甘蓝BoPID基因的克隆与分析

doi:10.16420/j.issn.0513-353x.2020-0806

摘要/Abstract

摘要：

在通过酵母双杂交文库获得了与钙响应蛋白BoSPx相互作用蛋白BoPID的基础上,对BoPID的编码基因进行了克隆、时空特异性表达分析及筛选与其互作的蛋白,以期为BoSPx通过BoPID参与自交不亲和性分子过程提供依据。结果表明,BoPID包含2个外显子和1个内含子,编码439个氨基酸。激酶磷酸化预测分析显示具有激酶活性;系统进化和共线性分析显示BoPID具有较强的植物种属特异性,与芜菁、拟南芥和甘蓝型油菜等十字花科植物聚类在一起,BoPID与AtPID在染色体水平上高度同源。组织特异性表达分析显示BoPID在柱头中的表达量高于花器官中的其他部位。荧光定量PCR分析表明BoPID在甘蓝柱头自花授粉15 min显著上调表达,而后下调表达。启动子元件分析发现BoPID含有ABA、IAA、脱落酸、茉莉酸、水杨酸和胁迫响应等多个应答元件。BoPID蛋白定位于细胞膜和细胞质中,可能是一种泊位于膜上并突触于胞质中的双栖蛋白。酵母双杂交和GST-Pull down结果表明BoPID与BoCML12、BoCaM2、BoPIN1能够发生相互作用。BoPID可能是BoSPx与BoCML12、BoCaM2之间相互作用的桥梁蛋白,共同通过生长素调节钙响应的方式参与了自交不亲和分子过程。

关键词: 甘蓝, 酵母文库, 自花授粉, BoSPx, BoPID

Abstract:

The BoPID was found as one of the interactive protein of BoSPx through yeast two-hybrid screening assay in Brassica oleracea. Its characters such as coding sequence,spatiotemporal expression and interactive proteins were further analyzed,with the purpose of studying its possible role in interacting with the calcium-responsive protein BoSPx in the process of self-incompatibility. The results showed that BoPID encodes 439 amino acids and contains two exons and one intron. BoPID may have kinase activity based on the kinase phosphorylation prediction analysis of its sequence,its sequence is highly homologous with AtPIDat the chromosome level and has strong species specificity when clustering with cruciferous plants such as turnip,Arabidopsis and Brassica napusin the phylogeny and collinearity analysis. Additionally,its promoter area contains multiple response elements such as ABA,IAA,abscisic acid,jasmonic acid,salicylic acid and stress response. The spatiotemporal expression analysis indicated thatBoPID expressed higher in the stigma than that in other parts of floral organs,while it was up-regulated after 15 minutes of self-pollination and then down-regulated. In the analysis of subcellular localization,we found BoPID protein localized in the cell membrane and cytoplasm,indicating that it might be an amphibious protein and anchored on the membrane and synapses in the cytoplasm. We further found the proteins of BoCML12,BoCaM2 and BoPIN1 interact with BoPID in the assays of yeast two-hybrid and GST-Pull down. Based on these results,we reason that BoPID might play a role in regulating calcium response to auxin through interacting with BoSPx,BoCML12 and BoCaM2 in the process of self-incompatibility.

Key words: Brassica oleracea, yeast library, self-pollination, BoSPx, BoPID

中图分类号:

S635.1

张贺翠, 王玉奎, 左同鸿, 刘倩莹, 张以忠, 胡燈科, 谢琴琴, 朱利泉. 甘蓝BoPID基因的克隆与分析[J]. 园艺学报, 2021, 48(6): 1123-1134.

ZHANG Hecui, WANG Yukui, ZUO Tonghong, LIU Qianying, ZHANG Yizhong, HU Dengke, XIE Qinqin, ZHU Liquan. Cloning and Expression Analysis of BoPID in Brassica oleracea[J]. Acta Horticulturae Sinica, 2021, 48(6): 1123-1134.

图/表 11

参考文献 28

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用途 Fuction	引物名称 Primer name	引物序列 Sequence	退火温度/℃ T_m
酵母双杂交载体 Yeast two-hybrid vector	BK-F BK-R AD-BoCML12-F AD-BoCML12-R AD-BoCaM2-F AD-BoCaM2-R AD-PIN1-F AD-PIN1-R	TCAGAGGAGGACCTGCATATGTTACGAGAATCAGACGGTGAG TCGACGGATCCCCGGGAATTCAAAGAAATCGAACGCCGCT GTACCAGATTACGCTCATATGTTACGAGAATCAGACAGTGAGAT ATGCCCACCCGGGTGGAATTCCGCTGGTTTGTTACTTCTACCAC GTACCAGATTACGCTCATATGATGGCGGATCAGCTGACC ATGCCCACCCGGGTGGAATTCCTTAGCCATCATGACTTTAACGAA GTACCAGATTACGCTCATGCTTTGCCTCAAGGAATTGTACC ATGCCCACCCGGGTGGAATTC GTAGTAGAGAAGGGTTATGGGC	58 58 60 60
启动子表达 Promoter expression	GUS-F GUS-R	CAAGCTTGGCTGCAGGTCGACAGACAAGCGAATACTTTTAGGG GGTGGACTCCTCTTAGAATTCGAACATAACGTGGGAAGAGAGTA	60
qRT-PCR	qRT-PCR-F qRT-PCR-R	ATGGCCACTGATTCAGCAAT ATACCGGCCGTATTCCTCCT	60
RT-PCR	RT-PCR-F RT-PCR-R	ATGGCCACTGATTCAGCAAT GACGGCATAAGTAAACTGTTCCGATG	60
亚细胞定位 Subcellular localization	1300-GFP-F 1300-GFP-R	GAGAACACGGGGGACTCTAGAATGGCGTCTCCTAAATCCTCA GAGCTTTTGCTCCATGAGCTCAAAGAAATCGAACGCCGCT	60

用途 Fuction	引物名称 Primer name	引物序列 Sequence	退火温度/℃ T_m
酵母双杂交载体 Yeast two-hybrid vector	BK-F BK-R AD-BoCML12-F AD-BoCML12-R AD-BoCaM2-F AD-BoCaM2-R AD-PIN1-F AD-PIN1-R	TCAGAGGAGGACCTGCATATGTTACGAGAATCAGACGGTGAG TCGACGGATCCCCGGGAATTCAAAGAAATCGAACGCCGCT GTACCAGATTACGCTCATATGTTACGAGAATCAGACAGTGAGAT ATGCCCACCCGGGTGGAATTCCGCTGGTTTGTTACTTCTACCAC GTACCAGATTACGCTCATATGATGGCGGATCAGCTGACC ATGCCCACCCGGGTGGAATTCCTTAGCCATCATGACTTTAACGAA GTACCAGATTACGCTCATGCTTTGCCTCAAGGAATTGTACC ATGCCCACCCGGGTGGAATTC GTAGTAGAGAAGGGTTATGGGC	58 58 60 60
启动子表达 Promoter expression	GUS-F GUS-R	CAAGCTTGGCTGCAGGTCGACAGACAAGCGAATACTTTTAGGG GGTGGACTCCTCTTAGAATTCGAACATAACGTGGGAAGAGAGTA	60
qRT-PCR	qRT-PCR-F qRT-PCR-R	ATGGCCACTGATTCAGCAAT ATACCGGCCGTATTCCTCCT	60
RT-PCR	RT-PCR-F RT-PCR-R	ATGGCCACTGATTCAGCAAT GACGGCATAAGTAAACTGTTCCGATG	60
亚细胞定位 Subcellular localization	1300-GFP-F 1300-GFP-R	GAGAACACGGGGGACTCTAGAATGGCGTCTCCTAAATCCTCA GAGCTTTTGCTCCATGAGCTCAAAGAAATCGAACGCCGCT	60

启动子顺式作用元件 Promotercis-acting element	元件序列 Element sequence	功能 Function	位置/aa Location
TGA-box	TGACGTAA	生长素响应元件 Auxin response element	-86
AAGAA-motif	GTAAAGAAA	脱落酸响应元件 Abscisic acid response element	-339、-203
ABRE	TACGGTC		-110、-132、-199、 -856
DRE	GCCGAC		-129
G-box	CACGTG		-73
ACE	GACACGTATG		-54、-1 356
ARE	AAACCA	厌氧诱导元件 Anaerobic induction element	-487、-224
G-BOX	CACGTG	光响应元件 Light responsive element	-69、-110
AE-box	AGAAACAA		-100
GT1-motif	GGTTAA		-517
GTGGC-motif	CATCGTGTGGC		-102
TCT-motif	TCTTAC		-120
CGTCA-motif	CGTCA		-320
ERE	ATTTCATA	乙烯响应元件 Ethylene-responsive element	-676、-868、-1 399
TGACG-motif	TGACG	茉莉酸响应元件MeJA-responsiveness	-305
AT-rich element	ATAGAAATCAA	水杨酸响应元件 Salicylic acid response element	-837
MBS	CAACTG	MYB结合位点 MYB binding site	-149
MYC	CATTTG	胁迫响应元件 Stress response element	-404
STRE	AGGGG	胁迫响应元件 Stress response element	-612、-713

启动子顺式作用元件 Promotercis-acting element	元件序列 Element sequence	功能 Function	位置/aa Location
TGA-box	TGACGTAA	生长素响应元件 Auxin response element	-86
AAGAA-motif	GTAAAGAAA	脱落酸响应元件 Abscisic acid response element	-339、-203
ABRE	TACGGTC		-110、-132、-199、 -856
DRE	GCCGAC		-129
G-box	CACGTG		-73
ACE	GACACGTATG		-54、-1 356
ARE	AAACCA	厌氧诱导元件 Anaerobic induction element	-487、-224
G-BOX	CACGTG	光响应元件 Light responsive element	-69、-110
AE-box	AGAAACAA		-100
GT1-motif	GGTTAA		-517
GTGGC-motif	CATCGTGTGGC		-102
TCT-motif	TCTTAC		-120
CGTCA-motif	CGTCA		-320
ERE	ATTTCATA	乙烯响应元件 Ethylene-responsive element	-676、-868、-1 399
TGACG-motif	TGACG	茉莉酸响应元件MeJA-responsiveness	-305
AT-rich element	ATAGAAATCAA	水杨酸响应元件 Salicylic acid response element	-837
MBS	CAACTG	MYB结合位点 MYB binding site	-149
MYC	CATTTG	胁迫响应元件 Stress response element	-404
STRE	AGGGG	胁迫响应元件 Stress response element	-612、-713

候选蛋白 Candidate protein	蛋白名称 Protein name	功能注释 Functional comment
BoCML12	类钙调蛋白12 Calciumodulin-like protein 12	编码类钙调蛋白12,含有6个EF-Hand结合基序,响应机械刺激反应表达 The calmodulin-like 12,which contains six EF-Hand binding motifs,is expressed in response to a mechanical stimulation response
BoCaM2	钙调蛋白2 Calmodulin 2	编码含有4个EF-Hand结合基序的钙结合蛋白2 Calcium binding protein 2 encoding four EF-Hand binding motifs
BoPIN1	生长素极性运输载体 Auxin efflux carrier 1	pin1突变体的特征在于不引发任何花的花序分生组织,导致形成针状花序 The pin1mutant is characterized by not inducing any inflorescence meristem of the flower,resulting in the formation of needles