黑果枸杞茎叶响应NaCl胁迫合成花色苷的转录组学分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1576

摘要/Abstract

摘要：

为深入理解黑果枸杞响应盐胁迫合成花色苷的过程，用300 mmol/L NaCl胁迫黑果幼苗，3 d后，收集茎叶，采用液相色谱-串联质谱联用（LC-MS/MS）及转录组测序技术，分别测定其花色苷含量及转录组。通过对花色苷种类、含量及差异表达基因的GO和KEGG分析，挖掘黑果枸杞茎叶响应NaCl胁迫合成花色苷的基因，利用RT-qPCR的方法验证转录组测序结果。结果表明，NaCl处理后，黑果枸杞茎叶中飞燕草素-3-O-芸香糖苷含量升高倍数最高（11.9倍），矮牵牛素-3-O-芸香糖苷含量最高（5.313 ± 0.286）μg/g。共筛选到差异表达基因1 416个（P<0.01），其中867个上调，549个下调，功能可归类于催化活性、光合作用、单一有机体代谢过程等GO条目，显著富集于14条KEGG代谢通路。NaCl胁迫后，有7个差异表达基因参与花色苷的合成，显著上调；27个差异表达基因参与植物激素信号转导，其中，15个属ABA信号转导通路；11个MYB和5个bHLH转录因子表达量显著变化。综上所述，ABA信号转导通路、MYB、bHLH转录因子及花色苷合成通路基因的表达变化在黑果枸杞茎叶响应NaCl胁迫合成花色苷的过程中发挥重要作用。

关键词: 黑果枸杞, 茎叶, NaCl, 花色苷, 分子调控

Abstract:

In order to analyze the process of Lycium ruthenicum Murr.（L. ruthenicum）shoots in anthocyanin biosynthesis response to salt stress，L. ruthenicum seedlings were treated with 300 mmol/L NaCl，and the shoots were collected at 3 d after treatment，and the anthocyanin content and transcriptome were analyzed by LC-MS/MS and RNA-seq. Combined with the analysis of species and contents of anthocyanin and differential expressed genes（DEGs）in GO and KEGG pathway，DEGs related to anthocyanin biosynthesis induced by salt stress were mined，and the results of transcriptome data were verified by RT-qPCR. The results showed delphinidin-3-O-rutinoside in L. ruthenicum shoots was the highest upregulated anthocyanin（11.9 times），and the content of petunia-3-O-rutinoside was the highest anthocyanin（5.313±0.286）μg/g. Total 1 416 DEGs（P<0.01）were screened，including 867 up-regulated and 549 down-regulated DEGs，which were classified into GO terms，such as catalytic activity，photosystem and single-organism metabolic process. DEGs were significantly enriched in 14 KEGG pathways. Meanwhile，there were 7 DEGs involved in anthocyanin biosynthesis and all of them were up-regulated，and27 DEGs were involved in phytohormone signal transduction，in which 15 DEGs were related to ABA signal transduction pathway. The expressions of 11 MYB and 7 bHLH transcription factors were significantly changed. Sum up，the results of this study suggested that DEGs related to ABA pathway，MYB，bHLH transcription factor and anthocyanin biosynthesis play important role in anthocyanin biosynthesis in L.ruthenicum shoot responses to NaCl stress.

Key words: Lycium ruthenicum Murr., shoots, NaCl, anthocyanin, molecular modulation

郭嫒, 姜牧炎, 哈力马提·巴合太力, 刘煜媛, 王静. 黑果枸杞茎叶响应NaCl胁迫合成花色苷的转录组学分析[J]. 生物技术通报, 2022, 38(10): 173-183.

GUO Ai, JIANG Mu-yan, Ha Li-ma-ti·Ba He-tai-li, LIU Yu-yuan, WANG Jing. Transcriptome Analysis of Lycium ruthenicum Murr. Shoots in Anthocyanin Biosynthesis Response to Salt Stress[J]. Biotechnology Bulletin, 2022, 38(10): 173-183.

图/表 11

参考文献 32

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基因ID Gene ID	序列Sequence（5'-3'）
LrH2B1	F：AGTGCTTCCTGGTGAATTGG
LrH2B1	R：TGGATAATACCTAGCCCTAGTTTCC
Cluster-45.236520	F：TTTGCCTCTGGCTTTGTCTACC
Cluster-45.236520	R：TGGATACTGCTGTTGCCTTCAT
Cluster-45.192144	F：TGCACTGTTTGTCTCCTAGCCTCC
Cluster-45.192144	R：ACGCCATTCTTCTCGTCTCCATC
Cluster-45.35768	F：AAAACAGGACATGGCCTTTATG
Cluster-45.35768	R：CTGAGTTTGTAGGCAAGGTGGA
Cluster-45.156739	F：GCACAAAGTCTCCGAGAAGG
Cluster-45.156739	R：GGGGGCTTCATATTTCTTGTT
Cluster-45.33457	F：GAAGCCAACAGGTGAAACGACG
Cluster-45.33457	R：TCTTCATCAAAGCTCCCAAATGCT
Cluster-45.168632	F：GGTTTGATGAAAGGGGTGAAGAA
Cluster-45.168632	R：CCTGACGTGGCAGTATCGCTGAG，
Cluster-45.207146	F：TGAAGAATTAAGAGAGTCGAGGTCG
Cluster-45.207146	R：AAGTTGGCAATGGTGATGGTGTC.
Cluster-45.136887	F：TCAGATGAACCCAATTCACCAA
Cluster-45.136887	R：ACCCTAAAGAGCTGTCGGATGT
Cluster-45.146553	F：GGCACAGTAGCTGAATCCAAGA
Cluster-45.146553	R：CCTTTACCAACTTCTGCCCATA
Cluster-45.150170	F：GAGCAAGGCAGCAATAGCAGAT
Cluster-45.150170	R：AAGGTGGTGACATTGAGGTGAAG
Cluster-45.146155	F：GGTCAAGCCACTGATAGCCACA
Cluster-45.146155	R：GCATTCCTGCTTTCCCTGTGAT
Cluster-45.117764	F：TATCTTTCTTAGGCCCTTCCCTC
Cluster-45.117764	R：GCGTTATTGTTATTATTGTCGGTGT

基因ID Gene ID	序列Sequence（5'-3'）
LrH2B1	F：AGTGCTTCCTGGTGAATTGG
LrH2B1	R：TGGATAATACCTAGCCCTAGTTTCC
Cluster-45.236520	F：TTTGCCTCTGGCTTTGTCTACC
Cluster-45.236520	R：TGGATACTGCTGTTGCCTTCAT
Cluster-45.192144	F：TGCACTGTTTGTCTCCTAGCCTCC
Cluster-45.192144	R：ACGCCATTCTTCTCGTCTCCATC
Cluster-45.35768	F：AAAACAGGACATGGCCTTTATG
Cluster-45.35768	R：CTGAGTTTGTAGGCAAGGTGGA
Cluster-45.156739	F：GCACAAAGTCTCCGAGAAGG
Cluster-45.156739	R：GGGGGCTTCATATTTCTTGTT
Cluster-45.33457	F：GAAGCCAACAGGTGAAACGACG
Cluster-45.33457	R：TCTTCATCAAAGCTCCCAAATGCT
Cluster-45.168632	F：GGTTTGATGAAAGGGGTGAAGAA
Cluster-45.168632	R：CCTGACGTGGCAGTATCGCTGAG，
Cluster-45.207146	F：TGAAGAATTAAGAGAGTCGAGGTCG
Cluster-45.207146	R：AAGTTGGCAATGGTGATGGTGTC.
Cluster-45.136887	F：TCAGATGAACCCAATTCACCAA
Cluster-45.136887	R：ACCCTAAAGAGCTGTCGGATGT
Cluster-45.146553	F：GGCACAGTAGCTGAATCCAAGA
Cluster-45.146553	R：CCTTTACCAACTTCTGCCCATA
Cluster-45.150170	F：GAGCAAGGCAGCAATAGCAGAT
Cluster-45.150170	R：AAGGTGGTGACATTGAGGTGAAG
Cluster-45.146155	F：GGTCAAGCCACTGATAGCCACA
Cluster-45.146155	R：GCATTCCTGCTTTCCCTGTGAT
Cluster-45.117764	F：TATCTTTCTTAGGCCCTTCCCTC
Cluster-45.117764	R：GCGTTATTGTTATTATTGTCGGTGT

物质Compound	CK含量CK content/（μg·g^-1）	Na含量Na content/（μg·g^-1）	P值P -value	倍数变化Fold change
飞燕草素-3-O-葡萄糖苷 Delphinidin-3-O-glucoside	0.151±0.0409	1.13±0.0283	0.000027	7.5
飞燕草素-3-O-芸香糖苷 Delphinidin-3-O-rutinoside	0.432±0.0581	5.15±0.134	0.0000655	11.9
矮牵牛素-3-O-芸香糖苷 Petunidin-3-O-rutinoside	2.33±0.323	5.313±0.286	0.000659	2.28
柚皮素-7-O-葡萄糖苷 Naringenin-7-O-glucoside	0.112±0.0189	0.339±0.0457	0.0015	3.02

物质Compound	CK含量CK content/（μg·g^-1）	Na含量Na content/（μg·g^-1）	P值P -value	倍数变化Fold change
飞燕草素-3-O-葡萄糖苷 Delphinidin-3-O-glucoside	0.151±0.0409	1.13±0.0283	0.000027	7.5
飞燕草素-3-O-芸香糖苷 Delphinidin-3-O-rutinoside	0.432±0.0581	5.15±0.134	0.0000655	11.9
矮牵牛素-3-O-芸香糖苷 Petunidin-3-O-rutinoside	2.33±0.323	5.313±0.286	0.000659	2.28
柚皮素-7-O-葡萄糖苷 Naringenin-7-O-glucoside	0.112±0.0189	0.339±0.0457	0.0015	3.02

样品Sample	原始测序数据Raw reads	过滤后测序数据Clean reads	过滤后数据Clean bases	Error/%	Q20/%	Q30/%	GC content/%
CK1	55 697 476	54 900 826	8.24 G	0.02	98.48	95.29	42.65
CK2	60 551 930	59 757 658	8.96 G	0.03	97.2	91.95	42.48
CK3	55 743 892	54 933 528	8.24 G	0.03	97.25	92.05	42.53
Na1	61 583 804	61 024 066	9.15 G	0.03	96.83	91.12	42.21
Na2	46 367 542	45 803 662	6.87 G	0.02	98.39	95.06	42.32
Na3	47 635 904	46 871 318	7.03 G	0.02	98.48	95.32	42.31