镉胁迫下桑树幼苗的转录组分析

doi:10.11924/j.issn.1000-6850.casb2022-0374

摘要/Abstract

摘要：

为研究桑树幼苗对重金属镉胁迫应答的分子机制，挖掘与镉胁迫相关的功能基因，利用Illumina HiSeq测序方法对正常和CdCl₂胁迫条件下的桑树幼苗进行转录组学研究。基于从头转录组组装，从镉胁迫下的桑树幼苗中分离到39758个非冗余转录本。这些转录本的平均长度和N50分别为1135、1968 bp，平均GC含量为41.72%。从不同的镉胁迫时期鉴定得到15882个差异表达基因。KEGG途径富集分析表明，这些差异表达基因分别主要参与光合作用、次生代谢和能量代谢相关途径。此外，36个转录因子家族的148个转录因子在不同镉胁迫时期存在差异表达，包括bHLH、MYB、B3、NAC、MYB-related等。qRT-PCR显示差异表达基因的表达谱与RNA-Seq分析结果一致。研究结果可为了解桑树对镉的耐受机制提供依据。

关键词: 桑树, 镉胁迫, 转录组, 生物信息, 基因表达

Abstract:

To investigate the molecular mechanism of mulberry seedlings responding to cadmium (Cd) stress, and mine the genes closely related to Cd stress, the transcriptomes derived from mulberry seedlings grown under normal and CdCl₂ stress conditions were studied using the Illumina HiSeq method. In this study, 39758 non-redundant transcripts were isolated from mulberry seedlings based on de novo transcriptome assembly. The average length and N50 of these transcripts were 1135 and 1968 bp, respectively, and the average GC content was 41.72%. 15882 differentially expressed genes (DEGs) were identified from different Cd stress periods. KEGG enrichment analysis showed that these DEGs were mainly involved in photosynthesis, secondary metabolism and energy metabolism. In addition, 148 transcription factors (TFs) representing 36 TF families were differentially expressed in different Cd stress periods, including bHLH, MYB, B3, NAC and MYB-related. qRT-PCR showed that the expression profile of DEGs was consistent with RNA-Seq analysis. These results could provide a basis for further understanding of the Cd tolerance mechanism of mulberry.

Key words: mulberry, cadmium stress, transcriptome, bioinformatics, gene expression

王晓红, 韩世玉, 孙运朋, 张芳, 罗泽虎, 陈彪. 镉胁迫下桑树幼苗的转录组分析[J]. 中国农学通报, 2023, 39(13): 25-34.

WANG Xiaohong, HAN Shiyu, SUN Yunpeng, ZHANG Fang, LUO Zehu, CHEN Biao. Transcriptome Analysis of Mulberry Seedlings Under Cadmium Stress[J]. Chinese Agricultural Science Bulletin, 2023, 39(13): 25-34.

图/表 11

参考文献 28

[1]	ASGHER M, KHAN M I R, ANJUM N A, et al. Minimising toxicity of cadmium in plants-role of plant growth regulators[J]. Protoplasma, 2015, 252:399-413. doi: 10.1007/s00709-014-0710-4 URL
[2]	ALI H, KHAN E, SAJAD M A. Phytoremediation of heavy metals-Concepts and applications[J]. Chemosphere, 2013, 91(7):869-881. doi: 10.1016/j.chemosphere.2013.01.075 URL
[3]	YANG J, LI K, ZHENG W, et al. Characterization of early transcriptional responses to cadmium in the root and leaf of Cd-resistant Salix matsudana Koidz[J]. BMC Genomics, 2015, 16:705. doi: 10.1186/s12864-015-1923-4 URL
[4]	秦俭, 何宁佳, 黄先智, 等. 桑树生态产业与蚕丝业的发展[J]. 蚕业科学, 2010, 36(6):984-989.
[5]	徐宁, 俞燕芳, 毛平生, 等. 桑树修复土壤重金属污染的研究进展[J]. 农学学报, 2015, 5(1):37-40. doi: 10.11923/j.issn.2095-4050.2014-xb0350
[6]	张兴, 王冶, 揭雨成, 等. 桑树对矿区土壤中重金属的原位去除效应研究[J]. 中国农学通报, 2012, 28(7):59-63.
[7]	HUANG R Z, JIANG Y B, JIA C H, et al. Subcellular distribution and chemical forms of cadmium in Morus alba L.[J]. International journal of phytoremediation, 2018, 20(5):448-453. doi: 10.1080/15226514.2017.1365344 URL
[8]	秦芳, 胥晓, 刘刚, 等. 桑树幼苗对铅污染的生理耐性和积累能力的性别差异[J]. 环境科学学报, 2014, 34(10):2615-2623.
[9]	FAN W, LIU C, CAO B, et al. Genome-wide identification and characterization of four gene families putatively involved in cadmium uptake, translocation and sequestration in mulberry[J]. Frontiers in plant science, 2018, 9:879. doi: 10.3389/fpls.2018.00879 pmid: 30008726
[10]	CHEN Y, MAO H, WU N, et al. Different tolerance of photosynthetic apparatus to Cd stress in two rice cultivars with the same leaf Cd accumulation[J]. Acta physiologiae plantarum, 2019, 41(12):191. doi: 10.1007/s11738-019-2981-z
[11]	SINGH S, PARIHAR P, SINGH R, et al. Heavy metal tolerance in plants: Role of transcriptomics, proteomics, metabolomics, and ionomics[J]. Frontiers in plant science, 2015, 6:1143. doi: 10.3389/fpls.2015.01143 pmid: 26904030
[12]	王鹏云, 晁代印. 重金属污染的植物修复及相关分子机制[J]. 生物工程学报, 2020, 36(3):426-435.
[13]	UNAMBA C I N, NAG A, SHARMA R K. Next generation sequencing technologies: The doorway to the unexplored genomics of non-model plants[J]. Frontiers in plant science, 2015, 6:1074. doi: 10.3389/fpls.2015.01074 pmid: 26734016
[14]	YU R, LI D, DU X, et al. Comparative transcriptome analysis reveals key cadmium transport-related genes in roots of two pak choi (Brassica rapa L. ssp. chinensis) cultivars[J]. BMC Genomics, 2017, 18:587. doi: 10.1186/s12864-017-3973-2 URL
[15]	XU L, WANG Y, LIU W, et al. De novo sequencing of root transcriptome reveals complex cadmium-responsive regulatory networks in radish (Raphanus sativus L.)[J]. Plant science, 2015, 236:313-323. doi: 10.1016/j.plantsci.2015.04.015 URL
[16]	XU Z, DONG M, PENG X, et al. New insight into the molecular basis of cadmium stress responses of wild paper mulberry plant by transcriptome analysis[J]. Ecotoxicology and environmental safety, 2019, 171:301-312. doi: S0147-6513(18)31383-6 pmid: 30612018
[17]	李军, 赵爱春, 王茜龄, 等. 三个桑树肌动蛋白基因的克隆与组织表达分析[J]. 作物学报, 2011, 37(4):641-649.
[18]	VERBRUGGEN N, JURANIEC M, BALIARDINI C, et al. Tolerance to cadmium in plants: The special case of hyperaccumulators[J]. Biometals, 2013, 26(4):633-638. doi: 10.1007/s10534-013-9659-6 pmid: 23881358
[19]	GUO Z, ZENG P, XIAO X, et al. Physiological, anatomical, and transcriptional responses of mulberry (Morus alba L. ) to Cd stress in contaminated soil[J]. Environmental pollution, 2021, 284:117387. doi: 10.1016/j.envpol.2021.117387 URL
[20]	YUE R, LU C, QI J, et al. Transcriptome analysis of cadmium-treated roots in maize (Zea mays L.)[J]. Frontiers in plant science, 2016, 7:1298.
[21]	TAN M, CHENG D, YANG Y, et al. Co-expression network analysis of the transcriptomes of rice roots exposed to various cadmium stresses reveals universal cadmium-responsive genes[J]. BMC Plant biology, 2017, 17(1):194. doi: 10.1186/s12870-017-1143-y pmid: 29115926
[22]	KRETZSCHMAR T, BURLA B, LEE Y, et al. Functions of ABC transporters in plants[J]. Essays in biochemistry, 2011, 50:145-160. doi: 10.1042/bse0500145 pmid: 21967056
[23]	BRUNETTI P, ZANELLA L, DE PAOLIS A, et al. Cadmium-inducible expression of the ABC-type transporter AtABCC3 increases phytochelatin-mediated cadmium tolerance in Arabidopsis[J]. Journal of experimental botany, 2015, 66(13):3815-3829. doi: 10.1093/jxb/erv185 URL
[24]	IANNONE M F, GROPPA M D, BENAVIDES M P. Cadmium induces different biochemical responses in wild type and catalase-deficient tobacco plants[J]. Environmental and experimental botany, 2015, 109:201-211. doi: 10.1016/j.envexpbot.2014.07.008 URL
[25]	ZHOU C, ZHANG K, LIN J, et al. Physiological responses and tolerance mechanisms to cadmium in Conyza canadensis[J]. International journal of phytoremediation, 2015, 17(3):280-289. doi: 10.1080/15226514.2014.898021 URL
[26]	JALMI S K, BHAGAT P K, VERMA D, et al. Traversing the links between heavy metal stress and plant signaling[J]. Frontiers in plant science, 2018, 9:12. doi: 10.3389/fpls.2018.00012 pmid: 29459874
[27]	HU S, YU Y, CHEN Q, et al. OsMYB45 plays an important role in rice resistance to cadmium stress[J]. Plant science: An international journal of experimental plant biology, 2017, 264:1-8.
[28]	YANG G, WANG C, WANG Y, et al. Overexpression of ThVHAc1 and its potential upstream regulator, ThWRKY7, improved plant tolerance of cadmium stress[J]. Scientific reports, 2016, 6:18752. doi: 10.1038/srep18752

基因ID	上游引物	下游引物	扩增长度/bp
TRINITY_DN6126_c0_g1	CGGCAAGTCAGCAATCAGCAAATC	TGAAGCACTGGGCAAAACCTATGG	150
TRINITY_DN10621_c0_g2	GTGGTGGAAGGAATGTTGGGAAGG	GGCTCGGTGTGGAAATGGGATTG	126
TRINITY_DN9170_c2_g1	CCGACGAGGAATATGAGCCAATGG	TGTGCCCTTTACGAACCACCAATAG	80
TRINITY_DN7122_c0_g1	TCGAAGTTCCCAAGTTGCATCAGG	GGCTCTTCATGCTTTGGCTCCTC	85
MaACT3	GGTTCCCATCTACGAAGGCTATGC	CGGCTGTGGTGGTGAAAGAGTAAC	128

基因ID	上游引物	下游引物	扩增长度/bp
TRINITY_DN6126_c0_g1	CGGCAAGTCAGCAATCAGCAAATC	TGAAGCACTGGGCAAAACCTATGG	150
TRINITY_DN10621_c0_g2	GTGGTGGAAGGAATGTTGGGAAGG	GGCTCGGTGTGGAAATGGGATTG	126
TRINITY_DN9170_c2_g1	CCGACGAGGAATATGAGCCAATGG	TGTGCCCTTTACGAACCACCAATAG	80
TRINITY_DN7122_c0_g1	TCGAAGTTCCCAAGTTGCATCAGG	GGCTCTTCATGCTTTGGCTCCTC	85
MaACT3	GGTTCCCATCTACGAAGGCTATGC	CGGCTGTGGTGGTGAAAGAGTAAC	128

样本编号	原始序列/条	干净序列/条	干净序列占比/%	Q30/%
T1N	42387676	40262764	94.99	93.04
T2N	37398420	36139366	96.63	93.44
T3N	47838968	46868954	97.97	93.43
T1P	49460056	47208418	95.45	92.15
T2P	46339116	45219660	97.58	93.05
T3P	42929410	41838552	97.46	93.32

样本编号	原始序列/条	干净序列/条	干净序列占比/%	Q30/%
T1N	42387676	40262764	94.99	93.04
T2N	37398420	36139366	96.63	93.44
T3N	47838968	46868954	97.97	93.43
T1P	49460056	47208418	95.45	92.15
T2P	46339116	45219660	97.58	93.05
T3P	42929410	41838552	97.46	93.32

数据库	注释基因/个	占比/%
Pfam	14417	36.26
NR	26280	66.10
NT	25489	64.11
Swiss-prot	18589	46.76
KEGG	7201	18.11
KOG	13734	34.54
GO	18606	46.80
All	39758	100