‘金小童’大白菜BrcGASA3基因在盐碱胁迫下的表达分析及抗性鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2024-0799

摘要/Abstract

摘要：

目的对不同盐碱胁迫处理下耐盐碱性不同的大白菜（Brassica rapa ssp. pekinensis）品种中BrcGASA3的表达响应模式进行分析，对耐盐碱性相对较强的‘金小童’品种大白菜中BrcGASA3进行克隆，并在模式植物拟南芥（Arabidopsis thaliana）中进行遗传转化以鉴定BrcGASA3的盐碱胁迫响应特性。方法利用实时荧光定量PCR对盐碱耐性不同的大白菜品种在不同浓度NaCl和Na₂CO₃处理过程中BrcGASA3的表达模式进行检测；对‘金小童’品种大白菜中BrcGASA3的编码序列进行克隆；构建BrcGASA3过表达转基因拟南芥并进行盐碱胁迫处理，观察植株表型并进行生理生化指标测定及分析。结果在NaCl处理中，较低浓度处理时耐盐性较高的大白菜中BrcGASA3表达量较高，较高浓度处理时耐盐性较低的大白菜中BrcGASA3表达量较高；在Na₂CO₃处理中，低浓度处理下两种大白菜中BrcGASA3表达量差异相对较小，高浓度处理下耐盐性较高的大白菜中BrcGASA3表达量显著高于耐盐性较低的大白菜；成功克隆获得‘金小童’大白菜中BrcGASA3的编码序列并构建了多个BrcGASA3过表达转基因拟南芥纯合体株系，发现在NaCl和Na₂CO₃处理下BrcGASA3过表达转基因拟南芥显示出低于野生型拟南芥的胁迫耐性；测定出在不同浓度NaCl和Na₂CO₃处理下BrcGASA3过表达转基因拟南芥和野生型拟南芥的渗透调节物质的含量、丙二醛含量、叶绿素含量等生理生化指标，初步阐释了过表达BrcGASA3降低拟南芥植株盐碱胁迫耐性的生理响应情况。结论在抗盐碱性不同的大白菜品种中BrcGASA3在不同程度盐碱胁迫处理下的表达响应方式不同，BrcGASA3在拟南芥中过表达降低了植株在盐碱胁迫处理下的生长能力。

关键词: BrcGASA3, ‘金小童’大白菜, 盐碱胁迫, NaCl处理, Na₂CO₃处理, 表达分析, 抗性鉴定

Abstract:

Objective The expression patterns of BrcGASA3 responding to different saline-alkali stress treatments in Chinese cabbage (Brassica rapa L.ssp. pekinensis) cultivars with different saline-alkali tolerance were determined, BrcGASA3 of Chinese cabbage cultivar ‘Jinxiaotong’, which is with stronger saline-alkali tolerance, was cloned, and the genetic transformation in the model plant Arabidopsis thaliana was carried on to identify the saline-alkali stress response characteristics of BrcGASA3. Method Expression patterns of BrcGASA3 in Chinsese cabbage cultivars with different salinity tolerance during treatments with different concentrations of NaCl and Na₂CO₃ were detected using quantitative real-time PCR. The coding sequence of BrcGASA3 from the Chinese cabbage cultivar ‘Jinxiaotong’ was cloned. BrcGASA3 overexpressed transgenic A. thaliana was constructed and subjected to saline and alkaline stresses, then the phenotypes of the plants were observed and physiological and biochemical indexes were measured and analyzed. Result Under NaCl treatments, the expression of BrcGASA3 in Chinese cabbage with stronger salt tolerance was high when the NaCl concentration was low; while the expression of BrcGASA3 in Chinese cabbage with weaker salt tolerance was high when the NaCl concentration was high. Under Na₂CO₃ treatments, the expression of BrcGASA3 was similar in the two Chinese cabbage cultivars when the Na₂CO₃ concentration was low, but the expression of BrcGASA3 was significantly high in Chinese cabbage with stronger salt tolerance when the Na₂CO₃ concentration was high. The coding sequence of BrcGASA3 in the Chinese cabbage cultivar ‘Jinxiaotong’ was successfully cloned, and the overexpressed transgenic A. thaliana lines of BrcGASA3 were successfully constructed, and it was found that BrcGASA3-overexpressed transgenic A. thaliana showed lowered stress tolerance comparing with the wild-type A. thaliana under NaCl and Na₂CO₃ treatments. Physiological and biochemical indices including the contents of osmoregulatory substances, malondialdehyde content, and chlorophyll content of BrcGASA3-overexpressedtransgenic and the wild-type A. thaliana were determined under treatments with different concentrations of NaCl and Na₂CO₃, and the physiological responses reducing the saline-alkali stress tolerance in A. thaliana plants when overexpressing BrcGASA3 were preliminarily elucidated. Conclusion The expression response patterns of BrcGASA3 in the Chinese cabbage cultivars with different saline-alkali stress tolerance varied in different saline-alkali stress treatments, the growth ability of A. thaliana with the overexpression of BrcGASA3 decreased in the saline-alkali stress treatments.

Key words: BrcGASA3, Chinese cabbage ‘Jinxiaotong’, saline-alkali stress, NaCl treatment, Na₂CO₃ treatment, expression analysis, identification of resistance

马天意, 许家佳, 路文婧, 吴艳, 沙伟, 张梅娟, 彭疑芳. ‘金小童’大白菜BrcGASA3基因在盐碱胁迫下的表达分析及抗性鉴定[J]. 生物技术通报, 2025, 41(2): 127-138.

MA Tian-yi, XU Jia-jia, LU Wen-jing, WU Yan, SHA Wei, ZHANG Mei-juan, PENG Yi-fang. Expression Analysis and Resistance Identification of BrcGASA3 in Chinese Cabbage ‘Jinxiaotong’ Cultivar under Saline-alkali Stress[J]. Biotechnology Bulletin, 2025, 41(2): 127-138.

图/表 8

表1 本研究所用的引物

Table 1 Primers used in this study

引物名称 Primer name	序列Sequence （5′-3′）	用途Application
qPCR-BrcGASA3-F	AACCACATCCACCACAGTCC	qPCR
qPCR-BrcGASA3-R	GTCTTCCAGTTGTTGTAGCAAGG	qPCR
qPCR-BrcActin-F	AGCGTGGCAACCTGGGATG	qPCR
qPCR-BrcActin-R	TGATGAACAAGAAAGTAGGCATAGCG	qPCR
BrcGASA3-F	ATGCACCCAATACACCTGGAG	克隆载体构建Cloning vector construction
BrcGASA3-R	TCAAGGGCATTTAGGTCCACC	克隆载体构建Cloning vector construction
IN-BrcGASA3-F	ATGCCCGTCGACCCCATCGACCCAATACAC	表达载体构建Expression vector construction
IN-BrcGASA3-R	GGATCCGGTACCCCCTCAAGGGCATTTAGG	表达载体构建Expression vector construction
AtActin1-F	CGCTTAACCCGAAAGCTAAC	qPCR
AtActin1-R	TACGCCCACTAGCGTAAAGA	qPCR

图1 不同浓度NaCl和Na2CO3处理下大白菜BrcGASA3的表达量分析JT：‘金小童’品种大白菜；RC：‘日本春秋’品种大白菜；误差线表示标准偏差，误差线上不同字母表示差异显著（P<0.05），下同

Fig. 1 Expression analysis of BrcGASA3 in Chinese cabbage treated with different concertation of NaCl and Na2CO3JT: Chinese cabbage cultivar 'Jinxiaotong'‍; RC: Chinese cabbage cultivar 'Ribenchunqiu'. The error bars indicate the standard deviation, and the different letters on the error bars indicate significance (P<0.05), the same below

图2 BrcGASA3编码序列的PCR扩增结果和重组表达载体质粒的验证A：BrcGASA3编码序列的PCR扩增结果（M：DL 2000 DNA marker；-：水对照；1：BrcGASA3编码序列）；B：BrcGASA3重组表达载体质粒验证（M：DL 2000 DNA marker；-：水对照；+：克隆载体阳性对照；2：BrcGASA3大肠杆菌菌液）

Fig. 2 PCR amplification results of BrcGASA3 coding sequence and validation of recombinant expression vector plasmidA: PCR amplification results of the coding sequence of BrcGASA3 (M: DL 2000 DNA marker; -: water control; 1: BrcGASA3 coding sequence). B: Verification of BrcGASA3 recombinant expression vector plasmid (M: DL 2000 DNA marker; -: water control; +: cloning vector positive control; 2: BrcGASA3 E. coli suspension)

图3 BrcGASA3过表达转基因拟南芥的筛选及鉴定A：BrcGASA3过表达转基因拟南芥T1代植株的筛选；B：BrcGASA3过表达转基因拟南芥T2代植株的相对表达量检测

Fig. 3 Screening and characterization of BrcGASA3 overexpressed transgenic Arabidopsis thalianaA: Screening of T1 generation plants of transgenic Arabidopsis thaliana overexpressing BrcGASA3. B: Detection of relative expression of transgenic A. thaliana T2 plants overexpressing BrcGASA3

图4 T3代BrcGASA3过表达转基因拟南芥幼苗在不同浓度NaCl和Na2CO3处理中的表型CK：NaCl和Na2CO3浓度为0；OE1：brcgasa3-oe1；OE3：brcgasa3-oe3；OE31：brcgasa3-oe31

Fig. 4 Phenotypes of T3 generation BrcGASA3 overexpressed transgenic A. thaliana seedlings under different concentration of NaCl and Na2CO3 treatmentsCK: NaCl and Na2CO3 concentrations is 0; OE1: brcgasa3-oe1; OE3: brcgasa3-oe3; OE31: brcgasa3-oe31

图5 不同浓度NaCl和Na2CO3处理下Col-0和BrcGASA3过表达转基因拟南芥根长

Fig. 5 Root lengths of Col-0and BrcGASA3 overexpressed transgenic A. thaliana under NaCl and Na2CO3 treatments of different concentration

图6 不同浓度NaCl处理下Col-0和BrcGASA3过表达转基因拟南芥植株幼苗相关指标的变化

Fig. 6 Changes of related indexes in seedlings ofCol-0and BrcGASA3 overexpressed transgenic A. thaliana under NaCl treatments of different concentration

图7 不同浓度Na2CO3处理下Col-0和BrcGASA3过表达转基因拟南芥植株幼苗相关指标的变化

Fig. 7 Changes of related indexes in seedlings of Col-0 and BrcGASA3 overexpression transgenic A. thaliana under Na2CO3 treatments of different concentration

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