甘蓝自交不亲和相关基因BoPUB9的克隆及表达分析

doi:10.3724/SP.J.1006.2022.04276

作物学报 ›› 2022, Vol. 48 ›› Issue (1): 108-120.doi: 10.3724/SP.J.1006.2022.04276

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

甘蓝自交不亲和相关基因BoPUB9的克隆及表达分析

谢琴琴(), 左同鸿, 胡燈科, 刘倩莹, 张以忠, 张贺翠, 曾文艺, 袁崇墨, 朱利泉^*()

西南大学农学与生物科技学院, 重庆 400716

收稿日期:2020-12-16 接受日期:2021-04-14 出版日期:2022-01-12 网络出版日期:2021-06-09
通讯作者: 朱利泉
作者简介:E-mail: xieqq1995@163.com第一联系人：^**同等贡献
基金资助:
国家自然科学基金项目(31572127);中央高校基本科研业务费专项资金资助(XDJK2020D024)

Molecular cloning and expression analysis of BoPUB9 in self-incompatibility Brassica oleracea

XIE Qin-Qin(), ZUO Tong-Hong, HU Deng-Ke, LIU Qian-Ying, ZHANG Yi-Zhong, ZHANG He-Cui, ZENG Wen-Yi, YUAN Chong-Mo, ZHU Li-Quan^*()

College of Agronomy and Biotechnology, Southwest University, Chongqing 400716, China

Received:2020-12-16 Accepted:2021-04-14 Published:2022-01-12 Published online:2021-06-09
Contact: ZHU Li-Quan
About author:First author contact:^** Contributed equally to this work
Supported by:
National Natural Science Foundation of China(31572127);Fundamental Research Funds for the Central Universities(XDJK2020D024)

摘要/Abstract

摘要：

自交不亲和性(self-incompatibility, SI)是指植株的雌蕊对自身花粉和异体花粉进行识别从而抑制自身花粉萌发的特性, PUB (Plant U-Box)蛋白在植物抗逆及信号转导过程中起着重要的作用。本研究通过分析甘蓝自花授粉0~60 min的柱头转录组数据, 筛选到1个受自花授粉诱导上调表达的PUB蛋白编码基因BoPUB9。BoPUB9 cDNA序列长度为1368 bp, gDNA序列全长1720 bp, 含有1个臂重复结构域和1个U-box结构域。荧光定量PCR结果显示, BoPUB9基因在甘蓝的不同组织中均有表达, 在萼片中的表达量最高, 花瓣、雄蕊、柱头表达量次之, 在花粉花蕾中表达量相对较低, 同GUS染色结果保持一致。BoPUB9基因在自花授粉0~30 min内快速上调表达, 15 min后为异花授粉后表达量的20多倍, 在30 min后达到差异最大, 自花授粉后的表达量为异花授粉的40多倍。亚细胞定位结果显示, BoPUB9蛋白在细胞核和细胞质中均有表达, 且BoPUB9蛋白在大肠杆菌中被成功诱导表达, 相对分子质量为51 kD, 与预测结果一致。酵母双杂交与poll-down结果显示, SRK胞内域与PUB9蛋白存在相互作用。推测BoPUB9可能是一种参与甘蓝自交不亲和反应过程的新基因, 这为甘蓝自交不亲和的进一步研究和利用提供了新内容。

关键词: 甘蓝, 基因克隆, 自交不亲和, 表达分析, 酵母双杂交

Abstract:

Self-incompatibility (SI) refers to the characteristic that the pistil of a plant can recognize its own pollen and allogeneic pollen so as to inhibit the germination of its own pollen. PUB (Plant U-Box) proteins play an important role in plant stress resistance and signal transduction. In this study, through the analysis of stigma transcriptome data of Brassica oleracea self-pollination at 0-60 minute(s), a PUB protein coding gene BoPUB9 was screened and up-regulated by self-pollination. The BoPUB9 cDNA sequence was 1368 bp, gDNA of 1720 bp in length, containing one arm repeat domain and one U-box domain. The relative expression levels showed that BoPUB9 gene was expressed in different tissues of Brassica oleracea, and the relative expression level was the highest in sepals, followed by petals, stamens and stigmas, and relatively lower in pollen buds, which was consistent with the results of GUS staining. The relative expression level of BoPUB9 gene was up-regulated rapidly in self-pollination 0-30 minute(s), the expression level after 15 min was more than 20 times that after cross-pollination, and the difference was the biggest after 30 min, the expression level after self-pollination was more than 40 times that after cross-pollination. Subcellular localization revealed that BoPUB9 protein was expressed in both nucleus and cytoplasm, and BoPUB9 protein was successfully induced in E. coli, and the relative molecular weight of 51 kD was consistent with the predicted results. The results of yeast two-hybrid and poll-down indicated that the intracellular domain of SRK interacted with PUB9 protein. It was speculated that BoPUB9 might be a novel gene involved in the process of self-incompatibility in Brassica oleracea, which providing a new content for further research and utilization of self-incompatibility in Brassica oleracea.

Key words: Brassica oleracea, gene cloning, self-incompatibility, the relative expression level, yeast two-hybrid

谢琴琴, 左同鸿, 胡燈科, 刘倩莹, 张以忠, 张贺翠, 曾文艺, 袁崇墨, 朱利泉. 甘蓝自交不亲和相关基因BoPUB9的克隆及表达分析[J]. 作物学报, 2022, 48(1): 108-120.

XIE Qin-Qin, ZUO Tong-Hong, HU Deng-Ke, LIU Qian-Ying, ZHANG Yi-Zhong, ZHANG He-Cui, ZENG Wen-Yi, YUAN Chong-Mo, ZHU Li-Quan. Molecular cloning and expression analysis of BoPUB9 in self-incompatibility Brassica oleracea[J]. Acta Agronomica Sinica, 2022, 48(1): 108-120.

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引物名称 Primer name	引物序列 Primer sequence (5°-3°)	用途 Functions
BoPUB9-AD	F: GTACCAGATTACGCTCATATGATGGCAAAGACCGGATTTTTC	酵母双杂交引物
	R: ATGCCCACCCGGGTGGAATTCGGCTGTGTGAGTAAGATTCATA	Yeast two-hybrid primers
SRKj-BK	F: GTACCAGATTACGCTCATATGTCAGATCGTGAGGTTTTCAATTC	酵母双杂交引物
	R: ATGCCCACCCGGGTGGAATTCGCCTTGTTATCGTTCCATGACTA	Yeast two-hybrid primers
BoPUB9-PAN	F: AAGTCCGGAGCTAGCTCTAGAATGGCAAAGACCGGATTTTTC	基因亚细胞定位
	R: GCCCTTGCTCACCATGGATCCGGCTGTGTGAGTAAGATTCATA	Subcellular location
BoPUB9-PGEX	F: GATCTGGTTCCGCGTGGATCCATGGCAAAGACCGGATTTTTC	基因的原核表达
	R: GATGCGGCCGCTCGAGTCGACGGCTGTGTGAGTAAGATTCATA	Prokaryotic expression
BoPUB9-GUS	F: CAAGCTTGGCTGCAGGTCGACGTCTTCTTAACGACATGGCTTG	基因的启动子活性分析
	R: GGTGGACTCCTCTTAGAATTCCTCCGTCGGATCCACCG	Promoter activity analysis
dActin	F: GGCTGATGGTGAAGATATTCA	内参引物
	R: CAAGCACAATACCAGTAGTAC	Internal reference primers
qRT-PCR	F: TGGCTTCTGGTCAGACATACGA	荧光定量PCR引物
	R: GTGACAGCTTTCTTGGACTCG	qRT-PCR primers

软件或在线网站 Software and online websites	功能 Functions
Bio-soft (http://www.bio-soft.net/sms/index.html)和DNAMAN 8.0 软件	推导基因编码的氨基酸序列
Bio-soft (http://www.bio-soft.net/sms/index.html) and DNAMAN 8.0 software	Derive the amino acid sequence encoded by the gene
ExPASy-ProtParam tool (http://www.expasy.org/)	分析蛋白质理化性质
	Analysis of physical and chemical properties of protein
Signalp (http://www.cbs.dtu.dk/services/SignalP/)	预测蛋白质的信号肽
	Signal peptides for predicting proteins
TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/)	预测蛋白质的跨膜结构域
	Prediction of transmembrane domains of proteins
Netphos (http://www.cbs.dtu.dk/services/NetPhos/)	预测磷酸化位点
	Predicted phosphorylation site
NetNGlyc (http://www.cbs.dtu.dk/services/NetNGlyc/)	预测N-糖基化位点
	Prediction of N-glycosylation sites
ProtScale (https://web.expasy.org/protscale/)	预测疏水性/亲水性
	Prediction of hydrophobicity/hydrophilicity
SMART(http://smart.embl-heidelberg.de/)	蛋白质的高级结构域
	Advanced domain of protein
PROSITE (http://prosite.expasy.org/)	蛋白质的功能位点
	Functional sites of proteins
PlantCARE (http://bioinformatics.psb.ugentbe/webtools/plantcare/)	分析启动子的顺式作用元件
	Analysis of cis-acting elements of promoter
MEGA7软件和NCBI (http://www.ncbi.nlm.nih.gov/)	构建进化树
MEGA7 software and NCBI (http://www.ncbi.nlm.nih.gov/)	Construct an evolutionary tree
BRAD (http://brassicadb.org/brad/index.php)	分析基因启动子序列
	Analysis of gene promoter sequence

相关功能预测 Putative associated function	启动子顺式作用元件 Cis-elements in the promoter region
脱落酸应答 Abscisic acid (ABA) response	ABRE元件 ABRE
赤霉素应答 Gibberellin (GA) response	GARE基序, P-box元件 GARE-motif, P-box
生长素应答 Auxin (IAA) response	TGA元件 TGA-element
乙烯应答 Ethylene-response	ERELEE4元件 ERELEE4
水杨酸反应 Salicylic acid (SA) response	TCA元件 TCA-element
茉莉酸响应 MeJA-response	CGTCA基序, TGACG基序 CGTCA-motif, TGACG-motif
花粉特异性顺式作用元件 Pollen-specific cis-acting element	POLLENLELAT52元件 POLLENLELAT52
光响应 Light response	G-Box元件, GT1基序, TCT基序 G-Box, GT1-motif, TCT-motif
低温响应 Low-temperature response	LTR元件 LTR
干旱诱导 Drought induced response	MBS元件 MBS
压力与防御相关反应元件 Stress and defense related response elements	WBOXATNPR1元件 WBOXATNPR1

相关功能预测 Putative associated function	启动子顺式作用元件 Cis-elements in the promoter region
脱落酸应答 Abscisic acid (ABA) response	ABRE元件 ABRE
赤霉素应答 Gibberellin (GA) response	GARE基序, P-box元件 GARE-motif, P-box
生长素应答 Auxin (IAA) response	TGA元件 TGA-element
乙烯应答 Ethylene-response	ERELEE4元件 ERELEE4
水杨酸反应 Salicylic acid (SA) response	TCA元件 TCA-element
茉莉酸响应 MeJA-response	CGTCA基序, TGACG基序 CGTCA-motif, TGACG-motif
花粉特异性顺式作用元件 Pollen-specific cis-acting element	POLLENLELAT52元件 POLLENLELAT52
光响应 Light response	G-Box元件, GT1基序, TCT基序 G-Box, GT1-motif, TCT-motif
低温响应 Low-temperature response	LTR元件 LTR
干旱诱导 Drought induced response	MBS元件 MBS
压力与防御相关反应元件 Stress and defense related response elements	WBOXATNPR1元件 WBOXATNPR1

甘蓝自交不亲和相关基因BoPUB9的克隆及表达分析

Molecular cloning and expression analysis of BoPUB9 in self-incompatibility Brassica oleracea

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