菜心减数分裂相关基因BrASY2参与植株耐热性的功能分析

doi:10.3864/j.issn.0578-1752.2025.11.012

Abstract

Abstract:

【Objective】 Chinese flowering cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) was a heat-resistant and enjoy cold vegetable, high temperature is one of the main problems faced in the production of summer and autumn vegetable production. In the early stage, we discovered a gene fragment that may be closely related to the response to heat stress in the transcriptome database of Chinese flowering cabbage, named BrASY2. By studying its physicochemical properties, protein structure, systematic evolution, and expression characteristics, and exploring its function in heat tolerance regulation, this study provides a reference for molecular breeding of heat-resistant. 【Method】 The BrASY2 gene sequence was obtained by whole gene synthesis, the physicochemical properties, signal peptides, protein structure, and cis acting elements of BrASY2 were analyzed using ExPASy ProtParam, ProtScale, SignalP-5.0, SOPMA, Swiss Model, and PlantCARE, respectively. Construct phylogenetic tree using MEGA 6.0, and the expression characteristics under heat stress were analyzed using real-time fluorescence quantitative PCR (qRT-PCR) method. Construct yeast heterologous overexpression vector pYES2-NTB-BrASY2 and gene silencing vector pK7GWIWG2(I)-BrASY2, respectively, to obtain transgenic yeast and silenced expression cabbage plants, and perform heat tolerance functional analysis.Cytological observation of meiotic chromosome behavior in BrASY2 silenced plant cells using anther pressing method.【Result】BrASY2 with an ORF of 852 bp was obtained, encoding 283 amino acids, the relative molecular weight was 69.16 kDa with a theoretical isoelectric point being 5.09. The results of gene structure analysis indicate that it contains abundant alpha helices and random coi. Signal peptide prediction revealed that BrASY2 was a hydrophilic protein without signal peptides. Evolutionary analysis revealed that BrASY2 had the closest genetic relationship with Chinese cabbage and Brassica napus. Transcriptome data showed that the BrASY2 gene expression levels were low in both the heat sensitive material CX7-3 and the heat-resistant material CX1-7 before high temperature stress (0 h), and the expression level was rapidly increased in CX1-7 after 6 h of heat stress, however, there was no significant change in CX7-3. qRT-PCR results showed that the expression level of BrASY2 gene in the leaves of heat-resistant inbred line CX1-7 showed an upregulation trend with the prolongation of heat stress treatment time, the highest expression level was occurred at 24 h. However, in the thermosensitive inbred line CX7-3, the relative expression level remained in a low-level expression state with no significant change. Analysis of heterologous overexpression of yeast revealed that overexpression of yeast pYES2-NTB-BrASY2 was tolerant to high temperatures and can help the yeast overcome heat stress.The silencing results showed that the expression of BrHsfA2, BrHsp90, and BrMn-SOD in the silenced plants were inhibited after heat stress compared with the control plants, and the plant leaves color became lighter, chlorophyll content decreased, relative conductivity and H₂O₂ content increased, and SOD activity decreased. The cytological observation results of meiosis showed that, compared with the control plants, the chromosome behavior of BrASY2 silenced plants at all stages was normal, and there was no significant difference in the proportion of balanced splits cells and quaternary inmeiosis anaphase.【Conclusion】 This study successfully obtained the BrASY2 gene in Chinese flowering cabbage, and it plays a positive regulatory role in response to heat stress.

Key words: Chinese flowering cabbage, BrASY2, heat resistance, gene overexpression, gene silencing expression

PANG QiangQiang, SUN XiaoDong, CHEN YiSong, ZHOU Man, WANG YaQiang, SHI GuoBin. Heat Resistance Analysis of Meiosis Related Gene BrASY2 in Chinese Flowering Cabbage[J].Scientia Agricultura Sinica, 2025, 58(11): 2239-2252.

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引物名称Primer name	引物序列Primer sequence (5′-3′)	引物用途Primer purpose
BrASY2-F	GGCAGATGGGTTCAGATTCG	qRT-PCR
BrASY2-R	AAGAGGCGTGCTCATACTCA
BrHsfA2-F	GACCATTGCTCCAAGACACC
BrHsfA2-R	GGAAGAGACGGTGACCTACG
BrHsp90-F	CTGCAACAGTCTCACCCTCT
BrHsp90-R	CTGAGAGCAAGTGGTGGAAC
BrMn-SOD-F	AGATGAGTGCTGAAGGTGCT
BrMn-SOD-R	CTGATTGGCGGTTGTGTCAA
BrCKⅠ-F	GTCTAACTTCCTCGCCACCT	内参基因Reference gene
BrCKⅠ-R	GATTCGACGACAAGCCAGAC	内参基因Reference gene

材料 Material	后期ⅠAnaphaseⅠ			后期ⅡAnaphaseⅡ
材料 Material	观察细胞数 Observe the No. of cells	均衡分裂数 No. of balanced splits	比例 Proportion (%)	二分体 Binary	三分体 Ternary	四分体 Quaternary	四分体比例 Quaternary proportion (%)
BrASY2沉默植株 BrASY2 silent plants	51	48	94.12	2	0	26	92.86
对照植株 Control plant	49	46	93.88	2	0	24	92.31

Heat Resistance Analysis of Meiosis Related Gene BrASY2 in Chinese Flowering Cabbage

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