不同谷子品种喷施咪唑啉酮除草剂后的转录组分析
Transcriptome Analysis of Different Foxtail Millet (Setaria italica L.) Varieties Treated with Imazapic Herbicide
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收稿日期: 2023-01-27 修回日期: 2023-03-30 网络出版日期: 2023-03-31
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Received: 2023-01-27 Revised: 2023-03-30 Online: 2023-03-31
作者简介 About authors
宋慧,研究方向为谷子资源与遗传育种,E-mail:
咪唑啉酮类除草剂能有效防治谷子田的单、双子叶杂草,为探究谷子对咪唑啉酮类除草剂抗性的分子机制,于抗性品种(R)和敏感品种(S)出苗后15 d均匀地喷洒甲咪唑烟酸,通过高通量RNA-Seq测序分析抗性和敏感谷子品种中差异表达的基因和代谢途径。结果表明,2个品种在除草剂处理后,与光合作用和代谢途径相关的基因均被下调,特别是在敏感品种中下调更为显著。同样,敏感品种在除草剂处理后,与脂肪酸延伸相关基因表达也显著下调。5个随机选择基因的实时定量PCR(qRT-PCR)的结果与深度测序的结果一致。上述相关基因可能在谷子对咪唑啉酮类除草剂的抗性中起重要作用。
关键词:
Imidazolinone herbicides can effectively control monocotyledonous and dicotyledonous weeds in foxtail millet fields. In order to explore the molecular mechanism of millet resistance to imidazolinone herbicides, after the emergence of resistant varieties (R) and sensitive varieties (S) differentially expressed genes and metabolic pathways in resistant and susceptible foxtail millet cultivars were analyzed by high-throughput RNA-Seq sequencing after 15 d of uniform spraying with methamphetamine nicotinic acid. The results showed that genes related to photosynthesis and metabolic pathways were down-regulated after herbicide treatment in both cultivars. However, the down-regulation was more significant in the sensitive cultivar. Similarly, fatty acid elongation genes were significantly down-regulated after herbicide treatment in the sensitive cultivar. Quantitative real-time PCR (qRT-PCR) results of five candidate genes showed excellent agreement with those deep sequencing. These genes may play an important role in the imidazolinone herbicide resistance.
Keywords:
本文引用格式
宋慧, 王涛, 邢璐, 刘俊芳, 张扬, 刘金荣, 陈红旗, 冯佰利.
Song Hui, Wang Tao, Xing Lu, Liu Junfang, Zhang Yang, Liu Jinrong, Chen Hongqi, Feng Baili.
咪唑啉酮类除草剂是一种广谱除草剂,其特点是活性高、用量少,喷施剂量只有传统除草剂的1/10,常用于防治一年生禾本科杂草、阔叶杂草和多年生杂草。其机理是通过抑制乙酰羟酸合酶(ALS)活性,影响支链氨基酸的合成,引起杂草代谢紊乱,光合能力下降,植株变黄和生长迟缓,达到抑制杂草生长的目的[5-6]。然而,杂草以外的敏感作物也会受到除草剂的影响,喷施后同样发生光合能力下降[7]。因此,在育种过程中培育抗咪唑啉酮类除草剂品种降低除草剂对作物的影响成为行之有效的途径[8⇓-10]。抗咪唑啉酮类除草剂的农作物品种多通过靶位点的突变来实现,单核苷酸突变引起ALS基因编码的氨基酸发生变化,提高对咪唑啉酮类除草剂抗性;此外,也可以通过减少作物对除草剂的吸收或运输、促进除草剂的解毒以及降低除草剂活性等方式提高作物对咪唑啉酮类除草剂的抗性[11⇓⇓⇓-15]。现有抗咪唑啉酮类除草剂谷子品种主要是通过常规杂交育种方法选育,有关谷子对咪唑啉酮类除草剂的抗性数据仅限于生理指标和田间表型分析[16],关于其抗性的分子机制研究较少。因此,本研究通过高通量RNA测序(RNA-Seq)来挖掘和鉴定咪唑啉酮类除草剂在抗性和敏感谷子品种中差异表达的基因和代谢途径,解析咪唑啉酮类除草剂处理后抗性和敏感性谷子品种表型差异的原因,为后续使用分子技术提高谷子对咪唑啉酮类除草剂的抗性提供基因资源。
1 材料与方法
1.1 试验材料和试验处理
咪唑啉酮类除草剂抗性品种(R)5058和敏感品种(S)豫谷18来源于安阳市农业科学院谷子研究所资源库。咪唑啉酮类除草剂选用德国巴斯夫(中国)有限公司生产的甲咪唑烟酸水剂。在谷子出苗后15 d向谷子幼苗均匀地喷洒甲咪唑烟酸(1125 mL/hm2)进行处理,以清水喷施作对照,设置3个重复,采样时间为处理后0和48 h,取样部位为第3片幼叶,田间取样后放入液氮中带回,样本在-80 ℃冰箱中保存。
1.2 RNA提取、cDNA文库构建和测序
按照说明书的描述,使用RNA提取试剂盒RNAeasyminikit从抗性和敏感品种的幼叶组织中提取总RNA。使用不含RNA酶的DNase去除样品中的基因组DNA。使用NanoPhotometer®分光光度计评估RNA纯度。分别使用RNA分析试剂盒Qubit® RNAAssayKit和AgilentBioanalyzer 2100系统评估RNA浓度和完整性。使用poly-T寡核苷酸附着的磁珠从总RNA中纯化mRNA。按照说明书,使用NEBNext®UltraTMRNALibraryPrepKitfor Illumina®构建测序文库,在IlluminaHiseq 4000平台上对制备的文库进行测序,以生成150 bp的配对末端读数。测序数据保存在美国国家生物技术信息中心(NCBI)(BioProject登录号:PRJNA590609;BioSample登录号:SAMN13335985、SAMN13335986、SAMN13335987、SAMN133359858、SAMN13335989和SAMN13335990)。
1.3 转录组数据的预处理
1.4 差异表达分析
使用R语言的edgeR包识别差异表达基因。2组之间的倍数变化计算为logFC=log2(处理组/对照组)。logFC>1和q<0.05的基因被认为是差异表达基因(DEG)。
1.5 DEG的功能富集与分析
使用TermFinder软件对所有DEG进行GeneOntology(GO,
1.6 实时定量PCR分析
表1 qRT-PCR所用引物
Table 1
引物名称 Primer name | 引物序列 Primer sequence (5′-3′) |
---|---|
SiACTIN7-F | GGCAAACAGGGAGAAGATGA |
SiACTIN7-R | GAGGTTGTCGGTAAGGTCACG |
LOC101765996-F | CATTCACAGCCTGAGGTGTTTCC |
LOC101765996-R | CCATCTCCGACATCTCGCATT |
LOC101784847-F | GACATCCCGGAGGTGCTCAA |
LOC101784847-R | CGTCAGGCTCGGCATTCAA |
LOC101759205-F | AGACATCACCGACCTGTTCCAA |
LOC101759205-R | GCCCAGCACTTGTTCTCACG |
LOC101765796-F | ACGCCATCAACTTCCCCATC |
LOC101765796-R | GCCTTGTAGACGACGACCCA |
LOC101782898-F | AGACAACCGAAAATCAGCAGACAG |
LOC101782898-R | TGCCCTCAGGTATGCCCAGT |
2 结果与分析
2.1 不同抗性谷子品种的转录组测序概况
本研究共构建了18个cDNA文库,过滤原始读数后有27 620 386~45 776 540个有效数据(表2)。大约88.13%~93.53%的数据映射到谷子参考基因组,82.49%~90.81%的数据可以唯一匹配到谷子参考基因组。18个文库的Q20和Q30分别在96.40%和91.63%以上。
表2 谷子转录组数据
Table 2
样本 Sample | 有效数据 Clean reads | 映射数据 Mapped reads | 映射率 Mapped rate (%) | 唯一映射率 Unique mapped rate (%) | GC (%) | Q20 (%) | Q30 (%) |
---|---|---|---|---|---|---|---|
R0_1 | 35 066 474 | 31 574 750 | 90.04 | 87.36 | 56.00 | 96.67 | 91.78 |
R0_2 | 27 620 386 | 25 642 394 | 92.84 | 89.43 | 54.00 | 97.23 | 92.65 |
R0_3 | 30 454 968 | 28 403 582 | 93.26 | 89.87 | 55.50 | 97.36 | 92.86 |
S0_1 | 32 637 848 | 29 758 054 | 91.18 | 87.40 | 55.00 | 96.75 | 92.06 |
S0_2 | 32 213 222 | 29 246 420 | 90.79 | 86.69 | 55.00 | 96.76 | 92.11 |
S0_3 | 30 212 562 | 27 707 582 | 91.71 | 86.30 | 54.50 | 96.91 | 92.26 |
RT_1 | 41 040 330 | 38 384 804 | 93.53 | 90.81 | 53.50 | 97.48 | 93.26 |
RT_2 | 32 272 636 | 29 688 540 | 91.99 | 88.97 | 54.00 | 97.19 | 92.74 |
RT_3 | 31 474 482 | 28 009 464 | 88.99 | 86.24 | 53.50 | 96.55 | 91.90 |
ST_1 | 31 395 192 | 28 837 638 | 91.85 | 89.42 | 53.00 | 96.82 | 92.31 |
ST_2 | 32 216 306 | 29 179 166 | 90.57 | 87.82 | 53.50 | 96.92 | 92.42 |
ST_3 | 38 151 776 | 35 326 060 | 92.59 | 89.61 | 53.00 | 97.26 | 92.91 |
WRT_1 | 30 168 990 | 26 748 998 | 88.66 | 82.49 | 54.00 | 96.40 | 91.63 |
WRT_2 | 41 280 980 | 38 641 026 | 93.60 | 89.58 | 55.00 | 97.75 | 93.49 |
WRT_3 | 33 018 182 | 30 868 352 | 93.49 | 89.25 | 55.00 | 97.66 | 93.26 |
WST_1 | 42 602 542 | 38 785 788 | 91.04 | 87.82 | 55.50 | 97.80 | 93.64 |
WST_2 | 38 116 254 | 33 591 880 | 88.13 | 84.78 | 55.50 | 97.78 | 93.60 |
WST_3 | 45 776 540 | 41 381 022 | 90.40 | 87.53 | 56.50 | 97.85 | 93.66 |
R0表示未处理的抗性品种,S0表示未处理的敏感品种,RT表示除草剂处理的抗性品种,ST表示除草剂处理的抗性品种,WRT表示水处理的抗性品种,WST表示水处理的敏感品种,GC表示鸟嘌呤和胞嘧啶含量,Q20表示质量值≥20的核苷酸的百分比,Q30表示质量值≥30的核苷酸的百分比。
R0 indicates untreated resistant varieties, S0 indicates untreated sensitive varieties, RT indicates herbicide treated resistant varieties, ST indicates herbicide treated resistant varieties, WRT indicates water treated resistant varieties, WST indicates water treated sensitive varieties, GC indicates guanine and cytosine content, Q20 indicates percentage of nucleotides with mass value ≥20. Q30 indicates the percentage of nucleotides with a mass value ≥30.
2.2 不同比较组之间DEG的鉴定
为了鉴定不同基因谷子抗除草剂的关系,比较了不同分组下的基因情况,分组包括除草剂处理48 h的抗性品种与处理0 h的抗性品种比较(RT vs R0)、除草剂处理48 h的抗性品种与水处理48 h的抗性品种比较(RT vs WRT)、水处理48 h的抗性品种与水处理48 h的抗性品种比较(WRT vs R0)、除草剂处理48 h的敏感品种与水处理48 h的敏感品种比较(ST vs WST)、除草剂处理48 h的敏感品种与处理0 h的敏感品种比较(ST vsS0)、水处理48 h的敏感品种与水处理48 h的敏感品种比较(WST vs S0)。与R0相比,RT鉴定出1413个DEG(表3)。其中上调983个,下调430个。此外,与S0相比,ST鉴定出7453个DEG。其中上调3566个,下调3887个。与WRT相比,RT鉴定出743个DEG,其中上调615个,下调128个。与WST相比,ST鉴定出5298个DEG,其中上调2396个,下调2902个。与R0相比,WRT鉴定出186个DEG。其中上调71个,下调115个。与S0相比,WST鉴定出90个DEG。其中上调453个,下调467个。
表3 差异表达基因的数量
Table 3
比较组 Comparison group | DEG数 DEG number | 上调 Up-regulated | 下调 Down-regulated |
---|---|---|---|
RT vs R0 | 1413 | 983 | 430 |
ST vs S0 | 7453 | 3566 | 3887 |
RT vs WRT | 743 | 615 | 128 |
ST vs WST | 5298 | 2396 | 2902 |
WRT vs R0 | 186 | 71 | 115 |
WST vs S0 | 920 | 453 | 467 |
综合比较发现,用甲咪唑烟酸除草剂的处理分别有153个(RT vs WRT)和763个(ST vs WST)DEG在抗性和敏感品种中发生了变化(图1)。
图1
图1
除草剂和水处理0 h和48 h后抗性(a)和敏感(b)品种差异表达基因的维恩图
Fig.1
Venn diagram of differentially expressed genes in resistant (a) and sensitive (b) cultivars under 0 h and 48 h of herbicide or water treatments
2.3 DEG的GO注释分析
图2
图2
处理后谷子差异表达基因的GO分类
Fig.2
GO classifications of foxtail millet differentially expressed genes after treatment
图3
图3
20条KEGG通路显著富集的散点图
(a) RT vs WRT;(b) ST vs WST。富集因子是该通路注释的DEG数与该通路注释的所有基因数的比值。更大的富集因子意味着更大的密集度。q值是修正后的p值,取值范围在0到1之间。q值越低,富集越显著。
Fig.3
Scatter plot of 20 significantly enriched KEGG pathways
(a) RT vs WRT; (b) ST vs WST. Rich factor is the DEG numbers ratio annotated in this pathway term to all gene numbers annotated in this pathway term. Greater rich factor means greater intensiveness. q-value is corrected p-value ranging from 0 to 1. The lower the q value, the more significant the enrichment.
2.4 与除草剂反应差异相关的支链氨基酸合成相关基因
在处理后的2个品种中鉴定出17个与支链氨基酸合成相关的DEG(图4),包括3个乙酰羟酸合酶基因(ALS)、2个酮醇酸还原异构酶(KARI)、1个二羟酸脱水酶(DHAD)、2个2-异丙基苹果酸合酶(IPMS)、3个3-异丙基苹果酸脱氢酶(IMDH)。6个分支氨基酸转氨酶(BBCA)。除草剂处理后,几乎所有的基因在2个品种中都有所提高,其中ALS2、IMDH1、BBCA1和BBCA4在抗性品种中上调更明显。ALS3、IMPS3、BBCA2和BBCA6的表达在敏感品种中上调更明显。总体来说,支链氨基酸合成相关的DEG在2个品种中的表达趋势差异不大。
图4
图4
支链氨基酸合成相关差异表达基因热图分析
条形图表示热图中每个基因表达水平(log2 TPM)。基因的表达水平用不同的颜色表示。红色表示高表达,蓝色表示低表达。
Fig.4
Heatmap analysis of DEGs related to branched chain amino acid synthesis
The bar represents the scale of each gene's expression levels (log2 TPM) in the heat map. The expression level of the genes is denoted using different colours. Red denotes high expression while blue denotes low expression.
2.5 与除草剂反应差异相关的光合作用相关基因
处理后2个品种均有许多与天线蛋白、光系统和固碳相关的基因(图5)。在处理后的2个品种中鉴定出8个与天线蛋白相关的DEG(图5a),包括2个LHCI天线蛋白和6个LHCII天线蛋白。在2个品种中除草剂处理后这些基因的表达有所下降。然而,它们的表达在敏感品种中比在抗性品种中受到更多抑制。此外,与光系统相关的13个DEG的表达量在敏感品种中也更明显的下调(图5b)。同时,光合作用的暗反应也受到了除草剂的影响。在2个品种除草剂处理后,与碳固定相关的7个DEG的表达均下调。和上述结果一致,这些基因在敏感品种中受到更显著的抑制(图5c)。结果表明,相比于敏感品种,抗除草剂品种在除草剂胁迫下可以保持光合基因相对正常的表达。
图5
图5
光合相关DEG热图分析
(a) 与天线蛋白相关的DEG;(b) 与光系统相关的DEG;(c) 与碳固定相关的DEG。条形图表示热图中每个基因表达水平(log2 TPM)。基因的表达水平用不同的颜色表示。红色表示高表达,蓝色表示低表达。下同。
Fig.5
Heatmap analysis of DEGs related to photosynthesis
(a) DEGs associated with antenna protein; (b) DEGs associated with photosystem; (c) DEGs related to carbon fixation. The bar represents the scale of each gene's expression levels (log2 TPM) in the heat map. The expression level of the genes is denoted using different colours. Red denotes high expression while blue denotes low expression.The same below.
2.6 分析可能与除草剂反应相关的代谢途径基因
鉴定了与代谢途径相关的13个DEG。它们包括八氢番茄红素合酶(PSY)、香叶基香叶基二磷酸还原酶(GGDR)、单功能核黄素生物合成蛋白(RIBA3)、蔗糖合酶(SUS)、乙酰辅酶A羧化酶(ACC)、过氧化物酶27(PRX27)、过氧化物酶70(PRX70)、镁-原卟啉IX单甲酯[氧化]环化酶(MPMC)、原叶绿素还原酶(POR)、UTP-葡萄糖-1-磷酸尿苷酰转移酶(UGP1)、UDP-葡萄糖4-差向异构酶(UGE)、乙醇脱氢酶(ADH)和1-脱氧-D-木酮糖-5-磷酸合酶(DXS)。除草剂处理后,RIBA3在抗性品种中上调,但在敏感品种中下调(图6)。ACC的表达水平在抗除草剂品种中无明显变化,在敏感品种中显著下调。除草剂处理后,2个品种的其余11个基因均被下调。尽管如此,它们在敏感品种中的表达比在抗性品种中受到更多的抑制。
图6
图6
涉及代谢途径的DEG的热图分析
条形图表示热图中每个基因表达水平(log2 TPM)。
Fig.6
Heatmap analysis of DEGs involved in the metabolic pathway
The bar represents the expression level of each gene (log2 TPM) in the heat map.
2.7 可能与除草剂反应相关的脂肪酸延伸基因分析
在植物中,3-酮酰基-CoA合酶和3-氧代酰基-CoA还原酶是与超长链脂肪酸(VLCFA)生物合成相关的重要酶。抗除草剂品种的KCS5(LOC101785243)和KCS17(LOC101778317)在处理后的表达水平没有显著变化。然而,这2个基因在敏感品种中表达均显著下调(图7)。此外,KCS5(LOC101764380)、KCS6(LOC101784847)、2个KCS10(LOC101778938和LOC101759842)、KCS11(LOC101771353)、KCS12(LOM101770003)、KCS20(LOC101756089)和3个3-氧代酰基-CoA还原酶基因(LOC101758878,LOC101758878,LOC101785727)在除草剂处理后在2个品种中均下调。然而,这些基因在敏感品种中比在抗性品种中受到更多抑制。结果表明,一些与长链脂肪酸生物合成途径相关基因的表达在2个品种中存在显著差异。
图7
图7
超长链脂肪酸延长酶相关DEG的热图分析
Fig.7
Heatmap analysis of DEGs related to very-long-chain fatty acid elongases
2.8 RNA-Seq测序数据的qRT-PCR验证
使用qRT-PCR以验证RNA-Seq数据的可靠性。随机选择了5个DEG进行验证,包括LOC101765996(无机磷酸盐转运蛋白)、LOC101784847(3-酮脂酰辅酶A合酶6)、LOC101759205(乙烯响应转录因子)、LOC101765796(蛋白质解毒40)和LOC101782898(ATP结合盒式转运蛋白家族成员15)。它们的表达在除草剂处理后受到影响,与RNA-Seq结果一致,从而证实了RNA-Seq数据的可重复性(图8)。
图8
3 讨论
3.1 除草剂处理后支链氨基酸合成相关基因的表达情况
3.2 与光合作用相关的DEG在敏感品种中显著下调
植物的光合性能对除草剂等胁迫因素很敏感,光合作用及其相关参数是除草剂引起的参考指标之一[5]。前人研究认为,咪唑啉酮类除草剂会引起向日葵叶片萎黄[23],还对向日葵的光合氧化还原反应和叶片气体交换过程产生不利影响[22];甲氧咪草烟处理能降低小麦植株的CO2固定率[6],谷子敏感材料喷施甲咪唑乙烟酸后会显著谷子发芽率和存活率,甚至致死,谷子幼苗在喷药第7天,敏感材料的抗逆生理指标超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)均表现显著下降[16]。本研究发现,经咪唑啉酮除草剂处理后在敏感品种和抗性品种中均下调了天线蛋白相关的DEG,包括2个LHCⅠ天线蛋白和6个LHCⅡ天线蛋白,然而这些基因的表达在敏感品种中比在抗性品种中受到更多的抑制;与光合作用相关的13个DEG在敏感品种中也显著下调;在CO2固定阶段,除草剂处理后与碳固定相关的7个DEG在2个品种中均有所下调,敏感品种中比在抗性品种中下调的幅度更明显,这一发现与前人[16]研究结果一致。
3.3 与代谢过程相关的DEG的表达在敏感品种中受到更多抑制
除了破坏植物的光合作用和能量代谢过程外,咪唑啉酮除草剂还会影响作物的一系列代谢过程。许多与代谢过程相关的酶在植物对非生物胁迫的反应中发挥着关键作用。类胡萝卜素是植物抗氧化系统的成员,在清除植物中的活性氧物质、帮助植物抵抗与应激相关的损害方面发挥着至关重要的作用[25],PSY是类胡萝卜素生物合成途径中的一种必需酶,POD负责提高植物的非生物胁迫耐受性[26],ADH赋予植物非生物和生物胁迫抗性。此外,一些酶参与植物的叶绿素合成。单功能核黄素生物合成蛋白、镁―原卟啉IX单甲基酯氧化环化酶和原叶绿素还原酶的缺失会导致黄化幼苗的产生[27]。本研究认为,RIBA3的表达在抗性谷子品种中上调,但在除草剂处理后的敏感品种中下调,抗除草剂品种处理后ACC的表达水平无明显变化,但敏感品种中显著下调。此外,与植物代谢过程相关的其他基因同样在敏感品种中比在抗性品种中受到更多抑制。
3.4 与超长链脂肪酸合成相关的DEG的表达在敏感品种中受到更多抑制
4 结论
谷子抗咪唑啉酮除草剂转录组分析表明,谷子敏感品种和抗性品的差异表达基因影响了谷子对除草剂的抗性,包括与光合作用、代谢途径和脂肪酸延伸有关的基因,表明抗性和敏感的谷子品种之间存在着复杂的抗性机制。
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The herbicide imazamox may provoke temporary yellowing and growth retardation in IMI-R sunflower hybrids, more often under stressful environmental conditions. Although, photosynthetic processes are not the primary sites of imazamox action, they might be influenced; therefore, more information about the photosynthetic performance of the herbicide-treated plants could be valuable for a further improvement of the Clearfield technology. Plant biostimulants have been shown to ameliorate damages caused by different stress factors on plants, but very limited information exists about their effects on herbicide-stressed plants. In order to characterize photosynthetic performance of imazamox-treated sunflower IMI-R plants, we carried out experiments including both single and combined treatments by imazamox and a plant biostimulants containing amino acid extract. We found that imazamox application in a rate of 132 μg per plant (equivalent of 40 g active ingredient ha) induced negative effects on both light-light dependent photosynthetic redox reactions and leaf gas exchange processes, which was much less pronounced after the combined application of imazamox and amino acid extract.
A kaleidoscope of carotenoids
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Deficiency in riboflavin biosynthesis affects tetrapyrrole biosynthesis in etiolated Arabidopsis tissue
Saturated very-long-chain fatty acids promote cotton fiber and Arabidopsis cell elongation
Molecular caliper mechanism for determining very-long chain fatty acid length
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