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作物学报 ›› 2018, Vol. 44 ›› Issue (02): 177-184.doi: 10.3724/SP.J.1006.2018.00177

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自花授粉诱导的甘蓝功能基因BoSPI的克隆与表达分析

蒲敏1, 罗绍兰1, 廉小平2, 张贺翠1, 白晓璟1, 王玉奎1, 左同鸿1, 高启国2, 任雪松2, 朱利泉1,*()   

  1. 1西南大学农学与生物科技学院, 重庆400700
    2重庆市蔬菜重点实验室, 重庆400700
  • 收稿日期:2017-05-04 接受日期:2017-09-10 出版日期:2018-02-12 网络出版日期:2017-10-27
  • 通讯作者: 朱利泉
  • 作者简介:

    pm326001821@email.swu.edu.cn

  • 基金资助:
    本研究由国家自然科学基金项目(31572127)和重庆市研究生科研创新项目(CYS16076)资助

Cloning and Expression Analysis of BoSPI Induced by Self-pollination in Brassica oleracea L. var. capitata

Min PU1, Shao-Lan LUO1, Xiao-Ping LIAN2, He-Cui ZHANG1, Xiao-Jing BAI1, Yu-Kui WANG1, Tong-Hong ZUO1, Qi-Guo GAO2, Xue-Song REN2, Li-Quan ZHU1,*()   

  1. 1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    2 Key Laboratory in Olericulture of Chongqing, Chongqing 400715, China
  • Received:2017-05-04 Accepted:2017-09-10 Published:2018-02-12 Published online:2017-10-27
  • Contact: Li-Quan ZHU
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31572127) and the Research Project of Graduate Students in Chongqing (CYS16076).

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摘要：

自交不亲和性是甘蓝在长期进化过程中形成的防止自交衰败、促进杂交优势的一种复杂而完善的遗传机制。克隆自交不亲和性相关基因对甘蓝自交不亲和性的深入研究和利用有重要意义。本研究通过挖掘0~60 min自花和异花授粉的甘蓝柱头转录组数据, 筛选到一个受自花授粉诱导上调表达的基因, 命名为BoSPI。BoSPI开放阅读框534 bp, 编码177个氨基酸, 理论等电点为4.21, 不包含信号肽和跨膜区, 含有4个保守的EF-hand结构域。BoSPI基因起始密码子上游2000 bp的核苷酸序列中含有真菌诱导响应、代谢调节以及器官形成等应答元件。BoSPI基因在大肠杆菌中可诱导表达为17 kD的蛋白。BoSPI在柱头中表达量最高, 在花瓣、萼片、叶片、雄蕊表达量较低。BoSPI蛋白被定位在细胞膜和细胞质。自花授粉30 min后对BoSPI基因的诱导表达显著增强。表明BoSPI参与了柱头响应自花花粉刺激的分子过程, 可能是实现甘蓝自交不亲和性相关的某种新功能基因。

关键词: 自花授粉, 结球甘蓝, 基因BoSPI, 转录组测序

Abstract:

Self incompatibility is a complex and comprehensive genetic mechanism formed in the long-term evolution, which prevents inbreeding and promotes heterosis. Mining functional genes involving in self incompatibility has an important significance for the study of self incompatibility in Brassica oleracea L. var. capitata. In this study, we identified a gene named BoSPI which expression was up-regulated and induced by self-pollination based on the stigma transcriptome data in 0-60 min self-pollination and cross-pollination. BoSPI contains an open reading frame (ORF) with the length of 534 bp, encoding a protein of 177 amino acid residues without introns, which contains four conserved EF-hand domains without signal peptide and transmembrane domain, the theory isoelectric point of BoSPI is 4.21. In addition, diverse cis-acting promoter elements involved in fungal elicitor response, metabolic regulation and organ formation were discovered in the upstream 2000 bp of initial codon of BoSPI. BoSPI could be expressed as a 17 kD protein in E. coli BL21 (DE3). The expression level of BoSPI was the highest in stigmas and lower in petals, sepals, leaves and stamens of cabbage after self-pollination. Subcellular localization analysis revealed that BoSPI encoded a protein localized in the cell membrane and cytoplasm. The expression of BoSPI gene was significantly induced by self pollination after 30 minutes. These results suggest that BoSPI is involved in the molecular processes of the stigma response to self-pollen stimulation, which may be a new functional gene related to the self incompatibility of Brassica oleracea L. var. capitata.

Key words: self-pollination, Brassica oleracea L. var. capitata, gene (BoSPI), transcriptome technology

表1

基因克隆及定量PCR中的引物"

引物
Primer		 引物序列
Primer sequence (5′-3′)		 退火温度
Tm (ºC)		 引物用途
Primer description.
GST F: GATCTGGTTCCGCGTGGATCCATGGCAAGTGGTAACCCTGTAAC 60 基因的原核表达
Prokaryotic expression
R: GATGCGGCCGCTCGAGTCGACCTAGGCCGTCGAACCACCG
qRP F: GATCAGGACAAGAACGGGCTC 60 qRT-PCR
R: GTTGTTGCTGTTAGCGAGGGAG
Actin3 F: GGCTGATGGTGAAGATATTCA 58 内参引物
Internal reference
R: CAAGCACAATACCAGTAGTAC
GFP F: AAGTCCGGAGCTAGCTCTAGAATGGCAAGTGGTAACCCTGTAACC 63 基因亚细胞定位
Subcellular localization of gene
R: GCCCTTGCTCACCATGGATCCGGCCGTCGAACCACCGTTC

图1

经转录组测序分析甘蓝柱头内BoSPI响应自花和异花授粉后的表达情况 SI: 自交组; CP: 杂交组。SI: self-pollination; CP: cross-pollination."

图2

甘蓝BoSPI基因cDNA和gDNA序列扩增"

图3

BoSPI基因序列及其推导的氨基酸序列(A)、Ca2+结合核心结构分析(B)和BoSPI蛋白三维结构预测(C) A图中下画线指示4个EF-hand结构域, 红色字体表示12个氨基酸组成的Ca2+结合的核心区域; B图中下排数字表示对这12个氨基酸残基的编号, 数字0表示该核心区域上游第一个氨基酸残基。"

图4

BoSPI及其同源序列关系分析红框表示与CML27不同但与CML26相同的核苷酸位点。"

表3

BoSPI基因上游调控区顺式作用元件"

相关功能预测
Associated putative function		 启动子顺式作用原件
cis-elements in the promoter region
Light response ACE, ATCT-motif, Box 4, Box I, G-Box, GAG-motif, MRE
Salicylic acid response TCA-element
MeJA response CGTCA-motif, GARE-motif
Endosperm expression Skn-1_motif
Fungal elicitor response Box-W1
Gibberellin response GARE-motif
DNA binding protein (ATBP-1) AT-rich element
Anaerobic induction ARE
Abscisic acid response ABRE
Stress responsiveness HSE, LTR, MBS, TC-rich repeats

图5

BoSPI蛋白的原核表达 M: 蛋白分子量标准; 1: GST蛋白; 2: BoSPI-GST融合蛋白。"

图6

BoSPI-GFP融合蛋白的亚细胞定位绿色表示GFP蛋白在激光共聚焦显微镜下所发的绿色荧光。"

图7

BoSPI基因在甘蓝不同组织中的表达分析"

图8

BoSPI基因在不同授粉处理中的表达。 SI: 自交组; CP: 杂交组。"

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