宁夏枸杞TGA2转化拟南芥增强其耐盐性

doi:10.16420/j.issn.0513-353x.2024-0015

摘要/Abstract

摘要：

采用RACE方法克隆宁夏枸杞（Lycium barbarum）TGA2并进行序列分析，采用PCR法克隆其启动子序列，构建过表达载体pCAMBIA 2300EGFP-TGA2转化拟南芥，进行TGA2亚细胞定位，并分析转TGA2拟南芥对NaCl胁迫的响应。结果表明，宁夏枸杞TGA2全长2 281 bp，编码493个氨基酸；上游启动子序列为2 479 bp，存在多个参与植物激素和光信号响应顺式作用元件；宁夏枸杞TGA2的氨基酸序列与三分三（Anisodus acutangulus）TGA2的氨基酸序列相似性较高；TGA2定位于细胞核，并表现出转录激活活性；在不同浓度NaCl胁迫下，转TGA2拟南芥的TGA2表达量均高于野生型，且对NaCl胁迫的耐受能力明显增强；在相同浓度NaCl胁迫下，转TGA2拟南芥的ABA、水杨酸、蔗糖果糖基转移酶含量大多显著高于野生型；在0 ~ 100 mmol · L^-1 NaCl胁迫下，转TGA2拟南芥GST活性均高于野生型；在300 mmol · L^-1 NaCl胁迫下，转TGA2拟南芥的SOD、POD、CAT活性高于野生型，MDA含量低于野生型。以上结果说明过表达TGA2基因可以有效缓解拟南芥由盐胁迫导致的膜脂过氧化作用，增强拟南芥对盐胁迫的抵抗能力。

关键词: 宁夏枸杞, TGA2, 转基因, 盐胁迫

Abstract:

In this study，TGA2 was cloned by RACE method from Lycium barbarum and performed sequence analysis. The TGA2 promoter sequence was cloned by PCR，and the overexpression vector pCAMBIA 2300EGFP-TGA2 was constructed to transform Arabidopsis thaliana. TGA2 subcellular localization was performed，and the response of the transformed A. thaliana to NaCl stress was also analyzed. The results show that TGA2 has a total length of 2 281 bp and encoded 493 amino acids. The upstream promoter sequence of TGA2 is 2 479 bp，with multiple cis-acting elements involved in plant hormone and light signal response. Phylogenetic analysis shows that the amino acid sequence of TGA2 from L. barbarum is highly similar to that of TGA2 from Anisodus acutangulus. TGA2 is located in the nucleus and exhibits transcriptional activation activity. Under different concentrations of NaCl stress，the expression level of TGA2 in transgenic A. thaliana was higher than that in wild-type，and the tolerance of transgenic TGA2 A. thaliana to NaCl stress was significantly enhanced. Physiological level test showed that under the same concentration of NaCl stress，the content of ABA，SA，and SST in TGA2 transgenic A. thaliana was significantly increased compared to wild-type. Under the stress of 0-100 mmol · L^-1 NaCl，the GST activity of TGA2 transgenic A. thaliana was higher than that of wild-type. Under 300 mmol · L^-1 NaCl stress，TGA2 transgenic A. thaliana showed higher SOD，POD，and CAT activities and lower MDA content compared to wild-type. These indicate that TGA2 gene can effectively alleviate the membrane lipid peroxidation caused by salt stress in A. thaliana，and enhance its resistance to salt stress.

Key words: Lycium barbarum, TGA2, genetic transformation, salt stress

杨雪, 胡进红, 李晶晶, 章英才, 王玲霞, 梁文裕. 宁夏枸杞TGA2转化拟南芥增强其耐盐性[J]. 园艺学报, 2024, 51(12): 2829-2842.

YANG Xue, HU Jinhong, LI Jingjing, ZHANG Yingcai, WANG Lingxia, LIANG Wenyu. Genetic Transformation of Lycium Barbarum TGA2 Gene in Arabidopsis Enhances Its Salt Tolerance[J]. Acta Horticulturae Sinica, 2024, 51(12): 2829-2842.

图/表 12

表1 引物序列

Table 1 Primers sequence

用途 Purpose	引物名称 Primer name	引物序列（5′-3′） Primer sequence
载体构建和表达模式 Vector construction and expression mode	TGA2-F	ATGGAGAGTCAAAGAATTGG
	TGA2-R	TCACATATTTGAGAAATGG
	3′GSP1	CAAGGTGCTTCATGGTGATAGGGGAAT
	3′GSP2	CCCTATGGGCATCACGTCCTAGAGAGA
	5′GSP1	GCTGGTGGTTCTTGAT
	5′GSP2	GGAGGATTTAGGCCACGAA
	5′GSP3	AAGGCATGTTGTGATCAGC
	3′CDS	AAGCAGTGGTATCAACGCAGAGTAC(T)30 V N
基因启动子克隆 Gene promoter cloning	TGA2-R1	TCTCCTCCTCCTGATTTTGAGCTTCC
	TGA2-R2	TCATCATTGCTCATCTTAACTCCTTGCAG
	TGA2-R3	AGAAGTGCATAAGGCATGTTGTGATC
目的基因扩增引物 Target gene amplification primers	TGA2-2300-EGFP-F	ACGGGGGACGAGCTCGGTACC ATGGAGAGTCAAAGAATTGGAGAC
目的基因扩增引物 Target gene amplification primers	TGA2-2300-EGFP-F	CACCATGGTGTCGACTCTAGA CATATTTGAGAAATGGTGTTGGGATG
基因定量分析 Quantitative analysis of genes	TGA2-T-F	CCACTATCCTTCGCAAGACC
	TGA2-T-R	GCTGAACCTGAATCACTACTCT
	TGA2-F-Y	CCAAGAACAGGAGTGTCACATA
	TGA2-R-Y	GTCTTAGCATCAGTCACCCTATC
目的基因扩增引物 Target gene amplification primers	TGA2-QRT-F	GTCTTAGCATCAGTCACCCTATC
目的基因扩增引物 Target gene amplification primers	TGA2-QRT-R	CCAAGAACAGGAGTGTCACATA
内参引物 Internal reference primer	Actin-F	CCTAAGGCCAACAGAGAGAAA
内参引物 Internal reference primer	Actin-R	CGACCACTAGCATACAAGGAA

图1 TGA2基因的RACE克隆 A：中间片段；B：5′和3′片段克隆；M：DL2000。

Fig. 1 RACE cloning of TGA2 A：Intermediate segments；B：Cloning of 5′ and 3′ fragments；M：DL2000.

图2 宁夏枸杞TGA2与其他16个物种TGA2的系统进化树

Fig. 2 Phylogenetic tree of TGA2 from Lycium barbarum and TGA2 from other 16 species

图3 TGA2基因启动子的克隆 1、2：TGA2启动子；M：DL2000。

Fig. 3 Cloning of promoters of TGA2 1，2：TGA2 promoter；M：DL2000.

表2 TGA2基因启动子部分顺式作用元件

Table 2 cis-Acting elements of TGA2 promoters

名称 Name	特征序列 Characteristic sequence	元件功能 Component function	位置/bp Site
ABRE	ACGTG	ABA响应 ABA response	-5
ACE	CTAACGTATT	光响应 Photoresponse	+ 9
AE-box	AGAAACAA	光响应 Photoresponse	-8
Box4	ATTAAT	光响应 Photoresponse	-6
G-box	CACGTT	光响应 Photoresponse	+ 6
P-box	CCTTTTG	赤霉素响应 Gibberellin response	+ 7、-7
TCA-element	CCATCTTTTT	水杨酸响应 Salicylic acid response	+ 9
TGACG-motif	TGACG	茉莉酸甲酯响应 Response of methyl jasmonate	+ 5、-5
TCT-motif	TCTTAC	光响应 Photoresponse	-953
TC-rich repeats	GTTTTCTTAC	防御和应激反应响应 Defense and stress response	-622、+ 213
MYB	CAACCA	MYB结合位点；干旱响应；ABA响应 MYB binding site；Drought response；ABA response	-676、+ 1702、-1125、 -2263
MYC	CATTTG	MYC结合位点；干旱响应；ABA响应 MYC binding site；Drought response；ABA response	-81、-1587
as-1	TGACG		+ 1、-131

图4 拟南芥TGA2亚细胞定位分析

Fig. 4 Subcellular localization analysis of TGA2

图5 TGA2表达载体构建及转化 A：目的基因扩增；B：大肠杆菌PCR鉴定。M：Marker DL2000；1 ~ 10：TGA2基因PCR产物。

Fig. 5 Construction and transformation of TGA2 gene expression vector A：Target gene amplification；B：Identification of E. coli by PCR；1-10：TGA2 gene PCR product.

图6 过表达TGA2拟南芥T1代（A）和T2代（B）DNA鉴定 M：DL2000；1 ~ 14：转基因拟南芥；WT：阴性对照（野生型）；CK：空白对照（水）；P：阳性对照（农杆菌菌液）。

Fig. 6 Overexpression of TGA2 in Arabidopsis thaliana T1（A）and T2（B）DNA identification M：DL2000；1-14：Transgenic A. thaliana；WT：Negative control（wild type）；CK：Blank control（water）；P：Positive control（Agrobacterium tumefaciens）.

图7 NaCl胁迫下拟南芥野生型和转基因（TGA2-OE）株系表型及存活率变化不同小写字母表示差异显著（P < 0.05）。下同。

Fig. 7 Phenotypic changes and survival rate of wild-type and TGA2 transgenic Arabidopsis thaliana under different NaCl concentrations The different lowercase letters between groups indicate significant differences（P < 0.05）. The same below.

图8 NaCl胁迫下宁夏枸杞幼苗（A）和拟南芥野生型与转基因（TGA2-OE）植株（B）的TGA2表达量

Fig. 8 The expression levels of TGA2 in Lycium barbarum seedlings（A）and TGA2 in wild-type and transgenic（TGA2-OE）Arabidopsis thaliana（B）under different NaCl concentrations stress

图9 NaCl胁迫下野生型和转基因（TGA2-OE）拟南芥ABA和水杨酸含量的变化

Fig. 9 Changes of ABA and SA contents in wild type and transgenic Arabidopsis thaliana under NaCl stress

图10 NaCl胁迫下野生型和转基因（TGA2-OE）拟南芥GST、SOD、POD、CAT活性和SST、MDA含量的变化

Fig. 10 Changes of GST，SOD，POD，CAT activities and SST，MDA content in wild type and transgenic Arabidopsis thaliana under NaCl stress

参考文献 30

[1]	Despres C, DeLong C, Glaze S, Liu E, Fobert P R. 2000. The Arabidopsis NPR1/NIM1 IM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors. The Plant Cell, 12 (2):279-290.
[2]	Droge-Laser W, Snoek B L, Snel B, Weiste C. 2018. The Arabidopsis bZIP transcription factor family-an update. Curr Opin Plant Biol,45:36-49.
[3]	Du X, Du B, Chen X, Zhang S, Zhang Z,Z, Qu S. 2014. Overexpression of the MhTGA2 gene from crab apple(Malus hupehensis)confers increased tolerance to salt stress in transgenic apple(Malus domestica). The Journal of Agricultural Science,(4):634-641.
[4]	Fode B, Siemsen T, Thurow C, Weigel R, Gatz C. 2008. The Arabidopsis GRAS protein SCL 14 interacts with class II TGA transcription factors and is essential for the activation of stress-inducible promoters. The Plant Cell, 20 (11):3122-3135.
[5]	Fujita Y, Fujita M, Satoh R, Maruyama K, Parvez M M, Seki M, Hiratsu K, Ohme-Takagi M, Shinozaki K, Yamaguchi-Shinozaki K. 2006. AREB 1 is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance in Arabidopsis. The Plant Cell, 17 (12):3470-3488.
[6]	Gao Junfeng. 2006. Guidance on plant physiology experiments. Beijing: Higher Education Press:210-217. (in Chinese)
	高俊凤. 2006. 植物生理学实验指导. 北京: 高等教育出版社:210-217.
[7]	Han L L, ZouH Z, Zhou L, Wang Y. 2022. Transcriptome-based identification and expression analysis of the glutathione S-transferase (GST) family in tree peony reveals a likely role in anthocyanin transport. Horticultural Plant Journal, 8 (6):787-802.
[8]	Herrera-Vásquez A, Fonseca A, Ugalde J M, Lamig L, Seguel A, Moyano T C, Gutiérrez R A, Salinas P, Vidal E A, Holuigue L. 2021. TGA class II transcription factors are essential to restrict oxidative stress in response to UV-B stress in Arabidopsis. Journal of Experimental Botany, 72 (5),1891-1905. doi: 10.1093/jxb/eraa534 pmid: 33188435
[9]	Li Chunhong, Wang Kaituo, Lei Changyi, Xu Feng, Ji Nana, Jiang Yongbo. 2022. Identification of TGA gene family in peach and analysis of expression mode involved in a baba-induced disease resistance. Acta Horticulturae Sinica, 49 (2):265-280. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-1000 URL
	黎春红, 汪开拓, 雷长毅, 许凤, 季娜娜, 蒋永波. 2022. 桃TGA家族鉴定及BABA诱导的抗病表达分析. 园艺学报, 49 (2):265-280. doi: 10.16420/j.issn.0513-353x.2020-1000 URL
[10]	Li Lixian, Wang Shuo, Chen Ying, Wu Yingtao, Wang Yaqian, Fang Yue, Chen Xuesen, Tian Changping, Feng Shouqian. 2022. PavMYB10.1 promotes anthocyanin accumulation by positively regulating PavRiant in sweet cherry. Acta Horticulturae Sinica, 49 (5):1023-1030. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0732 URL
	李丽仙, 王烁, 陈莹, 邬滢涛, 王雅倩, 房月, 陈学森, 田长平, 冯守千. 2022. 甜樱桃PavMYB10.1促进PavRiant表达和花青苷积累. 园艺学报, 49 (5):1023-1030. doi: 10.16420/j.issn.0513-353x.2021-0732 URL
[11]	Li P, Zheng T C, Li L L, Wang J, Cheng T R, Zhang Q X. 2022. Genome-wide investigation of the bZIP transcription factor gene family in Prunus mume:Classification,evolution,expression profile and low-temperature stress responses. Horticultural Plant Journal, 8 (2):230-242.
[12]	Li Tao, Sayed R A S, Liu Lei, Zheng Zheng, Du Yongchen, Li Junming. 2015. Age-related resistance and the mechanism of Ph-3 gene to phytophthora infestans in tomato. Acta Horticulturae Sinica, 42 (6):1077-1084. (in Chinese) doi: 10.16420/j.issn.0513-353x.2014-0359
	李涛, Sayed Rashad Ali Shah, 刘磊, 郑峥, 杜永臣, 李君明. 2015. 番茄对晚疫病的株龄相关抗性及Ph-3的抗性机制. 园艺学报, 42 (6):1077-1084. doi: 10.16420/j.issn.0513-353x.2014-0359
[13]	Liang Bin, Xie Qi. 2016. Optimization of activity measurement conditions for GST fusion protein. Journal of Chengde Medical College, 33 (5):413-415. (in Chinese)
	梁斌, 谢琦. 2016. GST融合蛋白活性测定条件的优化. 承德医学院学报, 33 (5):413-415.
[14]	Liang Xiaojie, He Xinru, He Jun, Tian Jianwen, Dai Guoli, Qin Ken. 2023. The influence of different hedge tree shapes and harvesting periods on the fruit quality of new strains of Lycium barbarum L. Northern Horticulture,(2):104-111. (in Chinese)
	梁晓婕, 何昕孺, 何军, 田建文, 戴国礼, 秦垦. 2023. 不同篱架树形及采摘期对宁夏枸杞新品系果实品质的影响. 北方园艺,(2):104-111.
[15]	Liang Xiaojie, Li Yuekun, Wang Yajun, Duan Linyuan, An Wei. 2019. Evaluation of salt tolerance of main varieties of Lycium barbarum L. Ningxia Agriculture and Forestry Technology, 60 (6):17-21. (in Chinese)
	梁晓婕, 李越鲲, 王亚军, 段淋渊, 安巍. 2019. 枸杞主栽品种耐盐性评价. 宁夏农林科技, 60 (6):17-21.
[16]	Liu J N, Ma X M, Yan L P, Liang Q, Fang H C, Wang C X, Dong Y H, Chai Z J, Zhou R, Bao Y, Wang L C, Gai S S, Lang X Y, Yang K Q, Chen R, Wu D J. 2022. MicroRNA and degradome profiling uncover defense response of Fraxinus velutina Torr. to salt stress. Front Plant Sci,13:847853.
[17]	Liu Y G, Chen Y. 2007. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques, 43 (5):649-656.
[18]	Livak K J, Schmittgen T D. 2002. Analysis of relative gene expression data using real-time quantitative PCR. Methods, 25 (4):402-408.
[19]	Luo Xiuyun, Li Yuanyuan, Zhou Geng, Tian Yun, Lu Xiangyang. 2015. The regulatory role of TGA transcription factors in plant oxidative stress response. Chemical and Bioengineering, 32 (3):1-5. (in Chinese)
	罗秀云, 李园园, 周赓, 田云, 卢向阳. 2015. TGA转录因子在植物氧化胁迫应答中的调控作用. 化学与生物工程, 32 (3):1-5.
[20]	Qin Ken, Zhang Bo, Wang Danqing, Gao Yan, Dai Guoli, Huang Ting, He Xinru, Zhou Xuan, Duan Linyuan, Jiao Enning. 2024a. A new cultivar of Lycium barbarum‘Ningnongqi 15’. Acta Horticulturae Sinica, 51 (S1):235-236. (in Chinese)
	秦垦, 张波, 王丹青, 高燕, 戴国礼, 黄婷, 何昕孺, 周旋, 段淋渊, 焦恩宁. 2024a. 宁夏枸杞新品种‘宁农杞15号’. 园艺学报, 51 (S1):235-236.
[21]	Qin Ken, Zhang Bo, Wang Danqing, Gao Yan, Dai Guoli, Huang Ting, He Xinru, Zhou Xuan, Duan Linyuan, Jiao Enning. 2024b. A new cultivar of Lycium barbarum‘Keqi 6082’. Acta Horticulturae Sinica, 51 (S1):237-238. (in Chinese)
	秦垦, 张波, 王丹青, 高燕, 戴国礼, 黄婷, 何昕孺, 周旋, 段淋渊, 焦恩宁. 2024b. 宁夏枸杞新品种‘科杞6082’. 园艺学报, 51 (S1):237-238.
[22]	Shearer H L, Wang L P, DeLong C, Despres C, Fobert P R. 2009. NPR1 enhances the DNA binding activity of the Arabidopsis bZIP transcription factor TGA7. Botany,87:561-570.
[23]	Shi Guangzhen, Wang Zhaoye, Sun Qi, Zhu Xinxia. 2022. Cloning and activity analysis of SikCDPK1 promoter from Saussurea involucrata. Biotechnology Bulletin, 38 (9):191-197. (in Chinese) doi: 10.13560/j.cnki.biotech.bull.1985.2022-0570
	史光珍, 王兆晔, 孙琦, 朱新霞. 2022. 雪莲SikCDPK1启动子的克隆和活性分析. 生物技术通报, 38 (9):191-197. doi: 10.13560/j.cnki.biotech.bull.1985.2022-0570
[24]	Srivastava D, Verma G, Chauhan A S, Pande V, Chakrabarty D. 2019. Rice(Oryza sativa L.) tau class glutathione S-transferase(OsGSTU30)overexpression in Arabidopsis thaliana modulates a regulatory network leading to heavy metal and drought stress tolerance. Metallomics:Integrated Biometal Science, 11 (2):375-389.
[25]	Stotz H U, Findling S, Nukarinen E, Weckwerth W, Mueller M J, Berger S. 2014. A tandem affinity purification tag of TGA 2 for isolation of interacting proteins in Arabidopsis thaliana. Plant Signaling & Amp;Behavior, 9 (10):e972794.
[26]	Tian Luchen. 2020. Interaction analysis of key enzyme gene promoters for SmTGA2and salvianolic acid B synthesis[M. D. Dissertation]. Yangling: Northwest A & F University. (in Chinese)
	田露辰. 2020. SmTGA2与丹酚酸B合成关键酶基因启动子的互作分析[硕士论文]. 杨凌: 西北农林科技大学.
[27]	Wang Yanhong, Xi Keyong, Tian Ye, Liu Deqi, Zhou Kegui, Yin Junliang, Liu Yiqing, Zhu Yongxing. 2024. Identification and expression pattern analysis of GST in Zingiber officinale. Acta Horticulturae Sinica, 51 (8):1803-1822. (in Chinese)
	王艳红, 席克勇, 田野, 刘德麒, 周克贵, 尹军良, 刘奕清, 朱永兴. 2024. 姜GST基因家族成员鉴定与表达分析. 园艺学报, 51 (8):1803-1822.
[28]	Xiang C, Miao Z, Lam E. 1997. DNA-binding properties,genomic organization and expression pattern of TGA6,a new member of the TGA family of bZIP transcription factors in Arabidopsis thaliana. Plant Molecular Biology, 34 (3):403-415. doi: 10.1023/a:1005873500238 pmid: 9225852
[29]	Zhang J Y, Qu S C, Qiao Y S, Zhang Z, Guo Z R. 2014. Overexpression of the malus hupehensis MhTGA2gene,a novel bZIP transcription factor for increased tolerance to salt and osmotic stress in transgenic tobacco. International Journal of Plant Sciences,59:441-453.
[30]	Zhu Xi. 2021. Isolation and functional study of StMAPKs genes related to stress in potatoes[M. D. Dissertation]. Lanzhou: Gansu Agricultural University. (in Chinese)
	朱熙. 2021. 马铃薯中与胁迫相关StMAPKs基因的分离及功能研究[硕士论文]. 兰州: 甘肃农业大学.