冷胁迫下甘蓝型冬油菜表达蛋白及BnGSTs基因家族的鉴定与分析

doi:10.3724/SP.J.1006.2023.14207

Abstract

Abstract:

Glutathione S-transferases (GST) are involved in regulating many aspects of plant growth, development, and response to adversity stress. In this study, the differentially expressed proteins of ‘16VHNTS309’ under cold stress were analyzed using bidirectional electrophoresis and mass spectrometry, and proteins involved in the cold stress response, such as BE, APX, SOD, and GST, were identified based on GO and KEGG analyses. The key protein GST was identified using qRT-PCR and physiological indicators in response to cold stress, and the GST gene of ‘16VHNTS309’ was cloned using homologous cloning. This gene CDS length was 642 bp, and encoded 213 amino acids, which was an unstable protein and belonged to the GST_N_3 glutathione S-transferase family. The sequence similarity with Brassica napus ‘ZS11’ was 99.22%, and a comparison of the amino acid sequences of both ‘ZS11’ and ‘Vision’ revealed a mutation from leucine (L) to proline (P) at position 127. The gene family analysis showed that 153 BnGSTs members were identified in Brassica napus and were classified into seven main types according to their functions: Zeta, Phi, Theta, CHQ, DHAR, Lambda, and Tau. Most BnGSTs belonged to Phi and Tau types. Phylogeny divided BnGSTs into 12 subfamilies, subfamilies I and VIII contained more members. BnGSTs were unevenly distributed on 18 chromosomes, and the number of BNGSTs genes on C06 chromosome was the largest, it contained 10 conserved protein motifs. There were 99 pairs of genes in the BnGSTs gene family that were colinearly related, 131 genes from gene duplication events, and segmental duplication events played an important role in the evolution of BnGSTs genes. Significantly higher expression of BnaA02g35760D, BnaC06g20450D, BnaC06g35490D, BnaA02g03230D, and BnaA02g35980D in strong cold-resistant varieties was 7-12 times higher than that in weak cold-resistant varieties under cold stress. And the strong cold-resistant varieties had higher physiological enzyme activities. In addition, a number of key candidate genes were identified for transient and sustained expression under freezing stress. This result lays the foundation for further studies on the molecular regulation of BnGSTs genes for cold resistance in strong cold resistant for Brassica napus.

Key words: winter Brassica napus L., cold stress, protein mass spectrometry, GST gene family, expression pattern

MA Li, BAI Jing, ZHAO Yu-Hong, SUN Bo-Lin, HOU Xian-Fei, FANG Yan, WANG Wang-Tian, PU Yuan-Yuan, LIU Li-Jun, XU Jia, TAO Xiao-Lei, SUN Wan-Cang, WU Jun-Yan. Protein and physiological differences under cold stress, and identification and analysis of BnGSTs in Brassica napus L.[J].Acta Agronomica Sinica, 2023, 49(1): 153-166.

Figures/Tables 13

Table 1

Fig. 1

Fig. S1

Table S1

Identification of differential proteins in response to cold stress in winter Brassica napus"

蛋白点编号 Spot number	基因登录号 Accession number	蛋白质名称 Protein name	分子质量 Molecular weight (kD)	等电点 pI	亚细胞定位 Cellular location	差异倍数 Fold change
2	XP_013688985.1	RNA-binding protein CP29B	31,076	4.82	叶绿体Chloroplast	NDT
3	XP_013656707.1	glycine-rich RNA-binding protein 10-like	16,038	5.24	微体Microbody	1.87±0.06
4	XP_013656707.1	glycine-rich RNA-binding protein 10-like	16,038	5.24	微体Microbody	0.49±0.01
6	XP_013687602.1	bifunctional enolase 2/transcriptional activator	47,556	5.56	内质网 Endoplasmic reticulum	1.99±0.09
8	XP_013713332.1	oxygen-evolving enhancer protein 1-1	35,164	5.55	叶绿体Chloroplast	2.54±0.03
9	XP_013688985.1	RNA-binding protein CP29B	31,076	4.82	叶绿体Chloroplast	NDT
11	XP_013649537.1	cysteine protease inhibitor WSCP	23,891	8.66	细胞外Extracellular	1.9±0.04
12	XP_013673398.1	ATP synthase subunit beta-2	53,800	5.39	细胞质Cytoplasm	2.52±0.06
13	XP_013659948.1	cysteine protease inhibitor WSCP-like	24,956	6.52	细胞外Extracellular	NDC
14	XP_013678556.1	actin-7	41,665	5.31	细胞质Cytoplasm	2.01±0.11
15	XP_013641139.1	L-ascorbate peroxidase 1, cytosolic-like isoform X3	27,594	5.58	微体Microbody	2.63±0.06
16	XP_013697709.1	probable phospholipid hydroperoxide glutathione peroxidase 6	25,260	9.2	线粒体Mitochondrial	2.08±0.18
17	AIF75338.1	chloroplast ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit	19,852	7.59	细胞外Extracellular	2.21±0.05
18	XP_013720130.1	PREDICTED: superoxide dismutase [Cu-Zn] 2	21,343	6.79	细胞外Extracellular	2.75±0.11
19	XP_013649537.1	cysteine protease inhibitor WSCP	23,891	8.66	细胞外Extracellular	3.01±0.05
20	XP_013649391.1	nucleoside diphosphate kinase 1	16,384	6.29	微体Microbody	1.94±0.04
21	XP_013712048.1	CBS domain-containing protein CBSX3	22,650	8.71	线粒体Mitochondrial	NDC
23	AIF75338.1	chloroplast ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit	19,852	7.59	细胞外Extracellular	3.33±0.39
24	XP_013649614.1	kunitz trypsin inhibitor 1-like	23,553	6.6	细胞外Extracellular	NDC
25	XP_013641139.1	L-ascorbate peroxidase 1, cytosolic-like isoform X3	27,594	5.58	微体Microbody	3.41±0.22
26	AIF75338.1	chloroplast ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit	19,852	7.59	细胞外Extracellular	3.11±0.29
27	XP_013641880.1	glutathione S-transferase DHAR1, mitochondrial	23,533	5.76	细胞质Cytoplasm	2.1±0.1
28	XP_013666490.1	malate dehydrogenase 1, cytoplasmic-like	35,695	6.11	细胞外Extracellular	3.12±0.06
29	XP_013666490.1	malate dehydrogenase 1, cytoplasmic-like	35,695	6.11	细胞外Extracellular	2.91±0.21
30	XP_013649614.1	kunitz trypsin inhibitor 1-like	23,553	6.6	细胞外Extracellular	NDC

Table S1

Fig. 2

Fig. 3

Fig. 4

Fig. S2

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

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基因名称 Gene name	引物序列 Primer sequence (5'-3')	产物大小 Product size (bp)
BE	F: GATGGAACCCAGAACGAG; R: GGAATGCCACTGACAACG	110
SPI	F: CCCCTCTTTATCGGACAG; R: GGAACGAACCCAACTCTA	89
APX	F: GAGCGGTGAGAAGGAAGG; R: CTCGTCAGCAGCGTATTT	100
SOD	F: GCCACATTTCAACCCTAACA; R: ACCACAAAGGCTCTTCCAAC	174
GST	F: ATCAACATCTCCGACAAACC; R: CGTCAGAATCAGACACCCAC	100
RBP	F: CGTGTTTATGTCGGCAATC; R: GCCACTGTCCCTGTCGTAG	114
β-Actin	F: TGGGTTTGCTGGTGACGAT; R: TGCCTAGGACGACCAACAATACT	63

Protein and physiological differences under cold stress, and identification and analysis of BnGSTs in Brassica napus L.

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[3]	BAI Dong-Mei, XUE Yun-Yun, HUANG Li, HUAI Dong-Xin, TIAN Yue-Xia, WANG Peng-Dong, ZHANG Xin, ZHANG Hui-Qi, LI Na, JIANG Hui-Fang, LIAO Bo-Shou. Assessment of cold tolerance of different peanut varieties and screening of evaluation indexes at germination stage [J]. Acta Agronomica Sinica, 2022, 48(8): 2066-2079.
[4]	CHEN Chi, CHEN Dai-Bo, SUN Zhi-Hao, PENG Ze-Qun, Adil Abbas, HE Deng-Mei, ZHANG Ying-Xin, CHENG Hai-Tao, YU Ping, MA Zhao-Hui, SONG Jian, CAO Li-Yong, CHENG Shi-Hua, SUN Lian-Ping, ZHAN Xiao-Deng, LYU Wen-Yan. Characterization and genetic mapping of a classic-abortive-type recessive genic-male-sterile mutant ap90 in rice (Oryza sativa L.) [J]. Acta Agronomica Sinica, 2022, 48(7): 1569-1582.
[5]	YUAN Da-Shuang, DENG Wan-Yu, WANG Zhen, PENG Qian, ZHANG Xiao-Li, YAO Meng-Nan, MIAO Wen-Jie, ZHU Dong-Ming, LI Jia-Na, LIANG Ying. Cloning and functional analysis of BnMAPK2 gene in Brassica napus [J]. Acta Agronomica Sinica, 2022, 48(4): 840-850.
[6]	HUANG Cheng, LIANG Xiao-Mei, DAI Cheng, WEN Jing, YI Bin, TU Jin-Xing, SHEN Jin-Xiong, FU Ting-Dong, MA Chao-Zhi. Genome wide analysis of BnAPs gene family in Brassica napus [J]. Acta Agronomica Sinica, 2022, 48(3): 597-607.
[7]	YU Hui-Fang, ZHANG Wei-Na, KANG Yi-Chen, FAN Yan-Ling, YANG Xin-Yu, SHI Ming-Fu, ZHANG Ru-Yan, ZHANG Jun-Lian, QIN Shu-Hao. Genome-wide identification and expression patterns in response to signals from Phytophthora infestans of CrRLK1Ls gene family in potato [J]. Acta Agronomica Sinica, 2022, 48(1): 249-258.
[8]	JIAN Hong-Ju, SHANG Li-Na, JIN Zhong-Hui, DING Yi, LI Yan, WANG Ji-Chun, HU Bai-Geng, Vadim Khassanov, LYU Dian-Qiu. Genome-wide identification and characterization of PIF genes and their response to high temperature stress in potato [J]. Acta Agronomica Sinica, 2022, 48(1): 86-98.
[9]	SHI Lei, MIAO Li-Juan, HUANG Bing-Yan, GAO Wei, ZHANG Zong-Xin, QI Fei-Yan, LIU Juan, DONG Wen-Zhao, ZHANG Xin-You. Characterization of the promoter and 5'-UTR intron in AhFAD2-1 genes from peanut and their responses to cold stress [J]. Acta Agronomica Sinica, 2021, 47(9): 1703-1711.
[10]	XUE Xiao-Meng, WU JIE, WANG Xin, BAI Dong-Mei, HU Mei-Ling, YAN Li-Ying, CHEN Yu-Ning, KANG Yan-Ping, WANG Zhi-Hui, HUAI Dong-Xin, LEI Yong, LIAO Bo-Shou. Effects of cold stress on germination in peanut cultivars with normal and high content of oleic acid [J]. Acta Agronomica Sinica, 2021, 47(9): 1768-1778.
[11]	HUANG Xing, XI Jin-Gen, CHEN Tao, QIN Xu, TAN Shi-Bei, CHEN He-Long, YI Ke-Xian. Identification and expression of PAL genes in sisal [J]. Acta Agronomica Sinica, 2021, 47(6): 1082-1089.
[12]	MENG Yu-Yu, WEI Chun-Ru, FAN Run-Qiao, YU Xiu-Mei, WANG Xiao-Dong, ZHAO Wei-Quan, WEI Xin-Yan, KANG Zhen-Sheng, LIU Da-Qun. TaPP2-A13 gene shows induced expression pattern in wheat responses to stresses and interacts with adaptor protein SKP1 from SCF complex [J]. Acta Agronomica Sinica, 2021, 47(2): 224-236.
[13]	GAO Yun, ZHANG Yu-Xue, MA Quan, SU Sheng-Nan, LI Chun-Yan, DING Jin-Feng, ZHU Min, ZHU Xin-Kai, GUO Wen-Shan. Effects of low temperature in spring on fertility of pollen and formation of grain number in wheat [J]. Acta Agronomica Sinica, 2021, 47(1): 104-115.
[14]	HENG You-Qiang,YOU Xi-Long,WANG Yan. Pathogenesis-related protein gene SfPR1a from Salsola ferganica enhances the resistances to drought, salt and leaf spot disease in transgenic tobacco [J]. Acta Agronomica Sinica, 2020, 46(4): 503-512.
[15]	LI Xu-Kai,LI Ren-Jian,ZHANG Bao-Jun. Identification of rice stress-related gene co-expression modules by WGCNA [J]. Acta Agronomica Sinica, 2019, 45(9): 1349-1364.