甘蓝自交不亲和性相关基因BoGSTL21的克隆与表达分析

doi:10.3724/SP.J.1006.2020.04004

Abstract

Abstract:

Glutathione-S-transferase (GSTs) plays an important role in plant resistance to stress, cytotoxic release and plant growth and development. In this study, we identified an up-regulated gene named BoGSTL21 based on the stigma transcriptome data in 0-60 min self-pollination. BoGSTL21 had an open reading frame (ORF) with the length of 900 bp, encoded a protein of 299 amino acid residues, which contained GST-N and GST-C domains without signal peptide and transmembrane domain, the theory isoelectric point of BoGSTL21 was 8.49. The promoter of BoGSTL21 gene contained many cis-acting elements such as light response, auxin response, abscisic acid response, low temperature and drought response. BoGSTL21 expresses in different tissues of Brassica oleracea. The expression level in stigma varies with developmental time, and was mainly overexpressed in mature stigma. The results of qRT-PCR revealed that BoGSTL21 mRNA expression level after self-and cross-pollinations for 0 min to 60 min was consistent with that of RNA-seq data. It was found through yeast two-hybrid that BoGSTL21 protein interacted with pollen development-related protein BoFAB1C, auxin-related protein BoPATL2, and aldolase-type TIM barrel family protein BoF9N12_9. BoGSTL21 gene was successfully induced and expressed in E. coli BL21 (DE3) with a purified protein size of 34 kD, which was consistent with the predicted results. According to the above results, BoGSTL21 may be a novel protein involved in the SI response process, which provides a new content for further research and utilization of self-incompatibility in Brasscia oleracea.

Key words: Brassica oleracea, gene cloning, self-incompatibility, expression analysis, yeast two-hybrid

Tong-Hong ZUO, He-Cui ZHANG, Qian-Ying LIU, Xiao-Ping LIAN, Qin-Qin XIE, Deng-Ke HU, Yi-Zhong ZHANG, Yu-Kui WANG, Xiao-Jing BAI, Li-Quan ZHU. Molecular cloning and expression analysis of BoGSTL21 in self-incompatibility Brasscia oleracea[J].Acta Agronomica Sinica, 2020, 46(12): 1850-1861.

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引物名称 Primer name	引物序列 Primer sequence (5°-3°)	用途 Functions
BoGSTL21-PGEX-4T-1	F: GATCTGGTTCCGCGTGGATCCATGAGTGCCGGAGTGAGAGTTAG	基因的原核表达
	R: CTCGAGTCGACCCGGGAATTCGGGACGTGCTTCTGCTTGG	Prokaryotic expression
qRT-PCR	F: TTCCTTTGCCGATTTAGTTTGG	荧光定量PCR引物
	R: AGTGTTCATCTCCTTAAGCCAA	qRT-PCR
dActin	F: GGCTGATGGTGAAGATATTCA	内参引物
	R: CAAGCACAATACCAGTAGTAC	Internal reference primers
BoGSTL21-BK	F: TCAGAGGAGGACCTGCATATGAGTGCCGGAGTGAGAGTTA	酵母双杂交引物 Yeast two-hybrid primers
	R: TCGACGGATCCCCGGGAATTCGGGACGTGCTTCTGCTTG
BoFAB1C-AD	F: GTACCAGATTACGCTCATATGGGGATGGTGAAGTTCTCTGTG
	R: ATGCCCACCCGGGTGGAATTCGTTCCATGGCTCAGGAACC
BoPATL2-AD	F: GTACCAGATTACGCTCATATGGCTCAAGAAGAGATACAGAAG
	R: ATGCCCACCCGGGTGGAATTCTTATGCTTGCGTTTTGAACC
BoF9N12_9-AD	F: GTACCAGATTACGCTCATATGGTGGTGTCGCCAAAGATAG
	R: ATGCCCACCCGGGTGGAATTCTCAGGTGATGGGTTGGGC
1391	F: GAACTGATCGTTAAAACTGC	通用引物
	R: TGGTCTTCTGAGACTGTATC	Universal primer
BoGSTL21-GUS	F: CAAGCTTGGCTGCAGGTCGACATGTTATACGTTGCGAACGC	基因的启动子活性分析
	R: GGTGGACTCCTCTTAGAATTCACTCAATCGTTCTTCTTCCGT	Promoter activity analysis

相关功能预测 Associated putative function	启动子顺式作用元件 Cis-elements in the promoter region
脱落酸(ABA)应答 Abscisic acid response	ABRE
光响应 Light response	G-Box, GT1-motif, TCT-motif
低氧诱导 Anaerobic induction response	ARE
促进和增强基因转录 Promote and enhance gene transcription	CAAT-box
赤霉素应答 Gibberellin-response	GARE-motif, P-box
MYBHv1结合位点 MYBHv1 binding site	CCAAT-box
涉及光响应的MYBHv1结合位点 MYB binding site involved in light response	MRE
转录启动约-30的核心启动子 Core promoter element around -30 of transcription start	TATA-box
低温响应 Low-temperature response	LTR
干旱诱导 Drought induced response	MBS
水杨酸(SA)反应 Salicylic acid response	TCA-element
MeJA茉莉酸响应 MeJA-response	CGTCA-motif, TGACG-motif
生长素(IAA)应答 Auxin-response	TGA-element

候选蛋白Candidate protein	蛋白名称 Protein name	功能注释 Functional annotations
BoFAB1C	1-磷脂酰肌醇-3-磷酸5-激酶 1-phosphatidylinositol-3- phosphate 5-kinase	具有1-磷脂酰肌醇-3-磷酸5-激酶活性, ATP结合,参与磷脂酰肌醇磷酸化, 花粉发育, 气孔关闭的生物过程。 It has 1-phosphatidylinositol-3-phosphate 5-kinase activity, ATP binding, participates in the biological processes of phosphatidylinositol phosphorylation, pollen development, and stomatal closure.
BoPATL2	patellin-2	PATL属于具有高尔基动力学(GOLD)结构域和Sec14p-like结构域串联的蛋白质家族。PATL受生长素调节。 PATL belongs to a family of proteins with a Golgi dynamics (GOLD) domain and a Sec14p-like domain tandem. PATL is regulated by auxin.
BoF9N12_9	磷酸核糖3-差向异构酶 Ribulose-phosphate 3-epimerase	属于醛缩酶型TIM桶家族蛋白, 参与碳水化合物代谢过程, 磷酸戊糖途径, 氨基酸的生物合成。 Belongs to the aldolase type TIM barrel family protein, involved in carbohydrate metabolism, pentose phosphate pathway, amino acid biosynthesis.

编号 No.	酵母菌种(质粒) Yeast strain (plasmid)	培养基 Yeast medium	菌斑 Colony	颜色 Color
1	Y2HGold (pGADT7-T×pGBKT7-53)	SD/-Leu/-Trp	是Yes	白色White
2	Y2HGold (pGADT7-T×pGBKT7-Lam)	SD/-Leu/-Trp	是Yes	红色Red
3	Y2HGold (pGADT7-BoFAB1C×pGBKT7-BoGSTL21)	SD/-Leu/-Trp	是Yes	白色White
4	Y2HGold (pGADT7-BoPATL2×pGBKT7-BoGSTL21)	SD/-Leu/-Trp	是Yes	白色White
5	Y2HGold (pGADT7-BoF9N12×pGBKT7-BoGSTL21)	SD/-Leu/-Trp	是Yes	白色White
6	Y2HGold (pGADT7-T×pGBKT7-53)	SD/-Ade/-His/-Leu/-Trp	是Yes	白色White
7	Y2HGold (pGADT7-T×pGBKT7-Lam)	SD/-Ade/-His/-Leu/-Trp	是Yes	无色No
8	Y2HGold (pGADT7-BoFAB1C×pGBKT7-BoGSTL21)	SD/-Ade/-His/-Leu/-Trp	是Yes	白色White
9	Y2HGold (pGADT7-BoPATL2×pGBKT7-BoGSTL21)	SD/-Ade/-His/-Leu/-Trp	是Yes	白色White
10	Y2HGold (pGADT7-BoF9N12×pGBKT7-BoGSTL21)	SD/-Ade/-His/-Leu/-Trp	是Yes	白色White

Molecular cloning and expression analysis of BoGSTL21 in self-incompatibility Brasscia oleracea

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