外源硫诱导苦荞镉胁迫响应的比较转录组学分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0951

摘要/Abstract

摘要：

Cd（镉）含量超标是影响苦荞（Fagopyrum tararicum）安全生产的主要重金属污染。为揭示外源添加硫（S）对苦荞耐Cd胁迫影响的分子机理，通过比较转录组学分析手段，分析根施100 mg/kg（NH₄）₂SO₄（S）处理与5 mg/kg CdCl₂（Cd）处理后苦荞叶片的差异表达基因及其功能。结果显示，共获得393 983 500条干净序列，鉴定到19 875个基因，其中差异表达基因共有1 760个。通过GO注释和KEGG pathway对这些差异表达基因进行分析表明，外源S添加增强谷胱甘肽还原酶途径，抑制氧化还原途径，并诱导水杨酸响应、脱落酸响应以及生长素响应途径应对Cd毒害。转录组数据鉴定到苦荞FtbHLH68受Cd+S处理特异诱导表达，模式植物拟南芥（Arabidopsis thaliana）同源基因突变体Atbhlh68表现出Cd+S处理缺陷表型，且Atbhlh68中叶绿素含量、净光合、GR活性和GSH活性变化均不响应Cd+S处理。研究结果揭示了苦荞响应Cd胁迫转录水平调控过程，阐明外源添加S增强植物抗Cd胁迫的分子机理，为外源S在苦荞抗Cd胁迫中的应用奠定理论基础并提供了作用靶点。

关键词: 镉胁迫, 外源S, 苦荞, 比较转录组, 基因功能验证, 氧化还原途径, 脱落酸, 生长素

Abstract:

Excessive Cd（cadmium）content is the main heavy metal pollution to tartary buckwheat（Fagopyrum tararicum）. To investigate the molecular mechanism of adding exogenous S on the Cd content of tartary buckwheat, differentially expressed genes in the tartary buckwheat leaves and their functions were analyzed via comparative transcriptome while adding S（100 mg/kg（NH₄）₂SO₄）on tartary buckwheat root and Cd（5 mg/kg CdCl₂）treatment. A total of 3 93 983 500 clean sequences were obtained and 19 875 expressed genes were identified, and 1 760 differentially expressed genes were obtained under different treatments. The analysis of these differentially expressed genes by GO annotation and KEGG pathway demonstrated that the exogenous S enhanced glutathione pathway, inhibited oxidoreduction process, induced the responses to salicylic acid, abscisic acid response and auxin response pathway to Cd stress. Transcriptome data identified that tartary buckwheat FtbHLH68 was specifically induced by Cd+S treatment, and the Arabidopsis thaliana’s homologous gene mutant Atbhlh68 presented a deficient phenotype under Cd+S treatment, and changes in chlorophyll content, net photosynthesis, GR activity and GSH activity in Atbhlh68 did not respond to Cd+S treatment. The results revealed the transcriptional level regulation process of tartary buckwheat in response to Cd stress, and clarified the molecular mechanism of exogenous addition of S enhancing plant resistances to Cd stress, which provides theoretical basis and target for application of exogenous S in Cd resistance of tartary buckwheat.

Key words: cadmium stress, exogenous sulfur, tartary buckwheat, comparative transcriptome, verification of gene function, oxidoreduction pathway, abscisic acid, auxin

刘辉, 卢扬, 叶夕苗, 周帅, 李俊, 唐健波, 陈恩发. 外源硫诱导苦荞镉胁迫响应的比较转录组学分析[J]. 生物技术通报, 2023, 39(5): 177-191.

LIU Hui, LU Yang, YE Xi-miao, ZHOU Shuai, LI Jun, TANG Jian-bo, CHEN En-fa. Comparative Transcriptome Analysis of Cadmium Stress Response Induced by Exogenous Sulfur in Tartary Buckwheat[J]. Biotechnology Bulletin, 2023, 39(5): 177-191.

图/表 8

表1 荧光定量PCR所用引物

Table 1 Primers for RT-qPCR

Gene	Primer-F（5'-3'）	Primer-R（5'-3'）
FtPinG0001636600.01	CAAGCGCTTTCCAAGATCGG	GCACTCATGACTTCGTTCGC
FtPinG0007992900.01	TGTTGTGTGTCCCACTCTGT	TTGGCTTGAGTCCCATGCTC
FtPinG0004475100.01	GGCTTGATATTGCTGCGGTG	TTGCCAGCTCCTGCTTAGAC
FtPinG0002599400.01	CACTTCTCCTCGCTAACGCT	CAATGGGTGAGTGGCGTAGT
FtPinG0005827500.01	GTTCGGGAAGAAGTACGGGG	CCAGCTAACCCAACCTAGCC
FtPinG0000987900.01	AAGTCCGTGGAGCAACTGAG	TGTGACTTCCTCCATGACGC
FtPinG0002633300.01	AAGACCGCAGCGTACTGAAA	TCTCAAAGGCGCACAGCTTA
FtPinG0008441500.01	GCCTTGGAGTGTCGACAAGA	TCCCTAACCCGAATGCACAC
FtPinG0001390600.01	GCTGGCGAGGAATAATTGCG	GCGCAATCCTTGCACTTGAA
FtPinG0007214300.01	AGGCATCACCAACATGGGAG	CTTGTGTGCTGAAAGTGCCC
FtPinG0002002200.01	TCACGAGGCTGGTGTTTTGA	ATCTAGTGGCAACCGCGTAG
FtPinG0000414800.01	GGGGAGAGCTCCATGTTGTG	TCCATGGCCGAATTGGTGAA

表2 样品分析结果统计表

Table 2 Statistical table of sample analysis results

样品 Sample	原始读取序列 Raw reads/bp	干净读取序列总数 Total clean reads/bp	总序列中质量值大于30的碱基数的比例 Clean Q30 bases rate/%	比对到参考基因组序列比例 Mapping rate/%	检测到表达的基因数目 Total gene
CK1	41 740 092	40 412 240	91.84	67.42	19 224
CK2	46 423 990	45 073 886	92.23	68.31	19 362
CK3	45 942 460	43 884 754	92.46	64.74	18 854
Cd1	41 384 496	40 064 950	92.9	92.22	19 109
Cd2	43 464 826	42 209 070	92.14	81.49	19 593
Cd3	46 953 848	45 429 048	92.14	85.88	19 560
Cd+S1	45 970 716	44 405 584	92.48	78.95	19 875
Cd+S2	48 106 694	46 437 132	92.12	83.07	19 593
Cd+S3	48 272 452	46 066 836	92.27	78.48	19 614

图1 黔苦4号叶片Cd处理和Cd+S处理的转录组分析结果 A：Cd处理和Cd+S处理转录组热图；B：Cd处理和Cd+S处理差异表达基因数量的统计；C：Cd处理和Cd+S处理差异表达基因韦恩图

Fig. 1 Transcriptome analysis of Cd treatment and Cd+S treatment in Qianku 4's leaves A: Transcriptome heat map in Cd treatment and Cd+S treatment. B: Statistics of the number of differentially expressed genes in Cd treatment and Cd+S treatment. C: Wayne diagram of differentially expressed genes under Cd treatment and Cd+S treatment

图2 Cd处理差异表达基因GO分析和KEGG分析 A-C：Cd处理上调基因GO分析；A：生物过程；B：细胞组分；C：分子功能；D：Cd处理上调基因KEGG分析；E-G：Cd处理下调基因GO分析；E：生物过程；F：细胞组分；G：分子功能；H：Cd处理下调基因KEGG分析

Fig. 2 GO analysis and KEGG analysis of differentially expressed genes under Cd treatment A-C: GO analysis of up-regulated genes under Cd treatment. A: Biological processes. B: Cellular components. C: Molecular function. D: KEGG analysis of up-regulated gene under Cd treatment. E-G: GO analysis of down-regulated genes under Cd treatment. E: Biological processes. F: Cell components. G: Molecular function. H: KEGG analysis of down-regulated gene under Cd treatment

图3 Cd+S处理差异表达基因GO分析和KEGG分析 A-C：Cd+S处理上调基因GO分析；A：生物过程；B：细胞组分；C：分子功能；D：Cd+S处理上调基因KEGG分析；E-G：Cd+S处理上调基因GO分析；E：生物过程；F：细胞组分；G：分子功能；H：Cd+S处理上调基因KEGG分析

Fig. 3 GO analysis and KEGG analysis of differentially expressed genes under Cd+S treatment A-C: GO analysis of up regulated genes under Cd+S treatment. A: Biological processes. B: Cellular components. C: Molecular function. D: KEGG analysis of up regulated gene under Cd+S treatment. E-G: GO analysis of down regulated genes under Cd+S treatment. E: Biological processes. F: Cell components. G: Molecular function. H: KEGG analysis of down regulated gene under Cd+S treatment

图4 Cd+S处理对比Cd处理差异表达基因GO分析和KEGG分析 A-C：Cd+S处理上调基因GO分析；A：生物过程；B：细胞组分；C：分子功能；D：Cd+S处理上调基因KEGG分析；E-G：Cd+S处理上调基因GO分析；E：生物过程；F：细胞组分；G：分子功能；H：Cd+S处理上调基因KEGG分析

Fig. 4 GO analysis and KEGG analysis of differentially expressed genes in CD+S treatment compared with CD treatment A-C: GO analysis of up-regulated genes under Cd+S treatment. A: Biological processes. B: Cellular components. C: Molecular function. D: KEGG analysis of up-regulated gene under Cd+S treatment. E-G: GO analysis of down-regulated genes under Cd+S treatment. E: Biological processes. F: Cell components. G: Molecular function. H: KEGG analysis of down-regulated gene under Cd+S treatment

图5 外源S添加影响Cd处理不同途径转录水平变化分析 A：Cd响应；B：氧化还原过程；C：谷胱甘肽还原酶；D：水杨酸响应；E：脱落酸响应；F：生长素响应；G： RT-qPCR对相关途径基因的验证

Fig. 5 Effects of exogenous S addition on transcriptional levels in different pathways of Cd treatment A: Response to cadmium ion. B: Oxidation-reduction process. C: Glutaredoxin activity. D: Response to salicylic acid. E: Response to abscisic acid. F: Response to auxin.G: Real-time quantitative PCR validation of several selected genes response to Cd stress and exogenous sulfur

图6 苦荞FtbHLH68基因功能验证 A：苦荞FtbHLH68基因表达检测；B-C：野生型Col-0和bhlh68突变体在Cd胁迫和Cd+S处理下的地上部分表型（B）以及叶片绿苗率（C）；D-E：野生型Col-0和bhlh68突变体在Cd胁迫和Cd+S处理下的根长表型（D）以及统计结果（E）；F-I：野生型Col-0和bhlh68突变体在Cd胁迫和Cd+S处理下叶绿素含量（F）、净光合（G）、GR活性（H）和GSH活性（I）考察。图中*表示在0.05水平差异显著

Fig. 6 Functional verification of tartary buckwheat FtbHLH68 gene A: Analysis of FtbHLH68 gene expression in tartary buckwheat. B-C: Phenotype (B) and green leaves rate (C) of wild-type Col-0 and bhlh68 mutants under Cd stress and Cd+S treatment. D-E: Root length phenotype (D) and statistical results (E) of wild-type Col-0 and bhlh68 mutants under Cd stress and Cd+S treatment. F-I: Chlorophyll content (F), net photosynthesis (G), GR activity (H) and GSH activity (I) of wild-type Col-0 and bhlh68 mutants under Cd stress and Cd+S treatment. * indicate significant difference at 0.05 level

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