| 摘要: |
| 【 目的】基于高温胁迫处理后的菜心转录组数据，从中筛选并命名了一个可能与高温胁迫响应相关的 候选基因，即天冬氨酸激酶基因BrAK3，对该基因编码蛋白的生物信息学进行了分析，探究其在耐热性调控中的功 能，以期为菜心耐热分子育种提供理论依据与基因资源。【方法】采用全基因合成方法获得BrAK3全长序列，利用生 物信息学工具分析其理化性质、信号肽、蛋白结构与顺式作用元件，利用MEGA 6.0构建系统发育树。以耐热的菜心自交系CX1-7和热敏的菜心自交系CX7-3为材料，构建BrAK3过表达植株和BrAK3沉默表达植株，采用实时荧光定量PCR（RT-qPCR）技术，分析高温（37 ℃）胁迫下BrAK3以及与高温胁迫相关基因（BrHsfA2、BrHsp90和BrMnSOD）的表达模式以及相关生理指标的变化。【结果】BrAK3基因全长为681 bp，编码226个氨基酸，具有典型的天冬 氨酸激酶保守结构域，蛋白二级结构以 α-螺旋、无规则卷曲和延伸链结构为主，含有11个高温胁迫响应元件CAATbox，与白菜、甘蓝型油菜、野生甘蓝和萝卜的同源性较高。转录表达分析结果显示，高温胁迫前，热敏材料CX7-3与耐 热材料CX1-7的BrAK3基因表达水平均较低；高温胁迫6 h后，CX1-7中的BrAK3表达水平迅速上升，而CX7-3中的BrAK3无显著变化。随着高温胁迫处理时间的延长，CX1-7中的BrAK3表达水平持续上调，于12 h时达到峰值；而CX7-3始终维持较低水平。组织表达分析结果显示，与高温胁迫前相比，高温胁迫6 h后根和叶柄中的BrAK3表达量均无显著变化；CX1-7叶片中的BrAK3表达水平显著增加，CX7-3中的BrAK3表达水平仍处于低水平。高温胁迫下，BrAK3过表达植株中的BrHsfA2、BrHsp90和BrMn-SOD基因表达上调，植株表型变化不明显，叶绿素含量无显著变化，相对电导率降低，同时脯氨酸含量和SOD活性升高，耐热性增强；BrAK3沉默表达植株中的BrHsfA2、BrHsp90和BrMn-SOD表达均受到抑制，叶片颜色变浅且叶绿素含量下降，相对电导率升高，脯氨酸含量和SOD活性均降低，植 株耐热性明显减弱。【 结论】成功获得了菜心BrAK3基因，证实其表达受高温特异性诱导，并具有正向调控菜心耐热 性的生物学功能。 |
| 关键词: 菜心 BrAK3 耐热性 基因过表达 基因沉默表达 |
| DOI：10. 13207/j. jnwafu. 2026. 11. 008 |
| 分类号: |
| 基金项目:海南省科技专项-省属科研院所技术创新科研项目（SQKY2022-0011）；海南省重点研发项目（ZDYF2024XDNY242，ZDYF2024KJTPY051）；海南省农业科学院院本级科研项目（HAAS2025KJCX11，HAAS2024SJTCSCS22）；国家现代农业产业技术体系建设专项资金项目（CARS-23-G55） |
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| Heat resistance analysis of the aspartate kinase gene BrAK3 in Chinese flowering cabbage |
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PANG Qiangqiang, SUN Xiaodong, ZHOU Man, WANG Yaqiang, SHI Guobin, CHEN Yisong
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Institute of Vegetable，Hainan Provincial Academy of Agricultural Sciences，Key Laboratory of Genetic Resources Evaluation and Utilization of Tropical Fruits and Vegetable（s Co-construction by Ministry and Province），Hainan Province Key Laboratory of Vegetable Biology，Hainan Province Melon and Vegetable Breeding Engineering Technology Research Center，Haikou，Hainan 571100，China
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| Abstract: |
| 【 Objective】Chinese flowering cabbage，a cold-preferring vegetable with low heat tolerance，faces significant production challenges during summer and autumn due to high temperatures. Based on the tran-scriptome data obtained from Chinese flowering cabbage under heat stress，we screened and named a candidate gene BrAK3 that may be related to heat stress response. This study aimed to analyze its physicochemical proper-ties，protein structure，phylogenetic relationships，and expression patterns of the protein encoded by BrAK3，and further explore its function in heat tolerance regulation，in order to provide a theoretical basis and genetic re-sources for the molecular breeding of heat-tolerant Chinese flowering cabbages.【Method】The full-length sequence of BrAK3 was obtained by whole gene synthesis. Its physicochemical properties，signal peptides，protein structure，and cis acting elements were analyzed using bioinformatic tools such as ExPASy ProtParam，ProtScale，SignalP-5.0，SOPMA，Swiss Model，and PlantCARE. A phylogenetic tree was constructed using MEGA 6.0.Using a heat-tolerant inbred line（CX1-7） and a heat-sensitive inbred line（CX7-3） of Chinese flowering cabbage as materials，overexpression vector pBarleyGate 101-BrAK3 and gene silencing vector pK7GWIWG2（Ⅰ）-BrAK3 were constructed. Real time fluorescence PCR（RT-qPCR） technology was used to analyze the expression patterns of BrAK3 and heat stress-related genes（BrHsfA2，BrHsp90，BrMn-SOD） under high-temperature stress（37 °C），and the changes in relevant physiological indices.【Result】The fulllength squence of the BrAK3 gene was 681 bp，encoding 226 amino acids that contained a typical aspartate kinase conserved domain. The secondary structure of the BrAK3 potein was predominantly composed of alpha he lices，irregular coils，and extended chain structures，and it contained eleven CAAT box heat stress response ele-ments. Phylogenetic analysis showed that BrAK3 had the highest homology with Brassica rapa，Brassica napus，Brassica oleracea var.Oleracea and Raphanus sativus.Transcriptional expression analysis showed that before heat stress，the expression level of the BrAK3 gene were low in both the thermosensitive material CX7-3 and the heat-resistant material CX1-7.After 6 h of heat stress，the expression level of BrAK3 in CX1-7 rapidly in-creased，while there was no significant change in CX7-3. With prolonged stress，the BrAK3 expression in CX1-7 continued to rise，peaking at 12 h，whereas it remained consistently low in CX7-3. Tissue-specific expression analysis showed no significant changes in the expression level of BrAK3 in the root and petiole before and after 6 h of heat stress. The leaf expression of BrAK3 was significantly higher than that before stress，while in CX7-3，it remained at a low level. Functional analysis showed that the expression level of BrAK3 directly affected the heat tolerance of Chinese flowing cabbage. In the BrAK3-overexpressed plant，the expression of BrHsfA2，BrHsp90 and BrMn-SOD was upregulated. The phenotypic changes of the plants and chlorophyll content were not significant，the relative conductivity decreased，and the proline content and SOD activity increased. In con?trast，in BrAK3-silenced plants，the expression of the BrHsfA2，BrHsp90 and BrMn-SOD genes was inhibited. The leaf color became lighter，the chlorophyll content decreased，the relative conductivity increased，the proline content and SOD activity decreased，and the plant’s heat tolerance was significantly weakened.【Conclusion】This study successfully identified the BrAK3 gene in Chinese flowering cabbage，confirming that its expression is specifically induced by high temperature and that it has a biological function of positively regulating the heat tolerance of Chinese flowering cabbage. |
| Key words: Chinese flowering cabbage BrAK3 heat resistance gene overexpression gene silencing expression |
