Expression and Functional Analysis of OsPT1 Gene in Rice Under Cadmium Stress

doi:10.13560/j.cnki.biotech.bull.1985.2022-0489

Abstract

Abstract:

OsPT1-encoded rice phosphate transporter protein plays important regulatory roles in growth and abiotic stress response in rice. Previous data showed that the expression of OsPT1 gene was induced by cadmium（Cd）stress, however, the function and mechanism of OsPT1 under Cd stress remains unclear. Therefore, the aim of this study is to clarify the function of OsPT1 under Cd stress and lay a foundation for the breeding of low-Cd rice cultivars. By using bioinformatics methods, the sequence feature, structure and function of OsPT1 gene were analyzed or predicted. The expression profiles of OsPT1 in different tissues and at different time points under Cd stress were examined by quantitative real-time PCR（RT-qPCR）as well. Simultaneously, PCR was used to clone the encoding sequence of OsPT1, and the recombinant plasmid pGADT7-OsPT1 was constructed and transferred into the the Cd-sensitive yeast strain ∆ycf BY4741 for determining the effect of OsPT1 on the tolerance of yeast to Cd. The results showed that OsPT1 is a protein of 527 amino acids with the molecular weight of 57.46 kD, which was encoded by a CDS of 1 584 bp. In rice genome, the cis-acting elements such as environmental factors like light and anaerobic, as well as hormones including methyl jasmonate were found in the promoter region of this gene. The results of phylogenetic analysis showed that OsPT1 was most closely related with sorghum SbPT1. The results of Cd-responsive gene expression analysis showed that the expression levels of OsPT1 in rice shoots increased to 1.31, 1.34 and 2.46 folds comparing to the untreated control under 100 μmol/L Cd treatment for 1, 6 and 12 h, respectively. The expressions in the roots increased to 1.28 and 1.14 folds comparing the untreated control under 100 μmol/L Cd treatment for 1 or 6 h, Cd treatment, however, it was down-regulated to 0.62 folds after 12 h Cd treatment. The result of Cd tolerance examination showed that the yeast transfered with OsPT1 demonstrated the decreased tolerance to Cd when treated with 25 μmol/L Cd when compared with the control（0 Cd）. All together, OsPT1 may play a certain role in the respondses of rice to Cd stress.

Key words: rice, OsPT1, cadmium stress, yeast, gene expression

JIANG Nan, SHI Yang, ZHAO Zhi-hui, LI Bin, ZHAO Yi-hui, YANG Jun-biao, YAN Jia-ming, JIN Yu-fan, CHEN Ji, HUANG Jin. Expression and Functional Analysis of OsPT1 Gene in Rice Under Cadmium Stress[J]. Biotechnology Bulletin, 2023, 39(1): 166-174.

Figures/Tables 11

Table 1 Quantitative real-time PCR primer sequence

引物名称Primer name	引物序列Primer sequence（5'-3'）
OsPT1-qF	CTACTGGCGGATGAAGATG
OsPT1-qR	GACCTTGCTGAAGATGTCC
Ubiquitin-qF	ATCACGCTGGAGGTGGAGT
Ubiquitin-qR	AGGCCTTCTGGTTGTAGACG

Fig. 1 PCR amplification of OsPT1 gene and endonuclease digestion of the recombinant vector pGADT7-OsPT1 A: The PCR amplification of OsPT1 gene. B: Recombinant vector after digestion

Table 2 Physicochemical properties of OsPT1 protein

蛋白质Protein	氨基酸数Number of amino acid/aa	分子式 Formula	分子量Molecular weight/kD	等电点pI	不稳定指数Instability index	平均疏水性Hydropathy index	正/负电残基数Acid-base amino acid	脂溶指数Aliphatic index
OsPT1	527	C₂₆₅₆H₄₀₂₁N₆₆₇O₇₁₀S₂₅	57.46	8.57	31.09	0.361	38/34	90.09

Table 3 Amino acid composition of OsPT1 protein

氨基酸Amino acid	数量Quantity	比例Proportion/%	氨基酸Amino acids	数量Quantity	比例Proportion/%
Ala（A）	67	12.7	Arg（R）	20	3.8
Asn（N）	11	2.1	Asp（D）	18	3.4
Cys（C）	6	1.1	Gln（Q）	16	3.0
Glu（E）	16	3.0	Gly（G）	52	9.9
His（H）	12	2.3	Ile（I）	35	6.6
Leu（L）	48	9.1	Lys（K）	18	3.4
Met（M）	19	3.6	Phe（F）	40	7.6
Pro（P）	22	4.2	Ser（S）	30	5.7
Thr（T）	35	6.6	Trp（W）	11	2.1
Tyr（Y）	22	4.2	Val（V）	29	5.5

Fig. 2 Hydrophobicity analysis of OsPT1 protein Regions with values above 0 are hydrophobic in character. Regions with values less than 0 are hydrophilic in character

Fig. 3 Secondary and tertiary structure prediction of OsPT1 protein A: The secondary structure of OsPT1, where blue indicates α-helix, red indicates extended chain, green indicates β-sheet, purple indicates irregular curl, and the abscissa indicates the position of each residue in the amino acid sequence of OsPT1. B: The tertiary structure of OsPT1, where purple indicates α-helix, blue represents irregular curl, and red represents Ala

Fig. 4 potential phosphorylation sites analysis of OsPT1 protein Red line indicates potential phosphorylated serine residues; green line indicates potential phosphorylated threonine residues; blue line indicates potential phosphorylated tyrosine residues; pink horizontal line indicates threshold for modification potential; the abscissa indicates the position of each residue in the amino acid sequence of OsPT1, and the ordinate indicates predicted phosphorylation potential score

Fig. 5 Phylogenetic analysis of PT1 proteins TdPT1: Triticum dicoccoides, XP_037426503.1; TaPT1: Triticum aestivum, AIZ11178.1; HvPT1: Hordeum vulgare, AAN37900.1; ZmPT1: Zea mays, NP_001183901.1; OsPT1: Oryza sativa, XP_015631295.1; SbPT1: Sorghum bicolor, XP_002467158.1; VvPT1: Vitis vinifera, XP_002281264.1; AtPT1: Arabidopsis thaliana, NP_001319749.1; SlPT1: Solanum lycopersicum, CAA76075.1; GmPT1: Glycine max, NP_001240239.1; PtPT1: Populus trichocarpa, XP_002300153.3

Fig. 6 Cis-acting elements analysis of OsPT1 gene Blocks of different colors indicate light responsiveness elements（green and red）, anaerobic responsiveness elements（yellow）, MeJA responsiveness elements（pink）, Sal Icylic acid responsiveness elements（blue）and disease, stress responsiveness elements（grey）, respectively

Fig. 7 Expression analysis of OsPT1 under Cd stress treatment A: Shoot. B: Root. * indicate significant difference from 0 h at P=0.01

Fig. 8 Effects of OsPT1 on the sensibility of Cd in yeast

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