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作物学报 ›› 2019, Vol. 45 ›› Issue (12): 1773-1783.doi: 10.3724/SP.J.1006.2019.94040

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

甘蓝SI相关基因BoCDPK14的克隆与分析

白晓璟1,廉小平2,王玉奎1,张贺翠1,刘倩莹1,左同鸿1,张以忠1,谢琴琴1,胡燈科1,任雪松2,曾静3,罗绍兰1,蒲敏1,朱利泉1,*()   

  1. 1 西南大学农学与生物科技学院, 重庆 400716
    2 西南大学园艺园林学院, 重庆 400716
    3 长江师范学院现代农业与生物工程学院, 重庆 408100
  • 收稿日期:2019-03-15 接受日期:2019-06-12 出版日期:2019-12-12 网络出版日期:2019-06-12
  • 通讯作者: 朱利泉
  • 作者简介:白晓璟, E-mail: xiamoyanyu33@163.com
  • 基金资助:
    本研究由国家自然科学基金项目(31572127);西南大学基本科研业务费专项资金项目(XDJK2017D073)

Cloning and analysis of BoCDPK14 in self-incompatibility Brasscia olerace

Xiao-Jing BAI1,Xiao-Ping LIAN2,Yu-Kui WANG1,He-Cui ZHANG1,Qian-Ying LIU1,Tong- Hong ZUO1,Yi-Zhong ZHANG1,Qin-Qin XIE1,Deng-Ke HU1,Xue-Song REN2,Jing ZENG3,Shao-Lan LUO1,Min PU1,Li-Quan ZHU1,*()   

  1. 1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400716, China
    2 College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400716, China
    3 School of Life Advanced Agriculture Bioengineering, Yangtze Normal University, Chongqing 408100, China
  • Received:2019-03-15 Accepted:2019-06-12 Published:2019-12-12 Published online:2019-06-12
  • Contact: Li-Quan ZHU
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31572127);the Fundamental Research Fund of Southwest University(XDJK2017D073)

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摘要：

甘蓝自交不亲和性(self-incompatibility, SI)是柱头对相同单倍型的花粉产生的排斥或抑制反应。钙依赖蛋白激酶(calcium-dependent protein kinase, CDPK)是植物面对逆境信号时参与抗逆反应的重要元件。本文通过甘蓝自花授粉0~60 min的柱头转录组数据分析, 成功地筛选到一个受自花授粉诱导上调表达的基因BoCDPK14, 该基因与拟南芥中参与植物逆境信号传导的钙依赖蛋白激酶基因高度同源。BoCDPK14基因开放阅读框1599 bp, 编码一种具有533个氨基酸残基的亲水性蛋白, 可在大肠杆菌胞质中被诱导表达, 其相对分子质量为60.4 kD, 表明BoCDPK14为活性胞质蛋白。该基因起始密码子上游2000 bp的核苷酸序列中含有胁迫反应、激素反应、代谢调节等应答元件。BoCDPK14在甘蓝柱头、花粉、花蕾、花瓣和叶片中表达, 且柱头中的表达量低于花粉。荧光定量PCR结果证实, BoCDPK14在0~60 min的表达变化趋势与转录组分析结果一致。通过酵母双杂交发现, BoCDPK14蛋白激酶结构域与谷氨酸受体通道蛋白BoGLR2.8d存在相互作用, 表明BoCDPK14可能是参与SI反应过程的新蛋白。本研究结果表明BoCDPK14可能作为Ca 2+信号元件参与甘蓝柱头响应花粉刺激的分子过程, 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, BoCDPK14基因, 酵母双杂交, 自花授粉, 自交不亲和

Abstract:

Brasscia oleracea self-incompatibility (SI) is the reaction of rejection or inhibition of stigma to pollens from the same haplotype. Calcium-dependent protein kinase (CDPK) is an important component of plants involved in resistance to stress signals. In this study, we identified an up-regulated gene named BoCDPK14 based on the stigma transcriptome data in 0-60 min self-pollination. The gene was highly homologous to the calcium dependent protein kinase gene involved in stress signaling in Arabidopsis lyrata. BoCDPK14 gene contained an open reading frame (ORF) with the length of 1599 bp, encoding a protein with the length of 533 amino acid residues. It was a hydrophilic protein expressed in cytoplasm, and could be induced to express a 60.4 kD protein in the cytoplasm of E. coli Transetta (DE3). It was suggested that BoCDPK14 is an active cytoplasmic protein. The 2000 bp upstream of BoCDPK14 translation initial codon contained elements for responses to stress, hormone, and metabolic regulation. BoCDPK14 was expressed in stigma, pollen, flower bud, petal, and leaf, with lower expression level in stigma than in pollen. The results of qRT-PCR revealed that BoCDPK14 mRNA expression level after self- and cross-pollinations for 0 min to 60 min was consistent with that of RNA-seq data. The interaction between BoCDPK14 protein kinase domain and glutamate receptor channel protein BoGLR2.8d was identified by yeast two-hybrid, suggesting that BoCDPK14 might be a novel protein involved in SI reaction. These results suggest that BoCDPK14 may act as a Ca 2+ signal element to participate the process of response to pollen stimulation in the stigma of Brasscia oleracea, which provides a new insight into the further research and utilization of self-incompatibility of Brasscia oleracea.

Key words: Brasscia oleracea, BoCDPK14 gene, yeast two-hybrid, self-pollination, self-incompatibility

表1

基因克隆及定量PCR中的引物"

引物名称
Primer		 引物序列
Primer sequence (5°-3°)		 用途
Use
GST
 F: GATCTGGTTCCGCGTGGATCCATGGGGAATTGCTGTGGAAC
R: GATGCGGCCGCTCGAGTCGACCTCTGCATCGCGATTATTAGAA		 基因的原核表达
Prokaryotic expression
QRP
 F: TCAAGAAAAGAGCACTCAGGG
R: GATGTGTACAGATATGGCTACGAAC		 荧光定量PCR引物
qRT-PCR
DActin
 F: GGCTGATGGTGAAGATATTCA
R: CAAGCACAATACCAGTAGTAC		 扩增内参基因
For the internal control
CDPK14-GFP
 F: AAGTCCGGAGCTAGCTCTAGAATGGGGAATTGCTGTGGAAC
R: GCCCTTGCTCACCATGGATCCCTCTGCATCGCGATTATTAGAA		 基因亚细胞定位
Subcellular location
CDPK14Δ-BD
 F: AGGACCTGCATATGGCCATGGATGGGGAATTGCTGTGGAAC
R: CCGCTGCAGGTCGACGGATCCTAACCATGGATGTTCAAGCACTT		 酵母表达
Yeast expression
GLR2.8d-AD
 F: GTACCAGATTACGCTCATATGAGCCCGACAAGTGAAATTAAAGTAG
R: ATGCCCACCCGGGTGGAATTCTTTAAGGAACACCCATGTGTTCTTG		 酵母表达
Yeast expression

图1

甘蓝柱头内响应自花和异花授粉后BoCDPK14表达模式 SP: 自花授粉; CP: 异花授粉。"

图2

甘蓝BoCDPK14基因cDNA和gDNA序列扩增"

图3

甘蓝BoCDPK14 CDNA结构图及其推导的氨基酸序列 下划直线为酪蛋白激酶II磷酸化位点, 虚线框为蛋白激酶C磷酸化位点, 椭圆为N-糖基化位点, 实线框为cAMP或cGMP依赖性蛋白激酶磷酸化位点, 双线框表示N-肉豆蔻酰化位点, 圆点为EF-hand钙结合结构域, 双线表示蛋白激酶ATP结合区, 星星表示丝氨酸/苏氨酸蛋白激酶活性位点。"

图4

BoCDPK14 蛋白Ca2+结合核心区域(A)和BoCDPK14蛋白三维结构预测(B) A图下排数字表示对这12个氨基酸残基的编号。"

图5

BoCDPK14与其他物种CDPK14氨基酸序列的系统进化树"

"

相关功能预测
Associated putative function		 启动子顺式作用元件
cis-elements in the promoter region
Abscisic acid responsiveness ABRE
Anaerobic induction ARE
DNA binding protein (ATBP-1) AT-rich element
Meristem expression CAT-box, CCGTCC-box
Ethylene-responsive element ERE
Gibberellin-responsive element GARE-motif
Endosperm expression GCN4_motif, Skn-1_motif
MYB binding site MRE
Zein metabolism regulation O2-site
Stress responsiveness TC-rich repeats, HSE
Light responsive ACE, Box 4, Box I, G-Box, GA-motif, GATA-motif, GTGGC-motif, I-box, 3-AF1 binding site

图6

BoCDPK14蛋白的原核表达 M: 蛋白相对分子质量标准; 1: BoCDPK14-GST未诱导蛋白; 2: BoCDPK14-GST诱导蛋白; 3: BoCDPK14-GST纯化蛋白; 4: GST蛋白。"

图7

BoCDPK14-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8

甘蓝柱头内BoCDPK14响应自花和异花授粉后表达模式(A)和BoCDPK14基因在甘蓝不同组织中的表达分析(B) SP: 自花授粉; CP: 异花授粉。"

图9

典型钙依赖蛋白激酶(CDPK)结构 V: 可变域; J: 连接域; EF: EF-hand 结构域。"

图10

酵母双杂交验证CDPK14Δ和GLR2.8d之间的相互作用 第1排表示pGADT7-BoGLR2.8d×pGBDT7-BoCDPK14Δ; 第2排表示阳性对照(pGADT7-T×pGBDT7-p53); 第3排表示阴性对照(pGADT7-T×pGBDT7-Lam)。"

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