activity. B36: Oxidoreductase activity, acting on peroxide as acceptor. B37: Pyrophosphatase activity. B38: Peroxidase activity. B39: Nucleoside-triphosphatase activity. B40: GTPase activity.

Fig. 8 KEGG enrichment results of TOP 20 differential genes

Fig. 10 KEGG enrichment results of TOP 20 EvsD differently expressed genes

Fig. 10 Map of EvsD differential gene in KEGG pathway ko00195

Fig. 11 Verification of seven selected DEGs by RT-qPCR Comparison of RNA-seq data（blue line chart）with RT-qPCR data（orange bar graph）. Different lowercase letters indicate significant differences in the expression levels of the same gene in different morphology of micro-rhizome development at 0.05 level

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