盐穗木转录因子HcSCL13基因的时空表达和胁迫响应特性

doi:10.6048/j.issn.1001-4330.2022.04.025

新疆农业科学 ›› 2022, Vol. 59 ›› Issue (4): 990-1000.DOI: 10.6048/j.issn.1001-4330.2022.04.025

• 基因工程·草业·农产品分析检测·畜牧兽医 • 上一篇下一篇

盐穗木转录因子HcSCL13基因的时空表达和胁迫响应特性

地力努尔·艾力木江(), 冯肖莉, 王艳()

新疆大学生命科学与技术学院生物资源基因工程重点实验室,乌鲁木齐 830046

收稿日期:2021-03-24 出版日期:2022-04-20 发布日期:2022-04-24
通信作者: 王艳
作者简介:地力努尔·艾力木江(1997- ),女,新疆人,硕士研究生,研究方向为植物学,(E-mail) 1455636632@qq.com
基金资助:
新疆维吾尔自治区优秀科学人才项目“盐穗木金属硫蛋白基因应答镉胁迫的转录调控机制”(2019Q013);国家地区自然科学基金项目“具有RNase活性的盐穗木病程相关蛋白HcPR10非生物胁迫抗性机理的研究”(31860061)

Study of the Promoter properties of the HcSCL13 Gene for the Transcription Factor of Halostachys caspica (Bieb.) C. A. Mey.

Delinur Alimujiang(), FENG Xiaoli, WANG Yan()

The Key Laboratory of Genetic Engineering of Biological Resources, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China

Received:2021-03-24 Online:2022-04-20 Published:2022-04-24
Correspondence author: WANG Yan
Supported by:
Project of Outstanding Scientific Talent Program of Xinjiang Uygur Autonomous Region "Transcriptional Regulation of the Gene in Response to Cadmium Stress"(2019Q013);Project of the National Natural Science Foundation of China "Mechanism of Abiotic Stress Resistance of HcPR10 with RNase activity"(31860061)

摘要/Abstract

摘要：

【目的】研究盐生植物盐穗木HcSCL13基因的时空表达和胁迫响应特性。【方法】将该基因全长启动子(2 230 bp)及截短序列与GUS报告基因融合,检测遗传转化后植物的GUS组织化学染色及酶活力。【结果】在稳定整合HcSCL13启动子的转基因拟南芥中,从种子萌发到种苗形成的过程中,地上和地下部位均表现出较强的启动子活性;随着植物的生长发育,启动子活性表现出一定的组织特异性;HcSCL13基因启动子对光、盐及机械损伤等胁迫积极响应。启动子上游-1 124-1 450 bp是影响其活性的区域,而上游-1 730-2 230 bp为盐响应区域。【结论】盐穗木HcSCL13基因的表达具有时空特异性及对光、盐和机械损伤等因素的响应特性。

关键词: 盐穗木; HcSCL13启动子; 非生物胁迫; GUS组织化学染色; GUS酶活

Abstract:

【Objective】 To explore the spatiotemporal expression and stress response properties of HcSCL13 genes in Halophytes caspica (Bieb.) C. A. Mey.【Method】 The full-length promoter (2,230 bp) and truncated sequence of the gene were fused to the GUS reporter to detect the GUS histochemical staining and enzyme vitality of genetically transformed plants.【Results】 Both aerial and underground parts of PHcSCL13.GUS homozyous Arabidopsis showed strong GUS staining at the stage of seed germination and seedling. With the development of transgenic PHcSCL13.GUS plant, the promoter activity displayed spatial and temporal patterns. Meanwhile, the promoter of HcSCL13 gene was responsive to light, salt and mechanical damage by GUS staining and activity analysis. After transient transformation of different length promoter fragments into the tobacco, the results of GUS activity suggested that the region for -1,124-1,450 bp upstream of the HcSCL13 promoter was responsible for its activity, while -1,730-2,230 bp upstream of the promoter was the salt response region.【Conclusion】 The above finding shows that HcSCL13 genes in Halophytes caspica (Bieb.) C. A. Mey. express space-time specificity and response properties to light, salt and mechanical damage, which lays the foundation in molecular mechanism.

Key words: Halostachys caspica; HcSCL13 promoter; abious stress; GUS histochemical stain; GUS enzyme activity

中图分类号:

Q94-3

地力努尔·艾力木江, 冯肖莉, 王艳. 盐穗木转录因子HcSCL13基因的时空表达和胁迫响应特性[J]. 新疆农业科学, 2022, 59(4): 990-1000.

Delinur Alimujiang, FENG Xiaoli, WANG Yan. Study of the Promoter properties of the HcSCL13 Gene for the Transcription Factor of Halostachys caspica (Bieb.) C. A. Mey.[J]. Xinjiang Agricultural Sciences, 2022, 59(4): 990-1000.

图/表 10

图1 HcSCL13 截短启动子植物表达载体构建示意

Fig.1 Schematic diagram of PHcSCL13GUS promoter fragment vector construction with different length

表1 HcSCL13 截短启动子的引物设计

Table 1 Primer design of HcSCL13 truncated promoter

名称 Name	序列(5'- 3') Sequence
P_HcSCL13-SP1P1	GATTACGCCAAGCTTAATTAGATCT GTTTGCAACC
P_HcSCL13-SP2 P1	GATTACGCCAAGCTTTCCCAATAGC CTCATATCTC
P_HcSCL13-SP3 P1	GATTACGCCAAGCTTCAA AACCAGTCTA ATAGCCG
P_HcSCL13-SP4 P1	GATTACGCCAAGCTTTGAATTGACC AGCTTGATTG
P_HcSCL13-SP5 P1	GATTACGCCAAGCTTTTCAACTATT TCCGTACAATTT
P_HcSCL13P2	CCGGGGATCCTCTAGACCGAGAATAG CACAATCTGA

图2 HcSCL13 启动子的时空表达模式注:A:种苗阶段,B:成苗阶段,C:生殖阶段的不同组织部位

Fig. 2 Spatial-temporal expression patterns of HcSCL13 promoter Note:A:Seedling stage, B : Adult seedling, C : Different tissues at the reproductive stage

图3 HcSCL13 启动子对光的响应注:A:下胚轴,B:子叶,C:根

Fig.3 Response to light for HcSCL13 gene promoter Note:A:Hypocotyls, B : Cotyledons, C : Roots

图4 HcSCL13 启动子对机械损伤的响应

Fig. 4 Response to mechanical damage for HcSCL13 gene promoter

图5 HcSCL13 启动子对低温和干旱胁迫的响应注:A 低温胁迫下的GUS组织化学染色;B干旱胁迫下的GUS组织化学染色;C 低温胁迫下的GUS酶活;D干旱胁迫下的GUS酶活

Fig. 5 Response to low temperature and drought stress for HcSCL13 gene promoter Note: A:GUS staining under low temperature, B: GUS staining under drought condition, C: Activity analysis for GUS, D Activity analysis for GUSunder drought condition

图6 HcSCL13启动子下盐胁迫的响应变化注:A:GUS组织化学染色,B:GUS酶活;数值为3次平行实验的平均值±标准差,不同小写字母代表组间差异(P< 0.05),不同大写字母代表组内差异(P< 0.05)

Fig. 6 Response to salt stress for HcSCL13 gene promoter Note: A:GUS staining,B:Activity analysis for GUS; The value is the mean ± standard deviation of three parallel experiments. Different lowercase letters represent the difference between groups (P< 0.05), and different uppercase letters represent the difference within groups (P< 0.05)

图7 HcSCL13截短启动子植物表达载体的酶切鉴定注:M:5 000 bp Marker,1-5:HindⅢ和XbaⅠ分别双酶切重组质粒pBI121-PHcSCL13SP1, pBI121-PHcSCL13SP2, pBI121- PHcSCL13 SP3, pBI121-PHcSCL13SP4和pBI121-PHcSCL13SP5

Fig. 7 Identification of plant expression vector of truncated HcSCL13 promoter Note: M:5,000 bp marker; 1-5: Recombinant plasmids of pBI121-PHcSCL13SP1,pBI121-PHcSCL13SP2,pBI121 -PHcS CL13SP3, pBI121-PHcSCL13SP4 and pBI121-PHcSCL13SP5 were digested by Hind Ⅲ and Xba I, respectively

图8 HcSCL13截短启动子在瞬转染烟草中的活性注:A:GUS组织化学染色,B:酶活分析;数值为4次平行实验的平均值±标准差,不同大写字母代表组间差异(P< 0.05)

Fig.8 Analysis oftruncated HcSCL13 promoterin transiently transfected tobacco Note: A:GUS staining, B:Activity analysis for GUS;The value is the mean ± standard deviation of 4 parallel experiments, and different capital letters represent the difference between groups (P< 0.05)

图9 盐胁迫下截短启动子活性注:数值为4次平行实验的平均值±标准差,不同小写字母代表组间差异(P< 0.05),不同大写字母代表组内差异(P< 0.05)

Fig. 9 Analysis of truncated promoter activity under salt stress Note: The value is the mean ± standard deviation of four parallel experiments. Different lowercase letters represent the difference between groups (P< 0.05), and different uppercase letters represent the difference within groups (P< 0.05)

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盐穗木转录因子HcSCL13基因的时空表达和胁迫响应特性

Study of the Promoter properties of the HcSCL13 Gene for the Transcription Factor of Halostachys caspica (Bieb.) C. A. Mey.

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