甘蓝型油菜BnCNGC基因家族鉴定及其在核盘菌侵染和PEG处理下的表达特性分析

doi:10.3724/SP.J.1006.2022.14091

Abstract

Abstract:

CNGCs are common cyclic nucleotide-gated channel proteins in plants, which play important roles in plant stress response. In order to elucidate the biological function of the BnCNGC genes in Brassica napus, we identified the BnCNGC family members in the genome and analyzed the distribution, structure, evolution, and the expression of candidate members in B. napus exposed to Sclerotinia sclerotiorum or PEG-simulated drought. In this study, employing CNGC amino acid sequences of Arabidopsis thaliana and Brassica oleracea as references, the homologies were acquired using BLASTP software in Brassica napus Genome Browser, and the gene family members were identified according to the conserved domain and specific motif of BnCNGC in plants. The gene structure, chromosome distribution, protein physicochemical property, subcellular localization, phylogenetic evolution, and promoter cis-acting regulatory elements were analyzed. The relative expression profiles of the candidate BnCNGC genes, screened out based on the transcriptome data in B. napus challenged with S. sclerotiorum or PEG-simulated drought, were analyzed by qRT-PCR. As a result, a total of 49 BnCNGC genes were identified in B. napus genome. They were distributed at 17 pairs of chromosomes, except for A08 and C06, and generally had 5-10 introns. The 1500 bp region in the upstream sequence of BnCNGC contained lots of cis-acting regulatory elements involved in stress response. Most of the corresponding proteins were composed of 413-801 amino acids with molecular weight (MW) of 47.62-110.58 kD and isoelectric point (pI) of 6.10-9.88. All the identified family members had specific motif of CNGC in the CNBD region. Phylogenetic analysis showed that BnCNGC were divided into four categories, namely Group I, II, III, and IV. Transcriptome data analysis revealed that BnCNGC9, BnCNGC27, and BnCNGC48 were involved in stress response. The relative expression levels of the three genes were detected in B. napus leaves exposed to different stresses. All were down-regulated by 48.2%-99.1%, 79.4%-87.1%, and 39.7%-92.6% under inoculation with Sclerotinia sclerotiorum, respectively. The relative expression levels of BnCNGC9 and BnCNGC48 were increased 3.34 times and 6.27 times at 24 and 48 hours after simulated drought stress, respectively, thus BnCNGC27 was not sensitive to drought stress.

Key words: Brassica napus, CNGC gene family, Sclerotinia sclerotiorum, PEG-simulated drought, relative expression analysis

CHEN Song-Yu, DING Yi-Juan, SUN Jun-Ming, HUANG Deng-Wen, YANG Nan, DAI Yu-Han, WAN Hua-Fang, QIAN Wei. Genome-wide identification of BnCNGC and the gene expression analysis in Brassica napus challenged with Sclerotinia sclerotiorum and PEG-simulated drought[J].Acta Agronomica Sinica, 2022, 48(6): 1357-1371.

Figures/Tables 11

Table 1

Fig. 1

Fig. 2

Table 2

Physicochemical property and subcellular localization prediction of BnCNGC protein in Brasscia napus"

CNGC蛋白 CNGC protein	基因号 Gene ID	染色体 Chr.	起始位置 Initial position	终止位置 End position	氨基酸数 Amino acid amounts	等电点 pI	分子量 MW (kD)	总平均亲水性 GRAVY	亚细胞定位 Subcellular localization
BnCNGC1	BnaA01g06670D	A01	3095672	3099840	694	8.35	80.77	-0.205	质膜Plasma membrane
BnCNGC2	BnaA01g22170D	A01	14545425	14551189	801	9.21	90.74	0.028	质膜Plasma membrane
BnCNGC3	BnaA01g27850D	A01	19425000	19428539	760	9.57	86.26	-0.064	质膜Plasma membrane
BnCNGC4	BnaA01g27860D	A01	19430256	19433767	743	9.64	85.28	-0.138	质膜Plasma membrane
BnCNGC5	BnaA02g07990D	A02	3784851	3788310	721	9.13	82.42	-0.123	质膜Plasma membrane
BnCNGC6	BnaA02g09790D	A02	4902667	4907022	621	9.38	71.77	-0.008	质膜Plasma membrane
BnCNGC7	BnaA02g10440D	A02	5360323	5362868	711	9.38	82.56	-0.178	质膜Plasma membrane
BnCNGC8	BnaA03g26780D	A03	13180480	13184496	705	9.26	81.49	-0.212	质膜Plasma membrane
BnCNGC9	BnaA03g34680D	A03	16863304	16866599	680	8.91	77.60	-0.147	质膜Plasma membrane
BnCNGC10	BnaA03g34700D	A03	16872203	16875542	654	8.92	74.72	-0.053	质膜Plasma membrane
BnCNGC11	BnaA03g50250D	A03	26087831	26090699	741	9.29	84.40	-0.157	质膜Plasma membrane
BnCNGC12	BnaA04g14030D	A04	11848036	11851336	736	9.44	84.50	-0.199	质膜Plasma membrane
BnCNGC13	BnaA04g14530D	A04	12210464	12213711	703	9.33	81.07	-0.077	质膜Plasma membrane
BnCNGC14	BnaA04g15220D	A04	12665265	12668409	647	9.50	74.80	-0.028	质膜Plasma membrane
BnCNGC15	BnaA05g01380D	A05	808631	812078	702	9.14	81.03	-0.154	质膜Plasma membrane
BnCNGC16	BnaA05g22550D	A05	17157632	17161030	753	9.55	85.87	-0.081	质膜Plasma membrane
BnCNGC17	BnaA05g22560D	A05	17162980	17166118	751	9.19	86.39	-0.186	质膜Plasma membrane
BnCNGC18	BnaA05g22570D	A05	17167881	17171529	790	9.71	90.63	-0.185	质膜Plasma membrane
BnCNGC19	BnaA06g10680D	A06	5629756	5632689	722	9.50	82.80	-0.204	质膜Plasma membrane
BnCNGC20	BnaA06g16940D	A06	9550763	9553801	706	8.46	81.62	-0.247	质膜Plasma membrane
BnCNGC21	BnaA07g13760D	A07	12144138	12147369	684	9.68	79.43	-0.102	质膜Plasma membrane
BnCNGC22	BnaA07g17630D	A07	14705083	14708986	688	8.68	78.43	-0.092	质膜Plasma membrane
BnCNGC23	BnaA09g41300D	A09	28879650	28883074	715	8.85	82.39	-0.110	质膜Plasma membrane
BnCNGC24	BnaA09g41750D	A09	29111335	29114828	737	9.44	84.65	-0.181	质膜Plasma membrane
BnCNGC25	BnaA10g06480D	A10	4931547	4934085	720	9.57	83.90	-0.194	质膜Plasma membrane
BnCNGC26	BnaA10g07330D	A10	5759951	5765207	699	8.60	80.72	-0.173	质膜Plasma membrane
BnCNGC27	BnaA10g18740D	A10	13494368	13497571	719	9.56	82.00	0.012	质膜Plasma membrane
BnCNGC28	BnaA10g19030D	A10	13658839	13663652	973	7.24	110.58	-0.284	质膜Plasma membrane
BnCNGC29	BnaC01g08020D	C01	4234002	4238184	705	9.01	81.88	-0.185	质膜Plasma membrane
BnCNGC30	BnaC01g34960D	C01	34227372	34232651	734	9.20	82.98	0.033	质膜Plasma membrane
BnCNGC31	BnaC02g11090D	C02	6416508	6419623	721	9.21	82.52	-0.122	质膜Plasma membrane
BnCNGC32	BnaC02g14560D	C02	10092252	10095260	711	9.35	82.54	-0.179	质膜Plasma membrane
BnCNGC33	BnaC03g31720D	C03	19518056	19522572	704	9.18	81.33	-0.212	质膜Plasma membrane
BnCNGC34	BnaC03g40070D	C03	25130865	25135231	413	6.10	47.62	-0.098	质膜Plasma membrane
BnCNGC35	BnaC04g01250D	C04	984082	987459	701	9.40	80.79	-0.137	质膜Plasma membrane
BnCNGC36	BnaC04g16000D	C04	14008149	14011414	684	9.68	79.49	-0.108	质膜Plasma membrane
BnCNGC37	BnaC04g36270D	C04	37794024	37797281	714	9.16	82.28	-0.081	质膜Plasma membrane
BnCNGC38	BnaC04g36890D	C04	38310006	38313393	745	9.45	85.22	-0.184	质膜Plasma membrane
CNGC蛋白 CNGC protein	基因号 Gene ID	染色体 Chr.	起始位置 Initial position	终止位置 End position	氨基酸数 Amino acid amounts	等电点 pI	分子量 MW (kD)	总平均亲水性 GRAVY	亚细胞定位 Subcellular localization
BnCNGC39	BnaC04g38170D	C04	39436436	39439178	647	9.50	74.82	-0.016	质膜Plasma membrane
BnCNGC40	BnaC05g12210D	C05	7083786	7086776	722	9.50	82.84	-0.207	质膜Plasma membrane
BnCNGC41	BnaC05g35840D	C05	35081609	35084991	750	9.60	85.61	-0.072	质膜Plasma membrane
BnCNGC42	BnaC05g35850D	C05	35087166	35090279	750	9.20	86.57	-0.201	质膜Plasma membrane
BnCNGC43	BnaC05g35860D	C05	35092931	35096102	762	9.88	87.76	-0.163	质膜Plasma membrane
BnCNGC44	BnaC07g42730D	C07	42093754	42097033	741	9.29	84.49	-0.169	质膜Plasma membrane
BnCNGC45	BnaC08g33890D	C08	32189874	32193318	715	8.85	82.34	-0.111	质膜Plasma membrane
BnCNGC46	BnaC09g29350D	C09	31884192	31886408	600	9.45	70.24	-0.169	质膜Plasma membrane
BnCNGC47	BnaC09g30630D	C09	33625801	33632938	698	8.42	80.76	-0.192	质膜Plasma membrane
BnCNGC48	BnaC09g42460D	C09	44006544	44009743	719	9.58	82.17	0.002	质膜Plasma membrane
BnCNGC49	BnaC09g42720D	C09	44128700	44131819	714	8.60	81.30	-0.139	质膜Plasma membrane

Table 2

Fig. 3

Fig. 4

Fig. 5

Table 3

Cis-acting regulatory elements predicted in promoter of the BnCNGC gene family in Brassica napus"

元件类型 Element type	特性 Characteristic	基因 Gene
CGTCA-motif	茉莉酸甲酯响应元件 cis-acting regulatory element involved in the MeJA-responsiveness	BnCNGC1-BnCNGC6, BnCNGC10, BnCNGC12-BnCNGC15, BnCNGC17, BnCNGC18-BnCNGC20, BnCNGC23-BnCNGC26, BnCNGC29, BnCNGC31-BnCNGC39, BnCNGC42-BnCNGC45, BnCNGC49
MBS	干旱响应元件 MYB binding site involved in drought-inducibility	BnCNGC2, BnCNGC5, BnCNGC12, BnCNGC16, BnCNGC17, BnCNGC21, BnCNGC29, BnCNGC31, BnCNGC34, BnCNGC36, BnCNGC37, BnCNGC41, BnCNGC46, BnCNGC47
元件类型 Element type	特性 Characteristic	基因 Gene
ABRE	脱落酸响应元件 cis-acting element involved in abscisic acid responsiveness	BnCNGC3, BnCNGC4, BnCNGC6, BnCNGC8, BnCNGC10, BnCNGC12, BnCNGC13, BnCNGC18-BnCNGC25, BnCNGC27, BnCNGC28, BnCNGC30, BnCNGC33, BnCNGC34, BnCNGC36, BnCNGC39-BnCNGC41, BnCNGC43, BnCNGC46-BnCNGC49
GARE-motif	赤霉素响应元件 Gibberellin-responsive element	BnCNGC6, BnCNGC20-BnCNGC22, BnCNGC27, BnCNGC36, BnCNGC38, BnCNGC48
WUN-motif	损伤响应元件 Wound-responsive element	BnCNGC1, BnCNGC3, BnCNGC4, BnCNGC9, BnCNGC11, BnCNGC14, BnCNGC16, BnCNGC21, BnCNGC22, BnCNGC25, BnCNGC27, BnCNGC29, BnCNGC32, BnCNGC33, BnCNGC38, BnCNGC41-BnCNGC43, BnCNGC48
TCA-element	水杨酸响应元件 cis-acting element involved in salicylic acid responsiveness	BnCNGC15, BnCNGC16, BnCNGC18, BnCNGC23, BnCNGC25, BnCNGC27, BnCNGC28, BnCNGC31, BnCNGC35-BnCNGC39, BnCNGC41, BnCNGC43, BnCNGC46
TGA-element	生长素响应元件 Auxin-responsive element	BnCNGC1-BnCNGC3, BnCNGC11, BnCNGC20, BnCNGC21, BnCNGC26, BnCNGC30, BnCNGC32, BnCNGC39, BnCNGC40, BnCNGC46, BnCNGC47
TC-rich repeats	逆境与防御反应响应元件 cis-acting element involved in stress and defense responsiveness	BnCNGC1, BnCNGC7, BnCNGC8, BnCNGC11, BnCNGC14, BnCNGC19, BnCNGC21, BnCNGC25, BnCNGC29, BnCNGC30, BnCNGC33, BnCNGC36, BnCNGC38, BnCNGC40, BnCNGC43, BnCNGC44
AT-rich sequence	诱导子激活元件 Element for maximal elicitor-mediated activation	BnCNGC2, BnCNGC3, BnCNGC8, BnCNGC9, BnCNGC12, BnCNGC17, BnCNGC19, BnCNGC20, BnCNGC25, BnCNGC44

Table 3

Table S1

Fig. 6

Fig. 7

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基因名称 Gene name	基因号 Gene ID	正向引物序列 Forward sequence (5′-3′)	反向引物序列 Reverse sequence (5′-3′)
BnCNGC9	BnaA03g34680D	AGTTACCCTCTTTCCTCTACTCGT	GTGCGGCTTTGAGCAGCTT
BnCNGC27	BnaA10g18740D	ATAAATTCGCAAACGAGCGC	GAACCGCCTACACCACCAC
BnCNGC48	BnaC09g42720D	GAGCAGCAGCAGCAGCAGC	CGGGCTCGAACCGGGTC
BnActin7	BnaA01g27090D	CTGGAATTGCTGACCGTATGAG	ATCTGTTGGAAAGTGCTGAGGG

基因名称 Gene name	FPKM-0	FPKM-12	FPKM-24	FPKM-48
BnCNGC2	0.76	0.30	0.53	0.10
BnCNGC32	1.65	1.89	0.65	0.54
BnCNGC24	0.88	1.06	0.40	0.72
BnCNGC29	1.50	1.79	1.68	1.59
BnCNGC5	0.23	0.67	0.40	0.20
BnCNGC31	0.49	2.49	1.37	0.45
BnCNGC33	1.86	1.67	2.55	7.20
BnCNGC36	0.72	0.21	0.31	0.14
BnCNGC7	2.84	3.53	3.42	1.53
BnCNGC1	1.74	2.32	2.52	3.08
BnCNGC39	0.64	1.34	1.15	1.42
BnCNGC11	0.47	0.31	0.31	0.90
BnCNGC35	4.15	1.78	5.82	1.25
BnCNGC41	3.41	3.07	3.13	0.73
BnCNGC48	10.25	17.10	12.49	1.61
BnCNGC9	4.33	2.21	4.62	0.35
BnCNGC8	3.43	2.86	3.71	12.18
BnCNGC34	0.03	0.04	2.31	18.35
BnCNGC38	2.04	4.54	3.49	4.11
BnCNGC16	2.12	1.77	2.49	0.15
BnCNGC47	3.84	5.64	5.52	1.17
BnCNGC15	6.09	4.69	10.52	4.13

Genome-wide identification of BnCNGC and the gene expression analysis in Brassica napus challenged with Sclerotinia sclerotiorum and PEG-simulated drought

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