基于转录组测序的狗牙根抗旱根系关键代谢途径分析

doi:10.11686/cyxb2023193

摘要/Abstract

摘要：

根系是狗牙根响应干旱胁迫的重要器官，其响应不同程度干旱的分子机制有待进一步明确。以抗旱（C138）和敏旱（C32）2个狗牙根基因型为材料，对其正常灌溉（土壤相对含水量为田间持水量的80%~90%）、中度干旱（50%~60%）及重度干旱（20%~30%）下的根系进行取样，基于Illumina高通量测序平台进行转录组测序，共获得43581条单基因，有33025条得到注释。结果表明，与正常灌溉相比，中度及重度干旱下C138共7537个差异基因表达，其中上调1164个，下调6373个；C32有6731个差异基因表达，其中上调4304个，下调2427个。Top20 GO富集分析发现，中度干旱下C138主要富集于氧化还原酶活性、碳水化合物代谢和纤维素结合等，重度干旱下C138主要富集于磷酸代谢及蛋白磷酸化等，C32主要富集于过氧化物酶活性、氧化应激反应等；KEGG Pathway富集分析发现中度干旱下C138主要富集在谷胱甘肽代谢、苯丙基类生物合成和氮代谢途径；C32主要富集在氧化磷酸化、三羧酸循环（TCA）、核糖体和碳代谢途径；重度干旱胁迫下C138主要富集在谷胱甘肽代谢、花生四烯酸代谢、核糖体酶途径；C32主要富集在谷胱甘肽代谢、苯丙基类生物合成途径。总体而言，狗牙根中度干旱胁迫基因表达响应主要与氧化反应有关，重度干旱胁迫主要与Ca²⁺通路、脱落酸（ABA）信号通路和谷胱甘肽代谢有关，因而谷胱甘肽代谢可能是狗牙根响应干旱的主要KEGG通路。且与C32相比，C138响应干旱胁迫时有更多的钙依赖性蛋白激酶相关基因表达，因而抗旱性能更强。综上，谷胱甘肽代谢和MYB转录因子相关基因可能是狗牙根抗旱的关键，可作为其干旱胁迫基因研究的首选。

关键词: 干旱胁迫, 狗牙根, 根系, 转录组

Abstract:

The root system is the most important organ of bermudagrass in response to drought stress， and the molecular mechanism of its response to different degrees of drought needs to be further clarified. In this study， two bermudagrass genotypes with contrasting drought-resistant （C138） and drought-sensitive （C32） properties， were sampled under normal irrigation （soil relative water content is 80%-90% of the field capacity）， moderate drought （50%-60% of field capacity） and severe drought （20%-30% of field capacity）. From transcriptome sequencing with the Illumina High-throughput sequencing platform， 43581 Unigenes were obtained， 33025 of which were annotated. Compared with normal irrigation， there were 7537 genes differentially expressed in C138 under moderate and severe drought， comprising 1164 up-regulated and 6373 down-regulated， while in C32， there were 4304 up-regulated and 2427 down regulated unigenes. Top20 GO enrichment analysis found that C138 was mainly enriched in oxidoreductase activity， carbohydrate metabolism and cellulose binding under moderate stress； C138 was mainly enriched in phosphate metabolism and protein phosphorylation under severe stress， and C32 was mainly enriched in peroxidase activity and oxidative stress response. KEGG Pathway enrichment analysis found that C138 was mainly enriched in glutathione metabolism， phenylpropyl biosynthesis and the nitrogen metabolism pathway， while C32 was mainly enriched in oxidative phosphorylation， tricarboxylic acid cycle， ribosomal enzymes and carbon metabolism pathways under moderate stress. Under severe drought stress， C138 was mainly enriched in glutathione metabolism， arachidoic acid metabolism and the ribosomal enzyme pathway， while C32 was mainly enriched in the glutathione metabolism and phenylpropyl biosynthesis pathways. Overall， the gene expression response to moderate drought stress in bermudagrass was mainly related to oxidative response. Severe drought stress was mainly related to the Ca²⁺ pathway， abscisic acid signaling pathway， and glutathione metabolism. Thus， glutathione metabolism might be the main KEGG pathway affected in response to drought in bermudagrass. Moreover， compared with C32， C138 had more calcium-dependent protein kinase-related gene expression in response to drought stress and thus had stronger drought resistance. In conclusion， glutathione metabolism and MYB transcription factor-related genes may be critical for drought resistance in bermudagrass and from this work are indicated as the first choice for investigation in a drought-stress gene study.

Key words: drought stress, bermudagrass, root, transcriptome

李硕, 李培英, 孙宗玖, 李雯. 基于转录组测序的狗牙根抗旱根系关键代谢途径分析[J]. 草业学报, 2024, 33(4): 186-198.

Shuo LI, Pei-ying LI, Zong-jiu SUN, Wen LI. Transcriptome analysis-based bermudagrass root RNA sequencing data under drought stress[J]. Acta Prataculturae Sinica, 2024, 33(4): 186-198.

图/表 11

参考文献 32

1	Eriyagama N， Smakhtin V， Gamage N. Mapping drought patterns and impacts： A global perspective. Colombo Sri Lanka： International Water Management Institute （IWMI）（Research Reports 133）， 2009.
2	Huang B. Mechanisms and strategies for improving drought resistance in turfgrass. Acta Horticulturae， 2008， 783（6）： 221-232.
3	Bian S， Jiang Y. Reactive oxygen species， antioxidant enzyme activities and gene expression patterns in leaves and roots of Kentucky bluegrass in response to drought stress and recovery. Scientia Horticulturae， 2009， 120（2）： 264-270.
4	Merewitz E B， Gianfagna T， Huang B. Protein accumulation in leaves and roots associated with improved drought tolerance in creeping bentgrass expressing an ipt gene for cytokinin synthesis. Journal of Experimental Botany， 2011， 62（15）： 5311-5333.
5	Cao Y J， Wei Q， Liao Y， et al. Ectopic overexpression of AtHDG11 in tall fescue resulted in enhanced tolerance to drought and salt stress. Plant Cell Reports， 2009， 28（4）： 579-588.
6	Shi H T， Wang Y P， Cheng Z M， et al. Analysis of natural variation in bermudagrass （Cynodon dactylon） reveals physiological responses underlying drought tolerance. PLoS One， 2012， 7（12）： 12-20.
7	Lu S， Chen C， Wang Z， et al. Physiological responses of somaclonal variants of triploid bermudagrass （Cynodon transvaalensis×Cynodon dactylon） to drought stress. Plant Cell Reports， 2009， 28（3）： 517-526.
8	Zhou Y， Lambrides C J， Shu F K. Drought resistance of bermudagrass （Cynodon spp.） ecotypes collected from different climatic zones. Environmental and Experimental Botany， 2013， 41（5）： 505-519.
9	Shabier A， Abudureyimu A， Younusi Q. Comparisons of drought resistance of six Xinjiang bermudagrass varieties.Journal of Xinjiang Agricultural University， 2008（2）： 17-21.
	阿力木·沙比尔，阿不来提·阿不都热依木，齐曼·尤努斯. 6份新疆狗牙根抗旱性比较. 新疆农业大学学报， 2008（2）： 17-21.
10	Duan M M. Evaluation of drought resistance and physiological response of Xingjiang wild bermudagrass germplasm. Urumqi： Xinjiang Agricultural University， 2016.
	段敏敏. 新疆野生狗牙根种质抗旱性鉴定及抗旱生理的研究. 乌鲁木齐：新疆农业大学， 2016.
11	Zeng L S， Li P Y. Evaluation on drought resistance of 10 bermudagrass （Cynodon dactylon） germplasms from Xinjiang.Chinese Journal of Grassland， 2019， 41（3）： 22-29.
	曾令霜，李培英. 10份新疆狗牙根种质抗旱性评价. 中国草地学报， 2019， 41（3）： 22-29.
12	Zhou Y， Lambrides C J， Fukai S. Drought resistance of C₄ grasses under field conditions： genetic variation among a large number of bermudagrass （Cynodon spp.） ecotypes collected from different climatic zones. Journal of Agronomy Crop Science， 2013， 199（4）： 253-263.
13	Zeng L S， Li P Y， Sun X F， et al. A multi-trait evaluation of drought resistance of bermudagrass （Cynodon dactylon） germplasm from different habitats in Xinjiang province. Acta Prataculturae Sinica， 2020， 29（8）： 155-169.
	曾令霜，李培英，孙晓梵，等. 新疆不同生境狗牙根种质抗旱性综合评价. 草业学报， 2020， 29（8）： 155-169.
14	Zhang Y L， Yu Q K， Li W， et al. Aboveground and belowground phenotypic characteristics of Cynodon dactylon lines differing in drought resistance and endogenous hormone response to drought stress. Acta Prataculturae Sinica， 2023， 32（3）： 163-178.
	张一龙，喻启坤，李雯，等. 不同抗旱性狗牙根地上地下表型特征及内源激素对干旱胁迫的响应. 草业学报， 2023， 32（3）： 163-178.
15	Anders S， Huber W. Differential expression analysis for sequence count data. Nature Precedings， 2010， 11（10）： R106.
16	Hou Z H， Yin J L， Lu Y F， et al. Transcriptomic analysis reveals the temporal and spatial changes in physiological process and gene expression in common buckwheat （Fagopyrum esculentum Moench） grown under drought stress. Agronomy， 2019， 9（10）： 17-26.
17	Shao A， Wang W， Fan S G， et al. Comprehensive transcriptional analysis reveals salt stress-regulated key pathways， hub genes and time-specific responsive gene categories in common bermudagrass （Cynodon dactylon （L.） Pers.） roots. BMC Plant Biology， 2021， 21（1）： 18-26.
18	Guan S J， Wang N， Xu R R， et al. Photosynthesis，antioxidant enzyme activity， and transcriptome sequencing analyses of Glyeyrrrhiza uralensis seedlings in response to drought stress. Pratacultural Science， 2021， 38（11）： 2176-2190.
	关思静，王楠，徐蓉蓉，等. 甘草幼苗响应干旱胁迫的光合、抗氧化特性及转录组分析.草业科学， 2021， 38（11）： 2176-2190.
19	Zhang J J， Li J J， Nian H J. The role of calcium/calmodulin signaling pathways in the stresses： Progress in researches. Chinese Journal of Microecology， 2013， 25（7）： 858-860.
	张晶晶，李金金，年洪娟. 钙/钙调素信号途径在胁迫中的作用研究进展.中国微生态学杂志， 2013， 25（7）： 858-860.
20	He L. Relationship between 2C serine/threonine protein phosphatase activity and drought tolerance in maize. Chengdu： Sichuan Agricultural University， 2008.
	何亮. 玉米2C型丝氨酸/苏氨酸蛋白磷酸酶活性与耐旱性的关系.成都：四川农业大学， 2008.
21	Meskiene I， Baudouin E， Schweighofer A， et al. Stress-induced protein phosphatase 2C is a negative regulator of a mitogen-activated protein kinase. Journal of Biological Chemistry， 2003， 278（21）： 18945-18952.
22	Danquah A， De Zélicourt A， Colcombet J， et al. The role of ABA and MAPK signaling pathways in plant abiotic stress responses. Biotechnology Advances， 2014， 32（1）： 40-52.
23	Xiong L， Schumaker K S， Zhu J K. Cell signaling during cold， drought， and salt stress. Plant Cell， 2002， 14（5）： 165-183.
24	Lin Y F， Li W， Dai L Y. Research progress of antioxidant enzymes functioning in plant drought resistant process. Crop Research， 2015， 29（3）： 326-330.
	林宇丰，李魏，戴良英. 抗氧化酶在植物抗旱过程中的功能研究进展.作物研究， 2015， 29（3）： 326-330.
25	Foyer C H， Noctor G. Ascorbate and glutathione： The heart of the redox hub. Plant Physiology， 2011， 155（1）： 2-18.
26	Song G F， Fan W L， Wang J J， et al.Cloning and characterization of drought-stress responsive gene GhGR in Gossypium hirsutum L. Scientia Agricultura Sinica， 2012， 45（8）： 1644-1652.
	宋贵方，樊伟丽，王俊娟，等. 陆地棉干旱胁迫响应基因GhGR的克隆及特征分析. 中国农业科学， 2012， 45（8）： 1644-1652.
27	Shan C J， Han R L， Liang Z S.Responses to drought stress of the biosynthetic and recycling metabolism of glutathione and ascorbate in Agropyron cristatum leaves on the Loess Plateau of China.Chinese Journal of Plant Ecology， 2011， 35（6）： 653-662.
	单长卷，韩蕊莲，梁宗锁.黄土高原冰草叶片抗坏血酸和谷胱甘肽合成及循环代谢对干旱胁迫的生理响应. 植物生态学报， 2011， 35（6）： 653-662.
28	Kim C， Lemke C， Paterson A H. Functional dissection of drought-responsive gene expression patterns in Cynodon dactylon L. Plant Molecular Biology， 2009， 70（2）： 1-16.
29	Abe H， Urao T， Ito T， et al. Arabidopsis AtMYC2 （bHLH） and AtMYB2 （MYB） function as transcriptional activators in abscisic acid signaling. Plant Cell， 2003， 15（1）： 63-78.
30	Guo H， Wang Y， Wang L， et al. Expression of the MYB transcription factor gene BplMYB46 affects abiotic stress tolerance and secondary cell wall deposition in Betula platyphylla. Plant Biotechnology Journal， 2017， 15（1）： 107-121.
31	Hu L X， Li H Y， Chen L， et al. RNA-seq for gene identification and transcript profiling in relation to root growth of bermudagrass （Cynodon dactylon） under salinity stress. BMC Genomics， 2015， 16： 1-12.
32	Yuan X， Huang P， Wang R， et al. A zinc finger transcriptional repressor confers pleiotropic effects on rice growth and drought tolerance by down-regulating stress-responsive genes. Plant Cell Physiology， 2018， 59（10）： 2129-2142.

编号Code	采集地点Collection location	抗旱类型Drought resistance type
C32	新疆吐鲁番市托克逊县Tuokexun County， Turpan City， Xinjiang Autonomous Region	敏旱型Drought sensitive
C138	新疆喀什疏勒县Shule County， Kashgar City， Xinjiang Autonomous Region	抗旱型Drought resistant

编号Code	采集地点Collection location	抗旱类型Drought resistance type
C32	新疆吐鲁番市托克逊县Tuokexun County， Turpan City， Xinjiang Autonomous Region	敏旱型Drought sensitive
C138	新疆喀什疏勒县Shule County， Kashgar City， Xinjiang Autonomous Region	抗旱型Drought resistant

基因ID Gene ID	正向引物Forward primer （5′-3′）	反向引物Reverse primer （5′-3′）
Actin	GCTCAACCCCAAGGCTAACAG	AGCGTATCCCTCGTAGATG
c458080.graph_c0	CGGTGCTGGTTGGGTAGTCTG	AAATGGCGGCAATAGCGAAAGC
c452636.graph_c0	CCCTGCCCAACGACGAAGAG	CATTGGCTTGGCGTGCTTGAG
c422082.graph_c1	GCCCATCACCGCCAATCCG	CAAACGCATTAGCAACGCAACG
c448662.graph_c0	GTGAGGTCCAGGCGGTGTTG	CGGCGAGTCGGTGTTCCTTG
c443527.graph_c0	TGCCCGTTGACCTGTCTTTCTC	AGCACTTCCTCCGCCATTTCG
c460219.graph_c1	GCGGCGAGCAATGATGTTACAG	AGATGGCTGGCGTTCTTGGC

基因ID Gene ID	正向引物Forward primer （5′-3′）	反向引物Reverse primer （5′-3′）
Actin	GCTCAACCCCAAGGCTAACAG	AGCGTATCCCTCGTAGATG
c458080.graph_c0	CGGTGCTGGTTGGGTAGTCTG	AAATGGCGGCAATAGCGAAAGC
c452636.graph_c0	CCCTGCCCAACGACGAAGAG	CATTGGCTTGGCGTGCTTGAG
c422082.graph_c1	GCCCATCACCGCCAATCCG	CAAACGCATTAGCAACGCAACG
c448662.graph_c0	GTGAGGTCCAGGCGGTGTTG	CGGCGAGTCGGTGTTCCTTG
c443527.graph_c0	TGCCCGTTGACCTGTCTTTCTC	AGCACTTCCTCCGCCATTTCG
c460219.graph_c1	GCGGCGAGCAATGATGTTACAG	AGATGGCTGGCGTTCTTGGC

样品 Sample	过滤后序列长度Clean datas length （bp）	GC含量 GC content （%）	碱基质量>20 Q₂₀ （%）	碱基质量>30 Q₃₀ （%）	样品 Sample	过滤后序列长度Clean datas length （bp）	GC含量 GC content （%）	碱基质量>20 Q₂₀ （%）	碱基质量>30 Q₃₀ （%）
CR138-1	5751481130	52.51	97.67	93.70	MR32-1	7662111708	52.97	97.70	93.80
CR138-2	6361023526	52.89	97.81	94.00	MR32-2	7405754772	52.73	97.77	93.94
CR138-3	5963989470	53.19	97.69	93.80	MR32-3	7729678484	53.38	97.79	94.03
CR32-1	8694639522	51.96	98.29	95.02	SR138-1	6680468588	52.06	97.67	93.72
CR32-2	7119356674	52.58	98.21	94.90	SR138-2	6788905702	52.18	97.37	92.94
CR32-3	6517861366	51.93	97.87	94.05	SR138-3	5945423404	52.11	97.86	94.16
MR138-1	9409664642	52.57	97.75	93.90	SR32-1	6205146120	52.31	97.94	94.31
MR138-2	6314881976	52.21	97.70	93.72	SR32-2	6469804754	52.11	97.86	94.08
MR138-3	6298108628	52.30	97.67	93.69	SR32-3	6935579000	51.64	97.81	94.00