紫鸭跖草根组织应答铜胁迫的转录组分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0664

摘要/Abstract

摘要：

紫鸭跖草是一种高耐铜的超积累植物,本研究首次应用RNA-Seq技术对其转录组进行分析。通过全长转录组分析组装了紫鸭跖草耐铜相关候选基因,通过转录组分析,共获得82 471条N50长度为2 299 bp的高质量unigene,为紫鸭跖草的进一步研究提供了丰富的数据。对照组（CK）、300 µmol/L胁迫组（CT1）和1 000 µmol/L胁迫组（CT2）的测序数据已保存在NCBI的SRA数据库中,登录号为SAMN11265427。CT1相比于CK,共有5 028条unigene在根组织中有显著性差异表达,约占全部unigene的6.10%,其中富集上调和下调的基因分别为3 138和1 890条;CT2相比于CT1,根中共有6 813个unigene富集差异表达,占所有unigene的8.26％。其中富集上调和下调的基因分别为2 555和4 258。随机选取10个基因进行qRT-PCR荧光定量分析,结果与Illumina测序数据一致,验证了基因数据差异表达的有效性。上述实验从分子水平上为研究铜胁迫下铜耐受的分子机制提供了理论依据。

关键词: 紫鸭跖草, 根组织, 铜胁迫, 转录组

Abstract:

Setcreasea purpurea is a highly copper（Cu）tolerant and hyperaccumulator plant. It is the first time that RNA-Seq technology was applied to profile the transcriptome of it in this study. Candidate genes related to copper tolerance of S. purpurea were assembled based on de novo transcriptome analysis. A total of 82 471 high-quality unigenes with an N50 length of 2 299 bp were obtained through transcriptome profiling,which provided abundant data for future studies on S. purpurea. The sequencing data of the control group（CK）,300 mol/L stress group（CT1）,and 1 000 mol/L stress group（CT2）were deposited in the NCBI sequence read archive database under the accession number SAMN11265427. After comparing CT1 with CK,a total of 5 028 unigenes were significantly differentially expressed in the roots,accounting for 6.10% of all unigenes. Among them,the significantly up-regulated unigenes and down-regulated unigenes were 3 138 and 1 890,respectively. After comparing CT2 with CT1,a total of 6 813 unigenes were significantly differentially expressed in the roots,accounting for 8.26% of all unigenes. Among them,the significantly up-regulated unigenes and down-regulated unigenes were 2 555 and 4 258,respectively. The results were consistent with Illumina sequencing data based on the quantitative fluorescence analysis of 10 randomly selected genes by qRT-PCR,which confirmed the validity of differentially expressed gene data. The above experiment provides a theoretical basis for examining the molecular mechanism of copper tolerance under copper stress on the molecular level.

Key words: Setcreasea purpurea, root tissue, copper stress, transcriptome

彭国颖, 胡亮, 黄超, 杨坤, 万玮, 黄长干. 紫鸭跖草根组织应答铜胁迫的转录组分析[J]. 生物技术通报, 2022, 38(2): 83-94.

PENG Guo-ying, HU Liang, HUANG Chao, YANG Kun, WAN Wei, HUANG Chang-gan. Transcriptome Analysis of Response to Heavy Metal Copper Stress in Setcreasea purpurea Root Tissue[J]. Biotechnology Bulletin, 2022, 38(2): 83-94.

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参考文献 38

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序列号Unigene ID	注释Annotated	序列Sequence（5'-3'）	Tm/℃	长度Length/bp
c1920_g1	磷酸化酶Phosphorylase	F：ATGTGGGCTCTGTCGTT R：TGCCTGCTGTGCTAATG	58/60	178
c48219_g1	羧酸酯酶12 Carboxylesterase 12	F：TTTGCGAGGTTGGGTAT R：AAGAGTGGCCGAGTTGA	60/60	191
c1055_g1	铁通透酶基因1 Fe transporter permease 1	F：TACCTGCCCGCCGTGTT R：TGATGACTGCCGCCTCC	60/59	154
c1246_g1	金属尼古丁胺转运蛋白9 Metal nicotine amine transporter 9	F：ATGTTAGAGTGAGATACCCTGT R：AATGGTTATGGCGTTGG	60/60	177
c1178_g1	磷酸酯酶2C Protein phosphatase 2C	F：ATTACCTACGAGGAGCG R：CCAACCTTATCCCACAG	58/60	164
c51710_g1	谷胱甘肽转移酶 Glutathione transferase	F：GAGCAAAGTAGGCATCG R：ACTAACGGCTTGAGGGT	59/60	158
c18811_g1	生长素结合蛋白 Auxin binding protein	F：GCACTGGGTCTGCCTGAT R：TGGAGTTCGCCGATGAG	56/60	146
c2112_g1	硝酸根转运蛋白 Nitrate transporter	F：CAAAGCCAACATAATACAAG R：TTGGGAGACTGAGAAGGT	60/58	168
c30939_g1	水通道蛋白 Aquaporin	F：ACCCTTCTGGCTTAGTG R：AATGATGGTGATGTGGC	57/60	121
c113_g1	假定蛋白21 Hypothetical protein 21	F：GGCTCTTTCTGGCTCGTA R：CAAGTTCATTGGCACCC	56/60	138

序列号Unigene ID	注释Annotated	序列Sequence（5'-3'）	Tm/℃	长度Length/bp
c1920_g1	磷酸化酶Phosphorylase	F：ATGTGGGCTCTGTCGTT R：TGCCTGCTGTGCTAATG	58/60	178
c48219_g1	羧酸酯酶12 Carboxylesterase 12	F：TTTGCGAGGTTGGGTAT R：AAGAGTGGCCGAGTTGA	60/60	191
c1055_g1	铁通透酶基因1 Fe transporter permease 1	F：TACCTGCCCGCCGTGTT R：TGATGACTGCCGCCTCC	60/59	154
c1246_g1	金属尼古丁胺转运蛋白9 Metal nicotine amine transporter 9	F：ATGTTAGAGTGAGATACCCTGT R：AATGGTTATGGCGTTGG	60/60	177
c1178_g1	磷酸酯酶2C Protein phosphatase 2C	F：ATTACCTACGAGGAGCG R：CCAACCTTATCCCACAG	58/60	164
c51710_g1	谷胱甘肽转移酶 Glutathione transferase	F：GAGCAAAGTAGGCATCG R：ACTAACGGCTTGAGGGT	59/60	158
c18811_g1	生长素结合蛋白 Auxin binding protein	F：GCACTGGGTCTGCCTGAT R：TGGAGTTCGCCGATGAG	56/60	146
c2112_g1	硝酸根转运蛋白 Nitrate transporter	F：CAAAGCCAACATAATACAAG R：TTGGGAGACTGAGAAGGT	60/58	168
c30939_g1	水通道蛋白 Aquaporin	F：ACCCTTCTGGCTTAGTG R：AATGATGGTGATGTGGC	57/60	121
c113_g1	假定蛋白21 Hypothetical protein 21	F：GGCTCTTTCTGGCTCGTA R：CAAGTTCATTGGCACCC	56/60	138

样本 Sample	原始数据 Raw reads/Mb	过滤数据 Clean reads /Mb	碱基总数 Total number of bases/Gb	Q20/%	Q30/%	过滤数据率 Clean reads ratio/%
CK-1	46.39	42.94	7.00	96.91	92.50	92.58
CK-2	44.77	41.86	6.76	96.86	92.44	93.51
CK-3	43.28	40.72	6.54	96.88	92.36	94.07
CT1-1	44.58	41.71	6.73	96.82	92.34	93.56
CT1-2	50.21	47.17	7.58	96.89	92.36	93.95
CT1-3	44.60	41.94	6.73	96.67	91.91	94.03
CT2-1	47.85	44.95	7.23	96.79	92.17	93.93
CT2-2	40.67	38.21	6.14	96.92	92.43	93.95
CT2-3	45.70	42.69	6.90	96.91	92.42	93.40

样本 Sample	原始数据 Raw reads/Mb	过滤数据 Clean reads /Mb	碱基总数 Total number of bases/Gb	Q20/%	Q30/%	过滤数据率 Clean reads ratio/%
CK-1	46.39	42.94	7.00	96.91	92.50	92.58
CK-2	44.77	41.86	6.76	96.86	92.44	93.51
CK-3	43.28	40.72	6.54	96.88	92.36	94.07
CT1-1	44.58	41.71	6.73	96.82	92.34	93.56
CT1-2	50.21	47.17	7.58	96.89	92.36	93.95
CT1-3	44.60	41.94	6.73	96.67	91.91	94.03
CT2-1	47.85	44.95	7.23	96.79	92.17	93.93
CT2-2	40.67	38.21	6.14	96.92	92.43	93.95
CT2-3	45.70	42.69	6.90	96.91	92.42	93.40

数据库 Database	注释基因的数量Number of annotated unigenes	注释基因的比例Percentage of annotated unigenes/%
NR	68 227	82.73
GO	23 236	28.17
KEGG	35 123	42.59
Pfam	27 806	33.72
eggNOG	67 602	81.97
Swissprot	52 994	64.26
In all database	8 422	10.21