玉米油菜素甾醇生物合成关键酶基因ZmCYP90B1的克隆及其对逆境胁迫的响应

doi:10.3724/SP.J.1006.2018.00343

Abstract

Abstract:

Brassinosteroids (BRs) as an important phytohormone, play essential roles in plant growth, development and responses to adversity stresses. The key enzyme encoded by CYP90B1 is involved in the BRs biosynthesis. However, the characteristic of CYP90B1 gene response to stress has not been reported in maize. In this study, we cloned ZmCYP90B1 gene from Zea mays, GenBank accession number KY242373, via RT-PCR in combination of RACE technique. The full-length sequence of this gene is 2058 bp and the the complete open reading frame is 1518 bp, encoding 506 amino acid peptides. The predicted protein has the molecular weight of 57.66 kD and isoelectric point of 9.54, containing one transmembrane domain and one conserved domain of p450. Multiple sequences alignment analysis indicated the predicted protein shared high similarities with CYP90B1 from other plant species. The phylogenetic tree revealed a notable difference in the evolution of CYP90B1 between dicotyledons and monocotyledons. The qRT-PCR result showed that the expression of ZmCYP90B1 was induced by drought, high salt, low temperature, ABA, Spodoptera exigua attack, and methyl jasmonate (MeJA), suggesting that ZmCYP90B1 was involved in various abiotic stresses, insect resistance and response to MeJA. Overexpressing ZmCYP90B1 in tobacco seedlings could enhance drought resistance with less water loss rate and higher SPAD value in the transgenic lines. In addition, the activities of SOD, CAT, and POD and the content of proline increased significantly in all transgenic tobacco lines than in the wild type; both MDA and ABA contents were obviously lower in the transgenic lines than in the wild type. Through expression analysis of the down-stream stress-responsive genes, we demonstrated that drought tolerance enhancement by overexpressing ZmCYP90B1 might be involved in ABA-independent pathway and related to transcriptional regulation of antioxidant- related genes.

Key words: brassionsteroids, ZmCYP90B1 gene, real-time quantitative PCR, adversity stresses

Fang-Meng DUAN, Qiu-Lan LUO, Xue-Li LU, Na-Wei QI, Xian-Shun LIU, Wen-Wen SONG. Cloning of the Key Gene ZmCYP90B1 in Brassinosteroids Biosynthesis from Zea mays and Its Response to Adversity Stresses[J].Acta Agronomica Sinica, 2018, 44(03): 343-356.

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引物 Primer	序列 Sequence (5°-3°)
DP-F	GGCGGCATCCTGGGCAAGT
DP-R	CCCGCGAAGAGCAGGCTCA
5°-GSP	TGGACCACTTGCCCAGGATGCC
3°-GSP	GAAGATGGAGGACAGGCTTGAGA
FP-F	CTCTCTCCAACCCATCCGTC
FP-R	GGGAGGTAATGTTGGGGTTTG

基因名称 Gene name	GenBank登录号 GenBank number	引物序列 Primer sequence (5°-3°)	产物长度 Product size (bp)	退火温度 Annealing temperature (°C)
NtAPX	NM_001324874.1	F: CAAATGTAAGAGGAAACTCAGAGGA R: AGCAACAACTCCAGCTAATTGATAG	262	60
NtCAT	NM_001326196.1	F: AGGTACCGCTCATTCACACC R: AAGCAAGCTTTTGACCCAGA	151	60
NtSOD	AF443178.1	F: CCGTCGCCAAATTGCATAG R: CGATAGCCCAACCAAGAGAAC	238	62
NtNCED1	NM_001326185.1	F: CTATTTCCACTTCAAAACCAACCAC R: GGCACTTTCCACGGCATCT	131	62
NtERD10C	AB049337.1	F: ACGTGGAGGCTACAGATCGTGGTTT R: TCTCCACTGGTACAGCCGTGTCCTCAC	366	70
NtLEA5	AF053076.1	F: GAACCCAACAAGAGCGAGAGA R: CGACAGGAAGCATTGACGAG	350	62
Tob103	U60495.1	F: CAAGGAAATCACCGCTTTGG R: AAGGGATGCGAGGATGGA	106	60
ZmCYP90B1	KY242373	F: GGCTGTGGTCTCGTGTTTTATG R: ACCATCCTCCCCTTTGCTCT	104	62
Actin	NM_00115473.1	F: CTGAGGTTCTATTCCAGCCATCC R: CCACCACTGAGGACAACATTACC	133	62

类别 Category	物种 Species	氨基酸残基数 Residues	分子量 Molecular weight (kD)	等电点pI	负电荷氨基酸比例 Negatively charged residues (%)	正电荷氨基酸比例 Positively charged residues (%)	不稳定系数 Instability index	总平均亲水性 Hydropathicity
单子叶植物 Monocotyledons	Zea mays	506	57.66	9.54	10.47	13.44	46.74	-0.171
	Dichanthelium oligosanthes	507	57.87	9.25	10.65	12.62	48.82	-0.161
	Oryza brachyantha	504	57.12	9.27	10.52	12.70	52.69	-0.138
	Aegilops tauschii	501	57.01	8.11	11.58	11.98	52.53	-0.133
	Triticum urartu	525	59.75	8.09	11.62	12.00	51.61	-0.162
双子叶植物 Dicotyledons	Arabidopsis thaliana	513	58.87	6.62	12.87	12.28	49.50	-0.301
	Nicotiana attenuata	491	55.78	8.89	9.98	11.41	44.36	-0.166
	Brassica napus	495	56.71	8.17	11.72	12.12	48.90	-0.213
	Glycine soja	492	55.77	8.85	10.16	11.38	43.87	-0.128
	Theobroma cacao	496	56.75	8.64	10.89	11.90	46.51	-0.219
	Gossypium hirsutum	485	55.42	8.94	10.52	12.16	44.88	-0.163

Cloning of the Key Gene ZmCYP90B1 in Brassinosteroids Biosynthesis from Zea mays and Its Response to Adversity Stresses

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