Expression Analysis and Resistance Identification of BrcGASA3 in Chinese Cabbage ‘Jinxiaotong’ Cultivar under Saline-alkali Stress

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

Abstract

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

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.

Figures/Tables 8

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

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

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)

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

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

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

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

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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