荒漠草原优势植物牛枝子对干旱胁迫的生理响应与转录组分析

doi:10.11686/cyxb2022326

摘要/Abstract

摘要：

干旱是草地植物面对的最大威胁，对耐旱草地植物的研究将促进人们更好地理解植物对干旱适应性反应背后的调控机制。牛枝子是优质的多年生强旱生牧草，分布于广袤的荒漠草原地区。目前对牛枝子抗旱研究主要集中在渗透调节物质变化、功能基因序列分析，但其抗旱性的内在机制尚不清楚。本研究采用单因素控水试验，土壤水分含量为田间持水量的70%~80%设为对照组（CK），田间持水量的20%~30%为严重干旱胁迫组（Tr），处理4周后，开展牛枝子生物量分配、水分利用、渗透调节、根系分布等生理生化指标测定，同时采集叶片和幼根进行转录组测序分析。结果表明，干旱胁迫条件下，牛枝子通过增加脯氨酸（Pro）、可溶性蛋白（SP）、可溶性糖（Ss）、K⁺等含量进行渗透调节来保持体内水分，增加同位素碳13（δ¹³C）和丙二醛（MDA）含量以提高水分利用效率和抗氧化能力，降低相对含水量（RWC）、胞间二氧化碳浓度（C_i）、气孔导度（Cond），减少生物量，增加根冠比等响应干旱胁迫。通过RNA-Seq差异基因表达分析，在叶中发现4058个差异基因，在根中发现2172个差异基因，叶和根中共有差异基因744个。这些差异基因（DEG）能够对干旱做出积极（上调）或消极（下调）的反应。叶中上调差异基因主要富集在植物-病原体相互作用、植物激素信号转导，而下调差异基因主要富集在光合代谢包括碳固定、光合作用天线蛋白合成、光合过程产物代谢。在根中上调差异基因主要富集在精氨酸与脯氨酸代谢、内质网中的蛋白质加工，下调差异基因主要富集在淀粉和蔗糖代谢、异黄酮生物合成、类黄酮生物合成。在牛枝子叶中差异表达的转录因子主要有AP₂/ERF-ERF、NAC、bHLH、WRKY、C₂H₂，根中主要有 HSF、MYB、AP₂/ERF-ERF、WRKY。其中bHLH特异性下调表达，HSF特异性上调表达。根与叶中的吡咯啉-5-羧酸还原酶（P5CR）、脯氨酸亚氨肽酶（PLD）、脯氨酸-4-羟化酶（P4H）均上调表达，脯氨酸脱氢酶（ProDH）下调表达将产生更多的脯氨酸与4-羟基-脯氨酸，叶中天冬氨酸氨基转移酶（AST）的上调产生更多4-羟基-酮戊二酸，增强了牛枝子渗透调节能力，保证了牛枝子对水分的吸收利用。因此，牛枝子主要通过调控与激素信号转导、渗透调节、气体交换相关的差异基因表达，参与各项生理代谢活动以响应严重干旱胁迫。

关键词: 干旱胁迫, 牛枝子, 生理, 转录组分析

Abstract:

Drought is the greatest threat to grassland plants， and research on drought-tolerant grassland plants will contribute to a better understanding of the regulatory mechanisms behind the adaptive responses of plants to drought. Lespedeza potaninii is a high quality， perennial， strongly drought tolerant forage species that is widely distributed in desert grassland areas in some parts of China. Current research on drought resistance in L. potaninii focuses on changes in osmoregulatory substances and functional gene sequence analysis， but the underlying mechanism of its drought resistance is still unclear. In this study， a single-factor water control experiment was conducted with soil moisture content of 70%-80% of the field water holding capacity for the control group （CK） and 20%-30% of the field water holding capacity for the severely drought stressed group （Tr）. After 4 weeks of treatment， tests on physiological and biochemical indicators such as biomass distribution， water use， osmoregulation and root distribution were carried out on L. potaninii， while leaves and young roots were collected for transcriptome sequencing analysis. The results showed that under drought stress conditions， L. potaninii exhibited increased content of proline （Pro）， a higher soluble protein content （SP）， increased soluble sugar， increased K⁺ ions for osmoregulation to maintain hydration of the plant tissues， increased δ¹³C indicating improved water use efficiency and increased malondialdehyde indicating oxidant stress， reduced relative water content， reduced leaf internal CO₂ concentration and stomatal conductance， reduced biomass and increased root to crown ratio， among other changes， in response to drought stress. By RNA-Seq differential gene expression analysis， 4058 differential genes were found in leaves and 2172 differential genes in roots， making a total of 744 differential genes in leaves and roots. These differential genes （DEGs） include those responding positively （up-regulated） or negatively （down-regulated） to drought. Up-regulated differentially expressed genes in leaves are mainly related to plant-pathogen interactions and phytohormone signaling， while down-regulated differentially expressed genes are mainly related to photosynthetic metabolism including carbon fixation， photosynthetic antenna protein synthesis， and photosynthetic process product metabolism. In roots， up-regulated differential genes were mainly related to arginine and proline metabolism， protein processing in the endoplasmic reticulum， and down-regulated differential genes were mainly related to starch and sucrose metabolism， isoflavone biosynthesis， and flavonoid biosynthesis. The main transcription factors differentially expressed in L. potaninii leaves were AP₂/ERF-ERF， NAC， bHLH， WRKY， C₂H₂， and in roots were HSF， MYB， AP₂/ERF-ERF， WRKY. The transcription factor bHLH was specifically down-regulated and HSF was specifically up-regulated. The expression of P5CR， PLD and P4H was up-regulated in roots and leaves. In leaves， ProDH was down-regulated， which will produce more proline and 4-hydroxy-proline， and AST was up-regulated which produces more 4-hydroxy-ketoglutarate and enhances the osmoregulatory ability and ensures water uptake and use by L. potaninii. Therefore， the main response to drought stress in L. potaninii was to initiate various physiological and metabolic activities through differential expression of genes related to hormone signal transduction， osmoregulation and gas exchange.

Key words: drought stress, Lespedeza potaninii, physiological, transcriptome analysis

张浩, 胡海英, 李惠霞, 贺海明, 马霜, 马风华, 宋柯辰. 荒漠草原优势植物牛枝子对干旱胁迫的生理响应与转录组分析[J]. 草业学报, 2023, 32(7): 188-205.

Hao ZHANG, Hai-ying HU, Hui-xia LI, Hai-ming HE, Shuang MA, Feng-hua MA, Ke-chen SONG. Physiological response and transcriptome analysis of the desert steppe dominant plant Lespedeza potaninii to drought stress[J]. Acta Prataculturae Sinica, 2023, 32(7): 188-205.

图/表 11

表1 牛枝子差异基因引物序列

Table 1 Differential gene primer sequences of L. potaninii

叶Leaf		根Root
基因 Gene ID	引物序列 Primer sequences （5′→3′）	基因 Gene ID	引物序列 Primer sequences （5′→3′）
BMK_Unigene_037155	GTGGTGATGACAAGGAAGAGAA ACTGGCAACTTCTCCTTAACC	BMK_Unigene_039577	ATACCGTCCCATAGCAAGAATG TCTCCAGCCTCCACTCAATA
BMK_Unigene_111723	CTCACACAGATGCAGGGTATG CCTCCTGGAAGCTTCTCTTTAAT	BMK_Unigene_040501	AACCCTACCTTCCTTCACAATC CACTCTTCACTTCCACCATCA
BMK_Unigene_009299	CGCTTTGGTGTTGGAGATTG CAATTGCGATGGGAGCAATAG	BMK_Unigene_016501	GATGTCGGGTCTTGGGATAAA GCTCCTCCATTGATAGGTCATAA
BMK_Unigene_035523	GGTGTGGAATGAGGAAGAGAAA CCACTCCCGAAAGCCAATAA	BMK_Unigene_276197	ACTGGTGTGAAAGGTCTTGTAG GTCTATGACAGGAGCACTCAAC
BMK_Unigene_035906	GACCTTCAACACTCCTGCTATG CATCTCCAGAGTCCAGAACAATAC	BMK_Unigene_029018	GCCATTCATTTGTTCCCATCC GTGTTTCTGTGTTCTGGGAATTT

表2 干旱胁迫对牛枝子根系特征及器官生物量的影响

Table 2 Effects of drought stress on the root characteristics and organ biomass of L. potaninii

指标Index	对照CK	处理Tr	F检验F-test	P
总根长Total root length （cm）	1265.98±24.87a	933.40±6.64b	166.87	0.00
总根表面积Total root area （cm²）	230.64±44.39a	92.15±2.97b	9.69	0.04
总根体积Total root volume （cm³）	1.98±0.16a	0.81±0.04b	51.15	0.00
根尖数Root tips number	3702.00±329.57a	2578.00±64.73b	11.20	0.03
根分枝数Root forks number	4309.67±127.96a	3384.33±240.77b	14.69	0.02
根生物量Root biomass （g·plant^-1）	1.40±0.10a	1.32±0.24a	0.27	0.63
茎生物量Stem biomass （g·plant^-1）	1.50±0.16a	1.16±0.12b	8.61	0.04
叶生物量Leaf biomass （g·plant^-1）	1.39±0.06a	1.27±0.24a	0.76	0.43
根冠比Root-shoot ratio	0.48±0.02b	0.54±0.02a	10.13	0.03

图1 干旱胁迫下牛枝子生理生化指标的变化不同小写字母表示不同处理下差异显著（P<0.05）。Different lowercase letters indicate significant differences under different treatments （P<0.05）.

Fig.1 Changes of physiological and biochemical indexes of L. potaninii under drought stress

表3 牛枝子不同器官及不同处理测序组装数据

Table 3 Sequencing and assembly data of different organs and different treatments of L. potaninii

样品名称 Sample		基本数据 Base number	GC占比 GC content （%）	Q₂₀ （%）	Q₃₀ （%）	过滤后的数据 Clean reads	映射读取 Mapped reads	映射比率 Mapped ratio （%）
CK	L₁	6186621368	44.54	98.60	95.43	20723703	12840214	61.96
	L₂	6234665288	45.00	98.58	95.41	20868074	12951855	62.07
	L₃	6225191496	44.71	98.47	95.09	20846238	12614651	60.51
	R₁	5786751212	43.89	98.25	94.59	19357392	11709354	60.49
	R₂	6370728902	43.86	98.08	94.12	21294316	13130824	61.66
	R₃	6138978110	44.20	98.34	94.85	20547009	12650760	61.57
Tr	L₁	6225600476	44.79	98.49	95.15	20816470	12764270	61.32
	L₂	5816270068	44.10	98.56	95.34	19465715	11695609	60.08
	L₃	6358918316	44.00	98.55	95.32	21287781	12910160	60.65
	R₁	5959686196	44.03	98.50	95.26	19944009	12394261	62.15
	R₂	6326554166	44.16	98.54	95.38	21198666	13124275	61.91
	R₃	6560622132	43.93	98.68	96.02	21970365	13775359	62.70

图2 实时荧光定量PCR验证RNA-Seq数据a：对RNA-Seq数据和qRT-PCR数据进行线性相关分析； b：上调差异基因RNA-Seq的表达量与qRT-PCR的相对表达量； c：下调差异基因RNA-Seq的表达量与qRT-PCR的相对表达量。a： Linear correlation analysis of RNA-Seq data and qRT-PCR data； b： Up-regulation of differential gene RNA-Seq FRKM and relative expression level of qRT-PCR； c： Down-regulation of differential gene RNA-Seq FRKM and relative expression level of qRT-PCR. FPKM： Fragments per kilobase of exon model per million mapped fragments.

Fig.2 Real-time fluorescence quantitative PCR validation RNA-seq data

图3 干旱胁迫下根和叶片中差异基因表达分析a： CKRvsTrR和CKLvsTRL差异基因火山图。黑点代表对干旱胁迫没有响应的基因，上调与下调基因分别用红点与绿点表示。b：维恩图中显示了叶与根中上调与下调基因具有的相同差异基因的数量。a： Differential gene volcano map of the CKRvsTrR and CKLvsTRL. Black dots represent genes that do not respond to drought stress， up-regulated and down-regulated genes are indicated by red and green dots respectively. b： The Venn diagram shows the number of identical differential genes in leaves and roots that have up- and down-regulated genes.

Fig.3 Analysis of differential gene expression in leaf and root under drought treatment

图4 牛枝子叶和根中响应干旱胁迫的差异基因GO富集分析a：叶中上调与下调差异基因GO富集分析； b：根中上调与下调差异基因GO富集分析；将过程注释到分子功能、细胞成分、生物过程。a： GO enrichment analysis of up-regulated and down-regulated differentially genes in leaf； b： GO enrichment analysis of up- and down-regulated differential genes in root. Annotate processes to molecular function， cellular component， biological process.

Fig.4 GO enrichment analysis of differential genes in response to drought stress in leaf and root of L. potaninii

图5 牛枝子叶和根中响应干旱胁迫的差异基因KEGG富集分析富集路径沿y轴列出，x轴表示富集因子。红色代表高Q值，而蓝色代表低Q值。Enrichment paths are listed along the y-axis， the x-axis represents enrichment factors. Red represents high Q-values， while blue represents low Q-values.

Fig.5 KEGG enrichment analysis of differential genes in response to drought stress in leaf and roots of L. potaninii

表4 牛枝子叶与根差异基因KEGG显著富集通路

Table 4 KEGG pathway was significantly enriched in differential genes of leaf and root of L. potaninii

组别Group	代谢通路Metabolic pathway	数量Count	上调Up	下调Down
对照叶vs处理叶CKLvsTrL	植物-病原体相互作用Plant-pathogen interaction	175	117	58
	植物激素信号转导Plant hormone signal transduction	68	68	0
	内质网上蛋白质加工Protein processing in endoplasmic reticulum	59	54	5
	淀粉与蔗糖代谢Starch and sucrose metabolism	50	50	0
	碳代谢Carbon metabolism	118	19	99
	光合作用-天线蛋白Photosynthesis-antenna proteins	40	0	40
	光合作用Photosynthesis	48	2	46
	光合生物中的碳固定Carbon fixation in photosynthetic organisms	49	4	45
	氨基酸生物合成Biosynthesis of amino acids	88	28	60
对照根vs处理根CKRvsTrR	精氨酸与脯氨酸代谢Arginine and proline metabolism	28	21	7
	内质网上蛋白质加工Protein processing in endoplasmic reticulum	41	30	11
	糖酵解/糖异生Glycolysis/gluconeogenesis	27	25	2
	植物-病原体相互作用Plant-pathogen interaction	93	7	86
	MAPK信号通路MAPK signaling pathway	71	14	57
	淀粉与蔗糖代谢Starch and sucrose metabolism	70	24	46
	异黄酮生物合成Isoflavonoid biosynthesis	11	1	10

图6 牛枝子差异基因转录因子数量统计a：叶； b：根。横坐标表示分配给特定家族的基因数量；纵坐标表示转录因子类型。a： Leaf； b： Root； The horizontal axis represents the number of genes assigned to a particular family； The vertical axis represents the transcription factor type.

Fig.6 Quantitative statistics of differential gene transcription factors in L. potaninii

图7 脯氨酸代谢通路与耐旱相关基因差异表达热图

Fig.7 Heat map of differential expression of genes related to proline metabolism and drought tolerance

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