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作物学报

作物学报 ›› 2023, Vol. 49 ›› Issue (8): 2122-2132.doi: 10.3724/SP.J.1006.2023.24205

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

不同耐旱性紫花苜蓿干旱胁迫下生理响应和转录调控的差异研究

陈力1,2(), 王靖1,2, 邱晓3, 孙海莲3, 张文浩1, 王天佐1,*()   

  1. 1 中国科学院植物研究所, 北京 100093
    2 中国科学院大学生命科学学院, 北京 100049
    3 内蒙古自治区农牧业科学院, 内蒙古呼和浩特 010000
  • 收稿日期:2022-09-06 接受日期:2022-11-25 出版日期:2023-08-12 网络出版日期:2022-12-02
  • 通讯作者: 王天佐
  • 作者简介:E-mail: chenli125975@163.com
  • 基金资助:
    国家重点研发计划项目(2022YFF1003203);内蒙古自治区科技计划项目(2021GG0372)

Differences of physiological responses and transcriptional regulation of alfalfa with different drought tolerances under drought stresses

CHEN Li1,2(), WANG Jing1,2, QIU Xiao3, SUN Hai-Lian3, ZHANG Wen-Hao1, WANG Tian-Zuo1,*()   

  1. 1 Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
    2 College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
    3 Inner Mongolia Academy of Agricultural & Animal Husbandry Sciences, Huhhot 010000, Inner Mongolia, China
  • Received:2022-09-06 Accepted:2022-11-25 Published:2023-08-12 Published online:2022-12-02
  • Contact: WANG Tian-Zuo
  • Supported by:
    National Key Research and Development Program of China(2022YFF1003203);Science and Technology Program of Inner Mongolia Autonomous Region(2021GG0372)

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摘要：

苜蓿是最重要的豆科牧草, 常被种植在干旱/半干旱地区。为研究不同耐旱性紫花苜蓿应对干旱胁迫的调控机制, 本研究以中科1号紫花苜蓿(Medicago sativa ‘Zhongke 1’)为试验材料, 以三得利紫花苜蓿(M. sativa ‘Sanditi’)为对照, 采用自然干旱法进行处理, 比较干旱胁迫对其生长性状、光合作用、叶绿素含量、叶片相对含水量、渗透调节物质和抗氧化酶活性等指标的影响, 并使用转录组测序分析2个品种响应干旱基因的差异。结果表明, 干旱胁迫显著降低了2个紫花苜蓿品种的株高、地上地下生物量、叶片相对含水量、光合速率、蒸腾速率、气孔导度、胞间二氧化碳浓度和叶绿素含量; 与三得利相比, 干旱胁迫下中科1号紫花苜蓿丙二醛含量和电导率更低, 渗透调节物质积累和活性氧清除能力更强。中科1号和三得利分别有5308个和8053个响应干旱的基因; GO功能注释分析发现, 中科1号的346个干旱响应基因被显著富集在6个GO类别, 而三得利的1683个干旱响应基因被显著富集在29个GO类别; 此外, 我们还筛选到19个在中科1号中表达量显著高于三得利的抗旱关键基因。本研究发现紫花苜蓿可能通过上调抗旱基因SUS、P5CS、LEA、SOD、POD、PEPC、NCED等的表达, 提高渗透调节能力和抗氧化酶活性来维持相对较高的光合速率, 从而增强抗旱能力。研究结果为进一步挖掘紫花苜蓿响应干旱胁迫的候选基因和抗旱育种提供了理论依据。

关键词: 紫花苜蓿, 干旱胁迫, 生理特性, 转录组

Abstract:

Alfalfa is the most important legume forage, which is often planted in aird or semi-arid regions. The objective of this study is to explore the responsive mechanism of alfalfa with different drought tolerance to drought stress, which provides the theoretical basis for tolerant breeding of alfalfa. In this study, the new variety Medicago sativa ‘Zhongke 1’ was used as the test material, and M. sativa ‘Sanditi’ was used as the control. The effects of drought stress on growth traits, photosynthesis, chlorophyll concentration, leaf water content, osmotic adjustment substances, and antioxidant enzyme activities were determined by natural drought strategy in pots. The deferentially expressed genes were compared by transcriptome analysis. Drought stress significantly reduced plant height, biomass, relative water content of leaves, photosynthetic rate, transpiration rate, stomatal conductance, intercellular carbon dioxide and chlorophyll concentration of alfalfa. Compared with Sanditi, Zhongke 1 revealed the lower malondialdehyde concentration and electrical conductivity under drought stress, but stronger osmotic regulation ability and superoxide radical scavenging ability. Transcriptome sequencing identified 5308 and 8053 drought-responsive genes of Zhongke 1 and Sanditi, respectively. GO functional annotation demonstrated that 346 drought-responsive genes were significantly enriched in six GO item categories in Zhongke 1, while 1683 drought-responsive genes in Sanditi were significantly enriched in 29 GO item categories. The relative expression levels of 19 key drought-tolerant genes in Zhongke 1 were significantly higher than those in Sanditi. Alfalfa may maintain a relatively high photosynthetic rate by up-regulating the relative expression levels of drought tolerant genes SUS, P5CS, LEA, SOD, POD, PEPC, and NCED, thus improving osmotic regulation ability and antioxidant enzyme activity. The results provide a theoretical basis for further exploration of alfalfa’s candidate genes in response to drought stress and drought tolerant breeding.

Key words: alfalfa, drought stress, physiological characteristics, transcriptome

图1

干旱胁迫对不同耐旱性紫花苜蓿株高(A)、地上生物量(B)、地下生物量(C)和存活率(D)的影响 *代表同一处理不同品种间的显著性(*: P < 0.05; **: P < 0.01); #代表同一品种不同处理间的显著性(#: P < 0.05; ##: P < 0.01)。"

图2

干旱胁迫对不同耐旱性紫花苜蓿光合速率(A)、气孔导度(B)、胞间CO2浓度(C)、蒸腾速率(D)、水分利用效率(E)和叶绿素含量(F)的影响 *代表同一处理不同品种间的显著性(*: P < 0.05; **: P < 0.01); #代表同一品种不同处理间的显著性(#: P < 0.05; ##: P < 0.01)。"

图3

干旱胁迫对不同耐旱性紫花苜蓿相对电导率(A)和叶片相对含水量(B)的影响 *代表同一处理不同品种间的显著性(*: P < 0.05, **: P < 0.01); #代表同一品种不同处理间的显著性(#: P < 0.05, ##: P < 0.01)。"

图4

干旱胁迫对不同耐旱性紫花苜蓿叶片脯氨酸(A)和可溶性糖含量(B)的影响 *代表同一处理不同品种间的显著性(*: P < 0.05, **: P < 0.01); #代表同一品种不同处理间的显著性(#: P < 0.05, ##: P < 0.01)。"

图5

干旱胁迫对不同耐旱性紫花苜蓿丙二醛含量(A)和超氧化物歧化酶(B)、过氧化物酶(C)活性的影响 *代表同一处理不同品种间的显著性(*: P < 0.05; **: P < 0.01); #代表同一品种不同处理间的显著性(#: P < 0.05; ##: P < 0.01)。"

图6

在干旱胁迫和正常条件下的转录组数据分析 A: 转录组相关性分析热图; B: 转录组差异基因数量。"

表1

中科1号干旱与对照差异表达基因的GO注释分析"

类别
Categories		 基因本体ID
GO ID		 描述
Description		 多重假设检验校正后的P值
P-adj value		 基因数
No. of genes
CC GO:0005618 细胞壁Cell wall 0.02 21
CC GO:0030312 外封装结构External encapsulating structure 0.02 21
MF GO:0046906 四吡咯结合Tetrapyrrole binding 0.05 85
MF GO:0016788 水解酶活性, 作用于酯键Hydrolase activity, acting on ester bonds 0.05 94
MF GO:0020037 血红素结合Heme binding 0.05 82
MF GO:0004553 水解酶活性, 水解O-糖基化合物
Hydrolase activity, hydrolyzing O-glycosyl compounds		 0.05 85

表2

三得利干旱与对照差异表达基因的GO注释分析"

类别
Category		 基因本体ID
GO ID		 描述
Description		 多重假设检验校正后的P值
P-adj value		 基因数
No. of genes
CC GO:0009521 光系统Photosystem 0.01×10-5 32
CC GO:0034357 光合膜Photosynthetic membrane 0.01×10-5 32
CC GO:0009579 类囊体Thylakoid 0.01×10-5 33
CC GO:0044436 类囊体部分Thylakoid part 0.01×10-5 33
MF GO:0016757 转移酶活性, 转移糖基Transferase activity, transferring glycosyl groups 0.08×10-5 149
CC GO:0009523 光系统II Photosystem II 0.03×10-3 20
MF GO:0016758 转移酶活性, 转移己糖基团Transferase activity, transferring hexosyl groups 0.04×10-3 132
MF GO:0004185 丝氨酸型羧肽酶活性Serine-type carboxypeptidase activity 0.04×10-3 29
MF GO:0046906 四吡咯结合Tetrapyrrole binding 0.04×10-3 131
MF GO:0016705 氧化还原酶活性, 作用于配对供体, 结合或减少分子氧
Oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen		 0.04×10-3 113
BP GO:0015979 光合作用Photosynthesis 0.01×10-2 38
CC GO:0009654 光系统II析氧复合物Photosystem II oxygen evolving complex 0.02×10-2 14
CC GO:0042651 类囊体膜Thylakoid membrane 0.08×10-2 14
CC GO:1990204 氧化还原酶复合物Oxidoreductase complex 0.08×10-2 14
MF GO:0005506 铁离子结合Iron ion binding 0.09×10-2 119
CC GO:0019898 膜的外在成分Extrinsic component of membrane 0.04×10-1 13
MF GO:0003700 DNA结合转录因子活性DNA-binding transcription factor activity 0.07×10-1 116
MF GO:0016835 碳氧裂解酶活性Carbon-oxygen lyase activity 0.01 26
CC GO:0009522 光系统I Photosystem I 0.02 10
MF GO:0016747 转移酶活性, 转移除氨酰基以外的酰基
Transferase activity, transferring acyl groups other than amino-acyl groups		 0.02 64
MF GO:0008236 丝氨酸型肽酶活性Serine-type peptidase activity 0.02 64
MF GO:0020037 血红素结合Heme binding 0.02 129
MF GO:0004180 羧肽酶活性Carboxypeptidase activity 0.03 29
MF GO:0070008 丝氨酸型外肽酶活性Serine-type exopeptidase activity 0.03 30
MF GO:0016746 转移酶活性, 转移酰基Transferase activity, transferring acyl groups 0.03 76
MF GO:0017171 丝氨酸水解酶活性Serine hydrolase activity 0.03 64
MF GO:0016701 氧化还原酶活性, 作用于单个供体并结合分子氧
Oxidoreductase activity, acting on single donors with incorporation of molecular oxygen		 0.04 26
MF GO:0140110 转录调节活性Transcription regulator activity 0.04 122
MF GO:0008374 O-酰基转移酶活性O-acyltransferase activity 0.04 11

图7

干旱胁迫下的相关差异基因表达水平值热图 基因的表达(Z值)水平由绿(低)到红(高)的颜色表示。"

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