棉花学报 ›› 2021, Vol. 33 ›› Issue (6): 435-447.doi: 10.11963/cs20200083
• 研究与进展 • 下一篇
收稿日期:
2020-10-26
出版日期:
2021-11-15
发布日期:
2022-04-14
通讯作者:
简桂良
E-mail:helang_88@163.com;jianguiliang@126.com
作者简介:
贺浪(1994―),男,硕士研究生, 基金资助:
He Lang1(),Zhang Huachong2,Si Ning1,Jian Guiliang1,*(
)
Received:
2020-10-26
Online:
2021-11-15
Published:
2022-04-14
Contact:
Jian Guiliang
E-mail:helang_88@163.com;jianguiliang@126.com
摘要:
【目的】BZR1是油菜素内酯(Brassinosteroid,BR)信号通路中关键的转录因子,去磷酸化后可直接调控BR信号通路下游基因的表达,影响植物的生长发育和免疫反应。明确其功能对了解棉花的抗/感病分子机制具有重要意义。【方法】分析未接种和接种黄萎病菌(大丽轮枝菌,Verticilium dahliae)的中植棉KV-3的转录组测序结果,筛选到1个下调表达基因GhBZR1,并通过cDNA末端快速扩增(Rapid amplification of cDNA ends,RACE)、生物信息学分析、实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,qRT-PCR)、亚细胞定位、病毒诱导的基因沉默(Virus-induced gene silencing,VIGS)和超表达,对该基因进行了克隆与功能验证。【结果】在陆地棉中克隆出GhBZR1基因全长1 515 bp,开放阅读框为942 bp,编码313个氨基酸。生物信息学分析表明,GhBZR1蛋白与海岛棉中的BZR1蛋白同源性最高,具有BES1_N超家族结构域,属于亲水性蛋白,具有多个磷酸化位点,在其启动子区域存在3个与病原菌抗性相关的顺式作用元件,分别是TC-rich repeats、ABRE和MBSI元件。亚细胞定位显示,GhBZR1蛋白定位在细胞核。qRT-PCR检测结果表明,GhBZR1基因在茎中优势表达;大丽轮枝菌诱导后,该基因在不同抗病、感病品种中的转录水平不同,且能被茉莉酸和水杨酸诱导表达;抑制GhBZR1基因表达能增强感病陆地棉品种86-1对黄萎病菌的抗性;超表达GhBZR1基因使转基因拟南芥抗黄萎病菌能力减弱、病症加重。【结论】GhBZR1基因在棉花抗黄萎病的过程中起负调控作用。
贺浪,张华崇,司宁,简桂良. 陆地棉GhBZR1基因的克隆及功能研究[J]. 棉花学报, 2021, 33(6): 435-447.
He Lang,Zhang Huachong,Si Ning,Jian Guiliang. Cloning and functional analysis of GhBZR1 in Gossypium hirsutum L.[J]. Cotton Science, 2021, 33(6): 435-447.
表1
本研究所用引物"
引物名称 Primer name | 引物序列 Primer sequence | 用途 Purpose |
GhBZR1-F | ATGACATCGGATGGGGCGAC | 扩增中间片段 Amplification of middle segment sequence |
GhBZR1-R | TTAACACTGAGGCTTTCCGCTTC | |
3′OGhBZR1 | TGCCTCCCCAAAATTTGCAC | 3′ RACE |
GSP Outer | ATGTTTGGGAATATGCAGATCGGA | |
3′I GhBZR1 | CGCGGATCCTCCACTAGTGATTTCACTATAGG | |
GSP Inner | TGATAATGCCCTTATGGAGGATAG | |
5′O GhBZR1 | GATTACGCCAAGCTTCGGTGGGTAGGACTA- GCTGGGGC | 5′ RACE |
5′I GhBZR1 | GATTACGCCAAGCTTACGACCCAACCAGCC- TCCGAACAA | |
UPM | CTAATACGACTCACTATAGGGCAAGCAGT- GGTATCAACGCAGAGT | |
qGhBZR1-F | GCTTCCCTTTTCATCGTTTTCA | 扩增基因全长 Amplification of full-length GhBZR1 gene |
qGhBZR1-R | GCGATTAAGTTTGGATTGGATTG | |
B-F | TGCCTCCACTCAGAATCTCAAA | 棉花GhBZR1表达水平检测 Detection of GhBZR1 expression in cotton |
B-R | TGGGGCAGAAACAGCATAAA | |
Ubiquitin-F | GCTCGGATACGATTGATAAC | 检测Ubiquitin基因表达水平 Detection of Ubiquitin gene |
Ubiquitin-R | AAGACGAAGAACAAGGTGAA | |
YB-F | CC CCCGGGATGACATCGGATGGGGCGAC | 构建GhBZR1基因亚细胞定位载体 Vector construction for subcellular localization |
YB-R | GC TCTAGAACACTGAGGCTTTCCGCTTC | |
VIGS-GhBZR1-F | TC TTAATTAAACACCCCCACTTTCATCTCC | 构建GhBZR1基因沉默载体 Construction of GhBZR1 gene silencing vector |
VIGS-GhBZR1-R | GT ACTAGTCTCTCCTTCCCATGGTTTCAGA | |
O-GhBZR1-F | CGGGATCCGATGACATCGGATGGGGCGAC | 构建GhBZR1超表达载体 Construction of GhBZR1 overexpression vector |
O-GhBZR1-R | GCTCTAGATTAACACTGAGGCTTTCCGCTTC | |
AtBZR1-F | GGCACCACTTATCGCAAGGGATG | 检测拟南芥中GhBZR1的表达 Detection of GhBZR1 expression in Arabidopsis |
AtBZR1-R | GGAAGGGCTGACTTGACATGAAGG | |
Atactin-12-F | CCGAAGGCTAACCGTGAGAAGATG | 检测Atactin-12的表达 Detection of Atactin-12 expression |
Atactin-12-R | GAGGGAGAGAACAGCTTGAATGGC |
表2
GhBZR1启动子中与抗病性相关的顺式作用元件"
顺式作用元件 Cis-acting element | 序列 Sequence | 位置 Position/bp | 作用 Function |
TC-rich repeats | ATTCTCCAAC | (+)481 | 参与防御和应激反应 Participate in defense and stress response |
ABRE | TACGTGTC ACGTG | (-)389 (-)391 | 响应脱落酸 Participate in abscisic acid reaction |
MBSI | AAAAAAC(G/C)GTTA | (+)142 | MYB 结合位点,参与类黄酮生物合成基因调控 MYB binding site, involved in the regulation of flavonoid biosynthesis genes |
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