谷子SiSPX9基因的克隆及耐低磷分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0404

摘要/Abstract

摘要：

目的 SPX基因家族在植物磷的信号感知、吸收和运输中发挥着重要的功能。探究SiSPX9在响应低磷胁迫过程中的功能，为解析谷子耐低磷机制提供理论依据。方法从谷子根中克隆SiSPX9，并对其进行生物信息学分析。利用实时荧光定量PCR（real-time quantitative PCR, RT-qPCR）分析SiSPX9在不同组织中的表达及不同低磷胁迫时长处理的表达模式。通过农杆菌介导的遗传转化法在拟南芥中异源表达SiSPX9，并对转基因植株进行低磷处理下发芽率、根长、根系表面积的检测。结果 SiSPX9基因CDS全长759 bp，编码253个氨基酸，具有SPX家族特征保守结构域；蛋白质结构预测结果显示，其编码的蛋白质二级结构由68.38% α-螺旋、1.19% β-折叠、30.43%的延伸链构成，三级结构与二级结构相统一；通过同源进化分析发现，谷子SiSPX9与高粱和玉米相似性最高，在进化上属于同一分支。RT-qPCR分析结果表明，SiSPX9基因在各个组织的表达特征存在差异性，在根中表达量最高，在叶中表达量最低；在不同低磷胁迫时长处理下，SiSPX9随着低磷胁迫时间的增加表达量呈先上升后下降的趋势，在低磷胁迫12 h时其表达量达到最高值，是适磷处理的15.3倍。功能验证分析结果表明，在低磷胁迫下，野生型（Col-0）和SiSPX9过表达拟南芥株系的萌发率、根长和根系表面积均受到一定程度抑制，但SiSPX9过表达株系明显优于Col-0。结论 SiSPX9过表达显著增强拟南芥在低磷胁迫下的萌发率和根系发育，表明其通过调控磷信号通路提升耐低磷胁迫的能力。

关键词: 谷子, SiSPX9, 低磷胁迫, 生物信息学, 基因克隆, 转基因拟南芥

Abstract:

Objective SPX gene family plays an important role in signal perception, absorption and transport of phosphorus in plants. To explore the function of SiSPX9 in response to low-phosphorus stress would provide a theoretical basis for analyzing the mechanism of low-phosphorus tolerance in foxtail millet (Setaria italica). Method SiSPX9 was cloned from foxtail millet root and analyzed by bioinformatics. Real-time quantitative PCR (RT-qPCR) was used to analyze the expressions of SiSPX9 in different tissues and the expression patterns of different low-phosphorus stress durations. SiSPX9 was heterologously expressed in Arabidopsis thaliana by Agrobacterium-mediated method, and the germination rate, root length and root surface area of transgenic plants were detected under low-phosphorus treatment. Result The CDS of SiSPX9 gene was 759 bp in length, encoding 253 amino acids, with the conserved domain of SPX family. The results of protein structure prediction showed that the secondary structure of the encoded protein was composed of 68.38% α-helix, 1.19% β-sheet and 30.43% extended chain, and the tertiary structure was consistent with the secondary structure. Through homologous evolution analysis, it was found that millet SiSPX9 had the highest similarity with sorghum and corn, and belonged to the same branch in evolution. The results of RT-qPCR analysis showed that the expression characteristics of SiSPX9 gene in different tissues were different, with the highest expression in the roots and the lowest expression in the leaves. Under different low-phosphorus stress duration treatments, the expression of SiSPX9 increased first and then decreased with the increase of low-phosphorus stress time. The expression reached the highest value at 12 h of low-phosphorus stress, which was 15.3 times that of suitable phosphorus treatment. Functional analysis showed that under low-phosphorus stress, the germination rate, root length and root surface area of wild type (Col-0) and SiSPX9 overexpression Arabidopsis lines were inhibited to a certain extent, but SiSPX9-overexpressing lines were significantly better than Col-0. Conclusion The overexpression of SiSPX9 markedly augments the germination rate and root development of Arabidopsis in conditions of low-phosphorus stress. This suggests that SiSPX9 enhances the tolerance to phosphorus deficiency by modulating the phosphorus signaling pathway.

Key words: Setaria italica L., SiSPX9, low-phosphorus stress, bioinformatics, gene cloning, transgenic Arabidopsis

郭浩杰, 王成, 杨馥熔, 杜冰, 孟超敏. 谷子SiSPX9基因的克隆及耐低磷分析[J]. 生物技术通报, 2025, 41(12): 114-123.

GUO Hao-jie, WANG Cheng, YANG Fu-rong, DU Bing, MENG Chao-min. Cloning and Low-phosphorus Tolerance Analysis of SiSPX9 Gene in Foxtail Millet[J]. Biotechnology Bulletin, 2025, 41(12): 114-123.

图/表 9

表1 本试验用到的引物

Table 1 Primers used in this experiment

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	引物用途 Primer usage
SiSPX9-F	ATGAAGTTCGGCAAGCGG	SiSPX9 CDS扩增
SiSPX9-R	TCACTGCGTGGGGATGAG
SiSPX9-qF	TTCGTGGACTACTGCCTGCTC	SiSPX9 RT-qPCR 分析
SiSPX9-qR	GCTTGTCTGCTGCTGCTGTG
Actin-qF	GGCAAACAGGGAGAAGATGA	谷子内参基因 RT-qPCR 分析
Actin-qR	GAGGTTGTCGGTAAGGTCACG
SiSPX9-pBI121-F	CGCGGATCCATGAAGTTCGGCAAGCGG	pBI121-SiSPX9 载体构建
SiSPX9-pBI121-R	CGCGAGCTCTCACTGCGTGGGGATGAG
35S-R	GAGCTCCTTTTCCAGCGGACC	转基因拟南芥分子鉴定

图1 SiSPX9基因CDS扩增结果

Fig. 1 CDS amplified results of SiSPX9 gene

图2 SiSPX9基因结构分析

Fig. 2 Structure analysis of SiSPX9 gene

图3 SiSPX9蛋白的生物信息学分析A：二级结构分析；B：三级结构预测；C：蛋白质亲/疏水性预测；D：蛋白跨膜结构预测；E：磷酸化位点预测

Fig. 3 Bioinformatics analysis ofSiSPX9proteinA: Prediction of secondary structure. B: Tertiary structure of SiSPX9 protein. C: Protein hydrophilicity/hydrophobicity prediction. D: Protein transmembrane structure prediction. E: Phosphorylation site prediction

图4 SiSPX9蛋白序列比对和进化树构建A：不同植物SiSPX9蛋白序列比对，图中红色方框表示SPX蛋白的结构域；B：SiSPX9基因系统进化树

Fig. 4 SiSPX9 protein sequence alignment and phylogenetic tree constructionA: Alignment of SiSPX9protein sequences from different plants. The red box in the diagram contains the domains ofSPX protein. B: Phylogenetic tree analysis of SiSPX9 gene

图5 谷子不同组织及低磷胁迫下SiSPX9的表达分析A：SiSPX9在谷子不同组织中的表达；B：SiSPX9在不同低磷胁迫时间下根中的表达情况，NP：正常磷处理（1 mmol/L KH₂PO₄）；LP：低磷处理（1 μmol/L KH₂PO₄）；*P<0.05，**P<0.01，ns表示差异不显著（P>0.05），下同

Fig. 5 Tissue-specific and low-phosphorus stress-induced expression of SiSPX9 in foxtail milletA: Expressions of SiSPX9 in different tissues of foxtail millet (Setaria italica). B: Expression patterns of SiSPX9 in roots under low-phosphorus stress at different time points. NP: Normal phosphorus treatment (1 mmol/L KH₂PO₄); LP: Low phosphorus treatment (1 μmol/L KH₂PO₄). *P<0.05, **P< 0.01, ns indicates no significant difference (P>0.05). The same below

图6 转基因SiSPX9拟南芥鉴定结果及表达量分析A：转基因拟南芥PCR检测电泳图（M：DL 2000 DNA marker；WT：野生型拟南芥；OE#1-OE1#3：转pBI121-SiSPX9拟南芥）；B：转基因拟南芥qPCR检测，OE#1-OE#3代表过表达阳性植株；显著性差异通过Duncan新复极差法确定；不同字母表示差异显著（P<0.05）；误差线表示3个独立生物学实验获得的标准差；以Actin为内参对目的基因相对表达量进行标准化计算。下同

Fig. 6 Identificatied results of transgenic SiSPX9A. thaliana andexpression analysisA: PCR detection of transgenic Arabidopsis lines (M: DL2000 DNA marker; WT: wild-type Arabidopsis; OE#1-OE#3: Arabidopsis transformed with pBI121-SiSPX9). B: qPCR analysis of transgenic Arabidopsis lines. OE#1-OE#3 indicates overexpression-positive lines. Significant differences were determined by Duncan’s new multiple range test (P<0.05). Different lowercase letters indicate statistically significant differences. Error bars indicate the standard deviation (SD) of three independent biological replicates. Relative expression of the target gene is normalized to Actin. The same below

图7 SiSPX9转基因株系萌发率对每次测量使用3个生物学重复进行数据量化，每个重复50粒种子；每个数据点表示3次独立试验的平均值（±标准差）

Fig. 7 Germination rates of SiSPX9 transgenic linesThree biological replicates were used for data quantification for each measurement, with 50 seeds per replicate. Each data point indicates the mean of three independent experiments (± standard deviation)

图8 低磷胁迫下SiSPX9转基因拟南芥的表型鉴定（比例尺：15 mm）

Fig. 8 Phenotypic identification of SiSPX9 transgenic Arabidopsis under low phosphorus stress(Scale bar:15 mm)

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