日本落叶松LkNiR启动子的克隆及表达特性分析

doi:10.13560/j.cnki.biotech.bull.1985.2026-0173

摘要/Abstract

摘要：

目的亚硝酸盐还原酶（nitrite reductase, NiR）是植物氮同化关键酶，可催化亚硝酸盐还原为铵态氮，保障氮同化高效进行。研究日本落叶松（Larix kaempferi）LkNiR基因启动子的表达特性及亚硝酸盐胁迫响应规律，为选育氮高效利用型林木品种提供理论支撑。方法从日本落叶松基因组DNA中克隆LkNiR启动子序列，利用PlantCARE与PLACE数据库预测顺式作用元件；构建LkNiR-GFP报告载体，通过农杆菌介导的遗传转化获得转基因烟草（Nicotiana tabacum）及日本落叶松毛状根，结合PCR鉴定、荧光观察与RT-qPCR技术检测启动子活性；以100 mmol/L亚硝酸钠处理日本落叶松幼苗，采用RT-qPCR技术分析LkNiR基因的时序表达模式。结果 LkNiR基因启动子（登录号：PV700087）全长1 941 bp，含TATA-box、CAAT-box等核心元件及多种激素与胁迫响应元件，LkNiR启动子含9个氮同化响应顺式元件。该启动子在转基因烟草中呈现组织偏好性表达，在根中活性较CaMV35S启动子高92.1%，在落叶松毛状根中活性较CaMV35S启动子高103.3%。亚硝酸盐胁迫下，LkNiR基因表达呈时空分化，根中早期快速响应，茎中响应平缓，叶中诱导最强且持续。结论 LkNiR启动子具有明显的根部偏好表达特性，其活性受亚硝酸盐信号诱导且具有时空分化特征，在日本落叶松氮同化与逆境响应过程中发挥关键调控作用。

关键词: 亚硝酸盐还原酶, 启动子, 日本落叶松, 毛状根, 氮同化, 亚硝酸盐胁迫, 顺式作用元件, 表达分析

Abstract:

Objective Nitrite reductase (NiR) is a key plant nitrogen assimilation enzyme that catalyzes nitrite reduction to ammonium nitrogen and ensures efficient nitrogen assimilation. This study investigated Larix kaempferiLkNiR promoter expression features and nitrite stress response mechanisms and provided theoretical support for breeding forest trees with high nitrogen use efficiency. Method The LkNiR promoter sequence was cloned from L. kaempferi genomic DNA, and screened for cis-acting elements using PlantCARE and PLACE databases. The LkNiR-GFP reporter vector was constructed, and transgenic tobacco as well as L. kaempferi hairy roots were obtained via Agrobacterium-mediated genetic transformation. Promoter activity was detected by PCR identification, fluorescence observation, and RT-qPCR assays. L. kaempferi seedlings were treated with 100 mmol/L sodium nitrite, and the LkNiR gene temporal expression pattern was analyzed via RT-qPCR. Result The full-length LkNiR gene promoter (Accession No.: PV700087) was 1 941 bp, containing core elements (e.g., TATA-box, CAAT-box), multiple hormone-responsive elements, and stress-responsive elements. Notably, the LkNiR promoter harbored nine cis-acting elements involved in nitrogen assimilation. The promoter exhibited tissue-preferential expression in transgenic tobacco, with 92.1% higher activity in roots than the CaMV35S promoter, and 103.3% higher activity in L. kaempferi hairy roots than the CaMV35S promoter. Under nitrite stress, LkNiR expression showed spatiotemporal divergence: roots displayed a rapid early response, stems exhibited a moderate response, and needles had the strongest and most sustained induction. Conclusion The LkNiR promoter exhibits significant root-preferential expression characteristics, its activity is induced by nitrite signaling and shows spatiotemporal differentiation features, and it plays a key regulatory role in nitrogen assimilation and stress response in L. kaempferi.

Key words: nitrite reductase, promoter, Larix kaempferi, hairy root, nitrogen assimilation, nitrite stress, cis-acting elements, expression analysis

武郁洁, 王宇倩, 刘翠双, 郁羽宵, 耿静泽, 盖颖. 日本落叶松LkNiR启动子的克隆及表达特性分析[J]. 生物技术通报, 2026, 42(11): 222-229.

WU Yu-jie, WANG Yu-qian, LIU Cui-shuang, YU Yu-xiao, GENG Jing-ze, GAI Ying. Cloning and Expression Characteristics Analysis of LkNiR Promoter from Larix kaempferi[J]. Biotechnology Bulletin, 2026, 42(11): 222-229.

图/表 5

图1 LkNiR基因启动子顺式作用元件分布A：LkNiR基因启动子根特异性顺式作用元件分布；B：LkNiR基因启动子核心顺式作用元件数量

Fig. 1 Distribution of cis-acting elements in the LkNiR gene promoterA: Distribution of root-specific cis-acting elements in the LkNiR gene promoter. B: Number of core cis-acting elements in the LkNiR gene promoter

表1 不同物种NiR基因启动子中氮同化响应元件数量

Table 1 Number of nitrogen assimilation-responsive elements in NiR gene promoters from different species

序号 No.	物种 Species	元件数量 No. of elements
1	落叶松 Larix kaempferi	9
2	油松 Pinus tabuliformis	6
3	火炬松 Pinus taeda	10
4	挪威云杉 Picea abies	8
5	白云杉 Picea glauca	3
6	杨树 Populus trichocarpa	14
7	拟南芥 Arabidopsis thaliana	5
8	大豆 Glycine max	16
9	水稻 Oryza sativa	10
10	玉米 Zea mays	9

图2 转基因烟草中组织表达分析A：重组载体构建示意图；B：转基因烟草植株4个阶段（a：预培养，b：再生培养，c：生根培养，d：炼苗阶段）；C：转基因烟草PCR鉴定结果（+：阳性对照，WT：野生型对照，ddH2O：阴性对照，M：2 000 bp DNA marker）；D：转基因烟草根、茎、叶组织中GFP基因相对表达量。* P<0.05，** P<0.01，*** P<0.001，**** P<0.000 1（t-test）。下同

Fig. 2 Tissue expression analysis in transgenic tobaccoA: Schematic diagram of recombinant vector construction. B: Four stages of transgenic tobacco plants (a: pre-culture, b: regeneration culture, c: rooting culture, d: hardening stage). C: PCR identification result of transgenic tobacco (+: positive control; WT: wild-type control; ddH2O: negative control; M: 2 000 bp DNA marker). D: Relative expression level of GFP gene in roots, stems and leaves of transgenic tobacco.* P<0.05, ** P<0.01, *** P<0.001, **** P<0.000 1 (t-test). The same below

图3 转基因落叶松毛状根中LkNiR启动子活性分析A：落叶松毛状根表型观察，比例尺为5 mm；B：落叶松毛状根GFP荧光观察，比例尺为3 mm；C：转基因毛状根GFP荧光强度定量分析；D：转基因毛状根PCR鉴定结果；E：转基因毛状根GFP基因相对表达量

Fig. 3 Activity analysis of the LkNiR promoter in transgenic L. kaempferi hairy rootsA: Phenotypic observation of L. kaempferi hairy roots, scale bar=5 mm. B: GFP fluorescence observation of L. kaempferi hairy roots, scale bar=3 mm. C: Quantitative analysis of GFP fluorescence intensity in transgenic hairy roots. D: PCR identification result of transgenic hairy roots. E: Relative expression level of GFP gene in transgenic hairy roots

图4 LkNiR启动子响应亚硝酸盐胁迫的时空表达模式分析A：落叶松幼苗亚硝酸盐胁迫处理示意图；B：亚硝酸盐胁迫下LkNiR基因的时空表达模式分析

Fig. 4 Spatiotemporal expression pattern analysis of the LkNiR promoter in response to nitrite stressA: Schematic diagram of nitrite stress treatment on L. kaempferi seedlings. B: Spatiotemporal expression pattern analysis of the LkNiR gene under nitrite stress

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