Cloning and Low-phosphorus Tolerance Analysis of SiSPX9 Gene in Foxtail Millet

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

Abstract

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

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.

Figures/Tables 9

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

Fig. 1 CDS amplified results of SiSPX9 gene

Fig. 2 Structure analysis of SiSPX9 gene

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

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

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

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

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)

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

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