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作物学报 ›› 2023, Vol. 49 ›› Issue (7): 1882-1894.doi: 10.3724/SP.J.1006.2023.24172

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

油莎豆耐盐性评估及盐胁迫下幼苗根系转录组学分析

王会伟(), 张向歌, 李春鑫, 许欣然, 胡海燕, 朱雅婧, 王艳, 张新友*()   

  1. 河南省农业科学院经济作物研究所, 河南郑州 450002
  • 收稿日期:2022-08-01 接受日期:2022-10-10 出版日期:2023-07-12 网络出版日期:2022-10-24
  • 通讯作者: *张新友, E-mail: haasz@126.com
  • 作者简介:E-mail: whuiweiw@163.com
  • 基金资助:
    本研究由国家重点研发计划项目(2019YFD1002600);河南省重大科技专项项目(211100110100);河南省农业科学院优秀青年科技基金项目(2022YQ19)

Evaluation of salt tolerance in Cyperus esculentus and transcriptomic analysis of seedling roots under salt stress

WANG Hui-Wei(), ZHANG Xiang-Ge, LI Chun-Xin, XU Xin-Ran, HU Hai-Yan, ZHU Ya-Jing, WANG Yan, ZHANG Xin-You*()   

  1. Industrial Crop Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan, China
  • Received:2022-08-01 Accepted:2022-10-10 Published:2023-07-12 Published online:2022-10-24
  • Contact: *E-mail: haasz@126.com
  • Supported by:
    The National Key Research and Development Project(2019YFD1002600);The Henan Major Science and Technology Project(211100110100);The Excellent Youth Science and Technology Fund of Henan Academy of Agricultural Sciences(2022YQ19)

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摘要：

油莎豆是一种综合利用价值高的新型经济作物, 抗逆性极强, 具有在盐渍化土壤上种植发展的潜力。为明确其耐盐特性, 本研究设置5个NaCl浓度(0、0.3%、0.6%、0.9%和1.2%), 分析了NaCl胁迫对油莎豆茎豆萌发和幼苗生长过程中形态和生理指标的影响。结果表明, 在0.3%和0.6% NaCl胁迫下, 茎豆发芽率、幼苗根长和苗高受影响程度较小; 根系中膜受损程度指标物丙二醛(MDA)和氧化胁迫物过氧化氢(H2O2)的含量增加不显著, 而渗透调节物甜菜碱(GB)、脯氨酸(Pro)的含量和抗氧化酶超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)的活性显著提高, 整体上受到的盐胁迫伤害较小。而在0.9%和1.2% NaCl胁迫下, 盐胁迫伤害十分明显, 茎豆萌发和幼苗生长受到严重的抑制。为进一步鉴定油莎豆耐盐相关基因, 利用RNA-seq技术检测了0、0.3%和0.6% NaCl胁迫下油莎豆根系的基因表达情况。通过基因差异表达分析、加权基因共表达网络分析(WGCNA)以及GO富集分析发现, 24个主要与氧化还原、跨膜转运、几丁质水解相关的GO terms显著富集, 涉及了15个显著上调表达基因。其中, DN23985_c0_g1、DN2960_c0_g1、DN8384_c1_g1分别编码玉米黄质环氧酶、L-抗坏血酸过氧化物酶、谷胱甘肽转移酶, 具有抗氧化作用, 参与抗氧化调节; DN21785_c1_g1和DN6596_c0_g1二者均编码氨基酸转运蛋白, 可能通过积累氨基酸类小分子(如Pro)而增强渗透调节; DN14393_c0_g1编码几丁质酶, 能够水解几丁质而促进植物应对胁迫响应。本研究明确了油莎豆在0.6%及以下浓度NaCl胁迫时具有较好的耐盐性, 并进一步筛选出耐盐相关基因, 为油莎豆在盐渍化土壤的种植和耐盐品种的培育提供了重要的参考。

关键词: 油莎豆, 耐盐性, NaCl胁迫, 转录组学, 耐盐相关基因

Abstract:

Cyperus esculentus is a new industrial crop with the high comprehensive utilization value, which is a strongly resistant to stresses and has a great potential to grow in saline soil. In order to clarify its salt tolerance, five NaCl concentrations (0, 0.3%, 0.6%, 0.9%, and 1.2%) were set in this study to analyze the effects of NaCl stress on morphological and physiological indexes during germination and seedling growth. The results showed that the germination percentage, root length, and seedling height were less affected under 0.3% and 0.6% NaCl stresses. Meanwhile, the contents of indicator of membrane damage degree, malondialdehyde (MDA), and oxidative stress substance, hydrogen peroxide (H2O2), did not increase significantly. However, the contents of osmoregulation substances [glycine betaine (GB), and proline (Pro)] and the activities of antioxidant enzymes [superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)] were significantly increased, and the damage of salt stress was less on the whole. However, under 0.9% and 1.2% NaCl stresses, the salt stress injury was very obvious and the germination and seedling growth of Cyperus esculentus were severely inhibited. In order to further identify the genes related to salt tolerance, RNA-seq technology was used to detect the gene expression in roots under 0, 0.3%, and 0.6% NaCl stresses. 24 GO terms mainly related to oxidoreduction, transmembrane transport, chitin hydrolysis was significantly enriched, among which 15 significantly up-regulated genes were involved, through gene differential expression analysis, weighted gene co-expression network analysis (WGCNA), and GO enrichment analysis. Among them, DN23985_c0_g1, DN2960_c0_g1, and DN8384_c1_g1 encoded zeaxanthin epoxidase, L-ascorbate peroxidase, and glutathione S-transferase, respectively, which had antioxidant effects and participated in antioxidant regulation. Both DN21785_c1_g1 and DN6596_c0_g1 encoded amino acid transporters, which may enhance osmoregulation by accumulating small amino acid molecules such as Pro. DN14393_c0_g1 encoded chitinase, which hydrolyzed chitin and promoted plant response to stress. In this study, it was confirmed that Cyperus esculentus had a good salt tolerance under 0.6% or less NaCl stress, and the salt-tolerance related genes were further screened out, which provided an important reference for the cultivation in saline soil and the breeding of salt-tolerant varieties.

Key words: Cyperus esculentus, salt tolerance, NaCl stress, transcriptomics, salt-tolerance related gene

图1

不同浓度NaCl胁迫对油莎豆茎豆萌发的影响 A: 不同浓度NaCl胁迫下油莎豆茎豆的萌发形态。B: 不同浓度NaCl胁迫下油莎豆茎豆的发芽率及耐盐指数。标尺为2 cm。YYS2: 豫油莎2号; YYS3: 豫油莎3号。"

图2

不同浓度NaCl胁迫对油莎豆幼苗生长的影响 A: 不同浓度NaCl胁迫下YYS2幼苗生长特征。B: 不同浓度NaCl胁迫下YYS3幼苗生长特征。标尺为5 cm。YYS2: 豫油莎2号; YYS3: 豫油莎3号。"

表1

NaCl胁迫对两个油莎豆品种幼苗苗高和根长的影响"

NaCl胁迫处理
NaCl stress treatment		 苗高Seedling height (cm) 根长Root length (cm)
豫油莎2号
Yuyousha 2		 豫油莎3号
Yuyousha 3		 豫油莎2号
Yuyousha 2		 豫油莎3号
Yuyousha 3
0 (CK) 18.00 ± 1.16 a 15.75 ± 1.25 a 16.56 ± 1.37 a 19.28 ± 1.68 a
0.3% 15.26 ± 1.08 b 13.53 ± 1.17 b 14.16 ± 1.12 b 17.54 ± 1.34 b
0.6% 14.65 ± 0.96 b 13.56 ± 0.85 b 13.53 ± 1.03 c 14.56 ± 1.23 c
0.9% 12.92 ± 1.12 c 11.50 ± 1.23 c 12.85 ± 0.93 d 13.26 ± 1.08 c
1.2% 10.93 ± 0.84 d 9.75 ± 0.99 d 8.92 ± 0.78 e 9.58 ± 0.93 d

图3

不同浓度NaCl胁迫下油莎豆幼苗根系生理指标变化 *表示指标在YYS2和YYS3之间存在显著差异(P < 0.05)。YYS2: 豫油莎2号; YYS3: 豫油莎3号。"

表3

转录组测序数据统计分析"

处理
Treatment		 测序总读数
Total reads number		 质控后读数
Clean reads number		 比对率
Mapped ratio (%)		 Q20碱基百分比
Q20
(%)		 Q30碱基百分比
Q30
(%)		 非冗余基因数
Unigene
number
CK-1 46,067,738 42,556,954 92.37 97.55 93.77 30,191
CK-2 42,308,034 39,254,802 92.78 97.71 94.06
CK-3 50,830,434 47,129,824 92.71 97.59 93.79
0.3%-1 57,271,072 53,011,182 92.56 97.70 93.93
0.3%-2 43,325,228 40,182,946 92.74 97.76 94.00
0.3%-3 52,217,758 48,377,604 92.64 97.48 93.43
0.6%-1 43,209,368 40,018,352 92.61 97.59 93.73
0.6%-2 46,496,392 43,155,836 92.81 97.52 93.58
0.6%-3 41,707,820 38,775,376 92.96 97.39 93.37

图4

重复样品间基因表达量的相关性分析 红框表示同一处理下的3个重复。"

图5

不同浓度NaCl胁迫下基因差异表达分析 A: 不同处理组(0.3% vs CK和0.6% vs CK)差异表达基因数目; B: 不同处理组间差异表达基因的维恩分析。"

图6

耐盐相关基因的筛选 A: 基于超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化物酶(CAT)、甜菜碱(GB)和脯氨酸(Pro)的WGCNA。每行代表一个模块, 每列代表一种性状; 矩形框中上方数字代表模块与性状之间的相关系数, 括号内数字为相应的P值。B: 不同处理间共有的差异表达基因和耐盐相关的黄色模块基因之间的维恩分析。"

图7

耐盐相关基因的GO富集分析 A: 基于MF的GO富集气泡图。B: 显著富集GO terms中所有基因的表达分析及功能注释。"

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