Response of the Transcription Factor BnHY5-2 in Brassica napus L.to Salt and Alkali Stress

doi:10.7668/hbnxb.20194546

Abstract

Abstract:

The transcription factor BnHY5-2 is associated with plant stress resistance.In order to reveal the response of Brassica napus L.transcription factor BnHY5-2 to salt alkali stress in B.napus,the response of BnHY5-2 to light and salt and alkali was analyzed by transient overexpression,qRT-PCR analysis and subcellular localization.The results revealed that under light conditions,the expression level of the BnHY5-2 gene in B.napus leaves and stems was 29.22 and 3.15 fold higher,respectively,compared to dark conditions.The higher sensitivity to light in leaves suggested that they were the primary site for light signal response.Under light conditions,the expression of BnHY5-2 in leaves and stems was significantly downregulated by 53.1% and 31.0%,respectively,when B.napus was planted in Dalian coastal saline-alkali soil;after applying saline-alkali treatment under dark conditions,the expression of BnHY5-2 was downregulated by 48.2% in the stem,while the difference in expression in the leaves was not significant,indicating organ differences,indicating that the leaves had stricter requirements for light conditions.In B.napus leaves with transient overexpression of BnHY5-2,two out of six genes related to saline-alkali stress(BnNAC32 and BnGS)showed upregulation by 1.25,3.28 fold,respectively,while the other four genes(Bnamy,BnAsp,BnNHX7,BnTPS)were downregulated by 24.8%,25.4%,71.0%,and 82.0%,respectively.Meanwhile,the content of the resistance substance betaine in B.napus increased from 0.256 to 0.573 mg/g,indicating an enhancement by 1.24 fold,suggesting that the overexpression of BnHY5-2 gene could improve the saline-alkali tolerance of B.napus.Subcellular localization results showed that the transcription factor BnHY5-2 was localized in the nucleus and regulates the expression of functional genes.Therefore,BnHY5-2 is not only related to light signaling but also participates in the saline-alkali resistance of Brassica napus L.

Key words: Brassica napus L., BnHY5-2, Saline-alkali stress, Overexpression, Transcription factor

YANG Mingxuan, LI Mingyu, WANG Bo, WANG Ze, LIU Zhiqiang, ZHOU Guangsheng, YU Fang, LIU Zhiwen. Response of the Transcription Factor BnHY5-2 in Brassica napus L.to Salt and Alkali Stress[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(4): 47-53. doi: 10.7668/hbnxb.20194546.

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Figures/Tables 8

Tab.1 Main physicochemical and nutrient indicators of soil samples

名称 Name	对照组(金州) Control group (Jinzhou)	盐碱土(庄河) Saline-alkali soil (Zhuanghe)	差异倍数 Difference multiple
pH	6.900±0.740	6.890±0.810	1.00
电导率/(μS/cm) Conductivity	55.810±3.370	240.270±9.580	4.31
总溶解固体/(mg/L) Tds	28.140±1.640	120.580±3.170	4.28
碱解氮/(mg/kg) Alkali-N	65.800±2.130	91.470±5.380	1.39
速效磷/(mg/kg) Olsen-P	34.670±2.290	17.600±1.170	0.51
速效钾/(mg/kg) Avail-K	52.370±5.210	108.000±7.420	2.06
有机质/% Organic matter	1.920±0.340	1.320±0.160	0.69
可溶性总盐/% Total soluble salts	0.100±0.010	0.610±0.050	6.10
全N/(g/kg) Total N	0.072±0.008	0.093±0.011	1.29
全P/(g/kg) Total P	0.910±0.110	0.570±0.090	0.63
全K/(g/kg) Total K	13.090±0.910	20.670±1.730	1.58
Na⁺/(g/kg)	0.021±0.002	0.713±0.078	33.95
K⁺/(g/kg)	0.010±0.001	0.023±0.003	2.30
1/2Ca²⁺/(cmol/kg)	1.640±0.150	2.030±0.210	1.24
1/2Mg²⁺/(cmol/kg)	0.930±0.090	3.460±0.120	3.72
Cl^-/(g/kg)	0.003±0.000	0.070±0.001	21.24
1/2S $O 4 2 -$ /(cmol/kg)	1 586.110±13.120	11 944.440±76.570	7.53
HC $O 3 -$ /C $O 3 2 -$	未检出	未检出
N $O 3 -$	未检出	未检出

Tab.1 Main physicochemical and nutrient indicators of soil samples

名称 Name	对照组(金州) Control group (Jinzhou)	盐碱土(庄河) Saline-alkali soil (Zhuanghe)	差异倍数 Difference multiple
pH	6.900±0.740	6.890±0.810	1.00
电导率/(μS/cm) Conductivity	55.810±3.370	240.270±9.580	4.31
总溶解固体/(mg/L) Tds	28.140±1.640	120.580±3.170	4.28
碱解氮/(mg/kg) Alkali-N	65.800±2.130	91.470±5.380	1.39
速效磷/(mg/kg) Olsen-P	34.670±2.290	17.600±1.170	0.51
速效钾/(mg/kg) Avail-K	52.370±5.210	108.000±7.420	2.06
有机质/% Organic matter	1.920±0.340	1.320±0.160	0.69
可溶性总盐/% Total soluble salts	0.100±0.010	0.610±0.050	6.10
全N/(g/kg) Total N	0.072±0.008	0.093±0.011	1.29
全P/(g/kg) Total P	0.910±0.110	0.570±0.090	0.63
全K/(g/kg) Total K	13.090±0.910	20.670±1.730	1.58
Na⁺/(g/kg)	0.021±0.002	0.713±0.078	33.95
K⁺/(g/kg)	0.010±0.001	0.023±0.003	2.30
1/2Ca²⁺/(cmol/kg)	1.640±0.150	2.030±0.210	1.24
1/2Mg²⁺/(cmol/kg)	0.930±0.090	3.460±0.120	3.72
Cl^-/(g/kg)	0.003±0.000	0.070±0.001	21.24
1/2S $O 4 2 -$ /(cmol/kg)	1 586.110±13.120	11 944.440±76.570	7.53
HC $O 3 -$ /C $O 3 2 -$	未检出	未检出
N $O 3 -$	未检出	未检出

Tab.2 Primer sequences

引物名称Primers	序列(5'—3') Sequence(5'—3')	用途Purpose
BnHY5-2-F1	TTCATCATCTCTATCTTCATCACCAGC	巢式PCR第一轮
BnHY5-2-R1	GAGTTTCAAGTCACAGACACATACCAT
BnHY5-2-KpnⅠ-F	GGGGTACCATGCAGGAGCAAACGAC	巢式PCR第二轮
BnHY5-2-BamHⅠ-R	CGGGATCCTCAGAGGCTCGCATCAGCGTTAG
BnPP2A-F	GTCAACAATCCGCACTACCTA	qRT-PCR
BnPP2A-R	ACCACGACGGGAAGAAAC
BnNAC32-F	GACGCTTGATCCACTTGTTGTTC
BnNAC32-R	GCTGGGTCATACATAGAGTAGGCT
BnHY5-2-F	CGAGAACCAGATGCTTAGACAGATT
BnHY5-2-R	GAGTTTCAAGTCACAGACACATACC
BnAsp-F	GGCGACGCTTCAATAAAAACTC
BnAsp-R	TAAGCAACTGTTACACCGAGAATAGG
BnNHX7-F	TGATGAATGATGGGACGGC
BnNHX7-R	GATAATAGAGCCCCAGTCGGAAG
BnTPS-F	TGCAGCTCAAAGACGGGTT
BnTPS-R	AAGTCACCGGGCAAGAAGGTA
BnGS-F	GCACACACAAATTACAGCACCA
BnGS-R	TGTGTTCCTTGTGACGCAGT
Bnamy-F	ACTCACTAGAAAGAGTTCTCACTCA
Bnamy-R	CCGATACATACACAACCTTAGCC

Fig.1 Expression levels of alt-tolerant genes in leaves after saline-alkali stress compared with control group Difference lowercase letters indicate significant differences. The same as Fig.2,4—5.

Fig.2 BnHY5-2 gene expression

Fig.3 Cloning of BnHY5-2, construction of vector and transformation of Agrobacterium tumefaciens GV3101 A. PCR amplification of BnHY5-2; B. pGMT-BnHY5-2 positive double enzyme digestion identification;C. PCR verification of GV3101 colony of pBI121GD-BnHY5-2; D. Colony PCR verification of pBI121GD-GFP-BnHY5-2.

Fig.4 Transient overexpression of BnHY5-2 (A) and expression of saline-alkali tolerance candidate related genes (B)

Fig.5 Change of betaine accumulation

Fig.6 Subcellular localization of onion epidermal cells

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