中国农业科学 ›› 2022, Vol. 55 ›› Issue (16): 3200-3209.doi: 10.3864/j.issn.0578-1752.2022.16.011
收稿日期:
2021-11-23
接受日期:
2022-05-22
出版日期:
2022-08-16
发布日期:
2022-08-11
通讯作者:
逯明辉
作者简介:
刘瑞瑶,E-mail: 基金资助:
LIU RuiYao(),HUANG GuoHong,LI HaiYan,LIANG MinMin,LU MingHui(
)
Received:
2021-11-23
Accepted:
2022-05-22
Online:
2022-08-16
Published:
2022-08-11
Contact:
MingHui LU
摘要:
【背景】辣椒作为一种在世界范围内普遍栽培的蔬菜,具有喜温不耐热的特性。随着近些年极端高温天气出现日渐频繁,热胁迫已经成为影响辣椒生产的主要环境因素之一,明确辣椒的耐热机制进而培育耐热品种对辣椒生产具有重要的意义。【目的】热激转录因子HsfA2在植物耐热性中发挥重要作用,筛选辣椒CaHsfA2上游的转录因子,并分析后者在辣椒耐热性形成中的作用,为进一步阐明辣椒耐热分子机制提供理论依据。【方法】以CaHsfA2起始密码子上游的955 bp启动子序列为诱饵,利用酵母单杂交(Y1H)技术,筛选CaHsfA2的上游转录因子,并通过Y1H点对点杂交、双荧光素酶报告系统(Dual-Luciferase)与萤火素酶互补技术(LCA)进一步验证二者之间的互作关系。利用qRT-PCR技术分析热胁迫下CaHsfA2上游转录因子在辣椒耐热品系‘R9’中的动态表达模式;利用基因瞬时表达技术分析上游转录因子的亚细胞定位;利用病毒诱导的基因沉默技术(VIGS)分析CaHsfA2上游转录因子的耐热功能。【结果】筛选获得了CaHsfA2上游转录因子CaBES1,验证了二者的互作关系,通过分析双荧光素酶报告系统的结果及CaBES1沉默辣椒植株中CaHsfA2的表达量发现,转录因子CaBES1对CaHsfA2具有转录抑制作用。亚细胞定位结果表明,CaBES1在细胞膜和细胞核上均有表达,热胁迫处理后细胞核内的荧光信号增强,符合其发挥生物学功能时由细胞质向细胞核转移的特性;动态表达模式分析表明,热胁迫下,CaBES1表达水平呈现先降低后升高的变化趋势,这也说明CaBES1可以响应热信号,为下一步对其耐热功能研究提供了支撑。辣椒CaBES1被沉默后,通过对比分析沉默植株和对照植株的表型、相对电导率、叶绿素含量等指标发现,CaBES1的沉默表达提高了CaHsfA2表达量并增强了辣椒的耐热性。【结论】CaBES1通过负调控CaHsfA2表达而抑制辣椒的耐热性。
刘瑞瑶,黄国弘,李海艳,梁敏敏,逯明辉. 辣椒CaHsfA2上游转录因子的筛选及耐热功能分析[J]. 中国农业科学, 2022, 55(16): 3200-3209.
LIU RuiYao,HUANG GuoHong,LI HaiYan,LIANG MinMin,LU MingHui. Screening and Functional Analysis in Heat-Tolerance of the Upstream Transcription Factors of Pepper CaHsfA2[J]. Scientia Agricultura Sinica, 2022, 55(16): 3200-3209.
表1
研究中使用的PCR引物"
用途 Application | 名称 Name | 序列 Sequence |
---|---|---|
酵母单杂交 Y1H | T7 | TAATACGACTCACTATAGG |
3AD | GAGATGGTGCACGATGCACAGT | |
pCaHsfA2-F | CGAGCTCTAAAAGCAAATGTCTGAAACGAGT | |
pCaHsfA2-R | GCGTCGACTATCTTTTTTCTTCTTCAGTCGCT | |
AD-CaBES1-F | GCCATGGAGGCCAGTGAATTCATGACATCGGGAACAAGG | |
AD-CaBES1-R | CAGCTCGAGCTCGATGGATCCTCTTGTCTTAGAACTCCCAA | |
互作关系验证 Verification of interactive relationship | LUC-pHsfA2-F | GCGTCGACTAAAAGCAAATGTCTGAAACGAGT |
LUC-pHsfA2-R | CATGCCATGGTATCTTTTTTCTTCTTCAGTCGCT | |
SK-BES1-F | GCTCTAGAATGACATCGGGAACAAGGA | |
SK-BES1-R | GGAATTCTCTTGTCTTAGAACTCCCA | |
亚细胞定位 Subcellular localization | pART27-BES1-F | GATGAACTATACAAAGAATTCATGACATCGGGAACAAGG |
pART27-BES1-R | TTCAGGCCTCCCGGGGGTACCTCTTGTCTTAGAACTCCCAA | |
qRT-PCR | qCaBES1-F | TTCGCTACCCGTTCTTTC |
qCaBES1-R | GTGATAGACCCTCCATTTTG | |
qCaHsfA2-F | GTAGCA TCAGTAGCCACAGC | |
qCaHsfA2-R | CAAGCAACTCTTCCCAAATA |
表2
CaHsfA2启动子顺式作用元件分析"
元件 Element | 序列 Sequence | 位置 Position | 数量 Amount | 功能 Function |
---|---|---|---|---|
ABRE | ACGTG | 61(-) | 1 | 参与脱落酸信号转导途径 Involved in the signal transduction pathway of ABA |
HSE | GAANNTTC | 36(+), 437(+) 725(+), 822(+) | 4 | 响应热胁迫 Responding to heat stress |
E-box | CANNTG | 7(+), 465(+) 716(+), 805(+) | 4 | BES1和BZR1结合位点 BES1 and BZR1 binding site |
ARE | AAACCA | 53(-) 899(+) 236(-) | 3 | 厌氧诱导必需元件 Necessary for anaerobic induction |
CGTCA/TGACG-motif | CGTCA/ TGACG | 475(±) | 1 | 参与茉莉酸响应 Involved in jasmonic acid reponsiveness |
TCA-element | CCATCTTTTT | 804(+) | 1 | 参与水杨酸反应 Involved in salicylic acid reponsiveness |
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