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作物学报

作物学报 ›› 2023, Vol. 49 ›› Issue (7): 1808-1817.doi: 10.3724/SP.J.1006.2023.21050

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

外源6-BA调控孕穗期低温后小麦幼穗发育的转录组分析

李凌雨(), 周琦锐, 李洋, 张安民, 王贝贝, 马尚宇, 樊永惠, 黄正来*(), 张文静*()   

  1. 安徽农业大学农学院, 安徽合肥 230036
  • 收稿日期:2022-07-17 接受日期:2022-11-25 出版日期:2023-07-12 网络出版日期:2022-12-01
  • 通讯作者: *张文静, E-mail: zhangwenjing79@126.com; 黄正来, E-mail: xdnyyjs@163.com
  • 作者简介:E-mail: 171650776@qq.com
  • 基金资助:
    本研究由国家自然科学基金项目(31801285);安徽省重点研究与开发计划项目(202204c06020040);安徽省科技重大专项(202003a06020014)

Transcriptome analysis of exogenous 6-BA in regulating young spike development of wheat after low temperature at booting stage

LI Ling-Yu(), ZHOU Qi-Rui, LI Yang, ZHANG An-Min, WANG Bei-Bei, MA Shang-Yu, FAN Yong-Hui, HUANG Zheng-Lai*(), ZHANG Wen-Jing*()   

  1. College of Agronomy, Anhui Agricultural University, Hefei 230036, Anhui, China
  • Received:2022-07-17 Accepted:2022-11-25 Published:2023-07-12 Published online:2022-12-01
  • Contact: *E-mail: zhangwenjing79@126.com; E-mail: xdnyyjs@163.com
  • Supported by:
    The National Natural Science Foundation of China(31801285);The Anhui Key Research and Development Plan(202204c06020040);The Major Science and Technology Project of Anhui Province(202003a06020014)

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摘要：

小麦孕穗期对低温非常敏感, 低温胁迫后外源喷施6-苄氨基腺嘌呤(6-BA), 能够缓解低温胁迫对小麦造成的伤害, 通过转录组测序技术分析6-BA提高小麦抗寒性的分子机制。选用低温敏感型品种皖麦52和低温迟钝型品种烟农19为试验材料, 在孕穗期低温胁迫后喷施20 mg L-1的6-BA溶液, 以喷施等量蒸馏水处理为对照。观察幼穗形态, 并测定幼穗可溶性糖含量和淀粉含量。再通过转录组测序筛选并分析差异表达基因, 探究差异表达基因的功能及可能参与的调控通路, 采用qRT-PCR方法对测序结果进行验证。外源6-BA处理10 d后, 与对照相比小麦幼穗形态发育良好, 可溶性糖、淀粉含量均升高。转录组结果表明, 在皖麦52和烟农19中分别鉴定出22,770个和9866个差异基因, 其中有661个基因在两个品种中均上调, 从中筛选出ARF5、AGPL1、1-SST、SWEET15等基因, 这些基因与调节植物激素水平、淀粉合成、糖代谢等有关。对筛选出的差异基因进行GO和KEGG富集分析。GO注释表明皖麦52和烟农19差异基因的功能都主要富集于细胞结构稳定性、代谢、催化活性等。KEGG富集分析表示信号转导、内源激素水平的调节、碳代谢、膜结构和功能的改变等途径发生显著变化。选择部分候选基因对其表达模式进行qRT-PCR分析, 结果说明RNA-seq数据准确。综上所述, 6-BA可以通过调节小麦内部抗氧化物质代谢、激素信号转导、碳水化合物代谢、渗透调节等途径缓解低温损伤, 研究结果可为探索减轻春季低温对小麦伤害的栽培措施提供理论基础。

关键词: 小麦, 6-BA, 低温, 幼穗, 转录组分析

Abstract:

In recent years, due to the frequent occurrence of extreme weather, low temperatures (LT) became one of the main disasters restricting wheat production. LT has negative effects on wheat growth and yield formation, especially at booting stage. Exogenous spraying of 6-benzylamino adenine (6-BA) after LT at booting stage can alleviate the damage caused by LT in wheat, but the related molecular regulation mechanism is still unclear. In this study, transcriptome sequencing technology was used to analyze the molecular mechanism of 6-BA improving cold tolerance in wheat. The LT sensitive variety Wanmai 52 and insensitive variety Yannong 19 were selected as the experimental materials. The experiment was carried out in combination of potted and field planting. Two wheat cultivars were planted in plastic pots at a planting density of ten plants per pot. At booting stage, the pots were moved into an artificial climate chamber for low temperature treatment. At the end of the treatment, 20 mg L-1 6-BA solution was sprayed, and an equal volume of distilled water was sprayed as the control. The morphology of young spike, and the content of soluble sugar and starch in young spikes were determined. Some candidate differentially expressed genes (DEGs) were screened and their relative expression patterns were analyzed by qRT-PCR, and the results were verified by qRT-PCR. After 10 days, compared with the control, the morphological development of young spike was better, fuller and longer. The contents of soluble sugar and starch in young spikes increased after 6-BA treatment. The results showed that 22,770 DEGs were identified in Wanmai 52 and 9866 in Yannong 19, respectively, and 661 genes were up-regulated in the two cultivars. ARF5, AGPL1, 1-SST, SWEET15, and other genes were screened out, which were related to the regulation of plant hormone level, starch synthesis, and sugar metabolism. GO and KEGG enrichment analysis were performed on the selected differential genes. GO annotation revealed that the functions of the differential genes of the two varieties were mainly concentrated in cell structure stability, metabolism, and catalytic activity. KEGG enrichment analysis demonstrated that signal transduction, regulation of endogenous hormone levels, carbon metabolism, the changes of membrane structure and function had significant changes. In conclusion, 6-BA could alleviate cold damage by regulating the metabolism of antioxidant substances, hormone signal transduction, carbohydrate metabolism, osmotic adjustment, and other ways in wheat. The results provide a theoretical basis for exploring the cultivation measures to reduce the damage of low temperature on wheat in spring.

Key words: wheat, 6-BA, low temperatures, young spikes, transcriptome

表1

实时荧光定量PCR所用的引物"

基因名称
Gene name		 正向引物
Forward sequence (5′-3′)		 反向引物
Reverse sequence (5′-3′)		 基因ID
Gene ID
GAPDH CCACTAACTGCCTTGCTCCT CCAGTGCTGCTTGGAATGA
ARF5 TCGACAGAGTATGGGTTGGAA CGGAAACATCGGCTGAAG TraesCS6A02G111900
AGPL1 ACATCACGCAGAAACCTACCA AACGGATTCCAACGACACTG TraesCS5D02G484500
SWEET15 TATCAGTTGGTTTGTGTGTTCGA GTCGGGAGTGGTGAAAGGA TraesCS7B02G050500

图1

外源6-BA处理后幼穗表型变化 Wanmai 52 CK为皖麦52低温处理后喷施清水; Wanmai 52 6-BA为皖麦52低温处理后喷施6-BA溶液; Yannong 19 CK为烟农19低温处理后喷施清水; Yannong 19 6-BA为烟农19低温处理后喷施6-BA溶液。"

图2

幼穗可溶性糖含量及淀粉含量"

图3

差异表达基因的韦恩图"

表2

低温胁迫后喷施外源6-BA差异表达基因的GO注释"

GO号
GO ID		 GO功能
GO function		 皖麦52基因数目
及其所占百分比
Gene number and ratio in Wanmai 52		 烟农19基因数目
及其所占百分比
Gene number and ratio in Yannong 19		 GO类别
GO category
GO:0016021 膜的有机成分Integral component of membrane 3990 (23.22%) 1367 (18.73%) C
GO:0005524 ATP结合ATP binding 2195 (12.77%) 764 (10.47%) M
GO:0005515 蛋白质结合Protein binding 2092 (12.17%) 920 (12.61%) M
GO:0016020 细胞膜Membrane 1609 (9.36%) 566 (7.76%) C
GO:0055114 氧化还原过程Oxidation reduction process 1604 (9.33%) 708 (9.70%) B
GO:0005634 细胞核Nucleus 1576 (9.17%) 1076 (14.74%) C
GO:0004672 蛋白激酶活性Protein kinase activity 1503 (8.75%) 428 (5.86%) M
GO:0006468 蛋白磷酸化Protein phosphorylation 1500 (8.73%) 427 (5.85%) B
GO:0003677 DNA结合DNA binding 1410 (8.20%) 1034 (14.17%) M
GO:0006355 调控转录, DNA模板化
Regulation of transcription, DNA templating		 1083 (6.30%) 410 (5.62%) B
GO:0003824 催化活性Catalytic activity 1005 (5.85%) 372 (5.10%) M
GO:0046872 金属离子结合Metal ion binding 953 (5.55%) 432 (5.92%) M
GO:0016491 氧化还原酶活性Oxidoreductase activity 901 (5.24%) 385 (5.28%) M
GO:0055085 跨膜转运Transmembrane transport 799 (4.65%) 266 (3.64%) B
GO:0003700 DNA结合转录因子活性
DNA-binding transcription factor activity		 648 (3.77%) 232 (3.18%) M
GO:0020037 血红素结合Heme binding 617 (3.59%) 301 (4.12%) M
GO:0005975 碳水化合物代谢过程Carbohydrate metabolic process 535 (3.11%) 256 (3.51%) B

图4

KEGG通路富集散点图"

图5

IAA信号通路基因表达模式分析 红蓝色块代表相应的FPKM数值, 数值越大, 颜色越红; 从左到右样本依次为皖麦52 CK、皖麦52 6-BA、烟农19 CK、烟农19 6-BA。"

图6

糖类物质积累相关基因表达模式分析 色块所知内容同图5。"

图7

差异表达基因qRT-PCR验证"

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