镉胁迫下水稻OsPT1的表达及功能分析

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

摘要/Abstract

摘要：

OsPT1编码的水稻磷酸盐（Pi）转运蛋白在水稻生长发育、非生物胁迫应答等方面发挥重要的调控作用。前期研究表明OsPT1为镉（Cd）响应基因，但其在Cd胁迫下的功能及作用机制仍然未知。阐明OsPT1在Cd胁迫下的作用，并为低Cd水稻品种的选育奠定基础。通过生物信息学方法对该基因的序列特征、结构和功能进行分析和预测，利用实时荧光定量PCR（RT-qPCR）方法检测Cd胁迫下水稻不同组织、不同时间点OsPT1的相对表达量。此外，利用PCR的方法克隆OsPT1的编码序列，构建pGADT7-OsPT1重组质粒载体，并将其转入Δycf1 BY4741酵母菌株（Cd敏感酵母菌株）用以验证OsPT1对酵母Cd耐受性的影响。结果表明，OsPT1编码序列全长为1 584 bp，编码分子量为57.46 kD，由527个氨基酸构成的蛋白。在水稻基因组中该基因上游启动子区含有与光、厌氧、茉莉酸甲酯等环境和激素响应相关的调控元件。系统进化分析表明，水稻OsPT1与高粱SbPT1亲缘关系最近。基因的镉响应表达分析结果表明，与对照相比，经100 μmol/L Cd处理的水稻在1、6和12 h后，地上部分OsPT1的转录水平分别上调1.31、1.34和2.46倍；水稻根部OsPT1在处理1和6 h后分别上调1.28和1.14倍，但在Cd处理12 h后，其表达水平下调至处理前的0.62倍。转基因酵母Cd耐受性结果表明，与对照（0 μmol/L Cd）相比，经25 μmol/L Cd处理后，转OsPT1的酵母对Cd的耐受性有一定的下降。OsPT1可能在水稻应对Cd胁迫过程中发挥一定的作用。

关键词: 水稻, OsPT1, 镉胁迫, 酵母, 基因表达

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

姜南, 石杨, 赵志慧, 李斌, 赵熠辉, 杨俊彪, 闫家铭, 靳雨璠, 陈稷, 黄进. 镉胁迫下水稻OsPT1的表达及功能分析[J]. 生物技术通报, 2023, 39(1): 166-174.

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.

图/表 11

表1 RT-qPCR引物序列

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

图1 OsPT1的PCR扩增及pGADT7-OsPT1重组载体的酶切验证 A：OsPT1的PCR扩增；B：酶切后的重组载体

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

表2 OsPT1蛋白的理化性质

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

表3 OsPT1蛋白的氨基酸组成

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

图2 OsPT1蛋白亲疏水性分析得分大于0的区域代表OsPT1的疏水区，小于0的区域代表OsPT1的亲水区

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

图3 OsPT1蛋白质的二、三级结构预测 A：OsPT1的二级结构，蓝色表示α-螺旋，红色表示延伸链，绿色表示β-转角，紫色表示无规则卷曲，横坐标表示每个氨基酸残基在OsPT1上的位置；B：OsPT1的三级结构，紫色表示α-螺旋，蓝色表示无规则卷曲，红色表示丙氨酸

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

图4 OsPT1蛋白潜在的磷酸化位点分析红线代表潜在的磷酸化丝氨酸残基；绿线代表潜在的磷酸化苏氨酸残基；蓝线代表潜在的磷酸化酪氨酸残基；粉红色水平线表示具有磷酸化潜力的阈值；横坐标表示每个氨基酸残基在OsPT1上的位置；纵坐标表示预测的磷酸化潜能得分

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

图5 PT1蛋白系统进化分析 TdPT1：二粒小麦，XP_037426503.1；TaPT1：小麦，AIZ11178.1；HvPT1：大麦，AAN37900.1；ZmPT1玉米，NP_001183901.1；OsPT1：水稻，XP_015631295.1；SbPT1：高粱，XP_002467158.1；VvPT1：葡萄，XP_002281264.1；AtPT1：拟南芥，NP_001319749.1；SlPT1：番茄，CAA76075.1；GmPT1：大豆，NP_001240239.1；PtPT1：毛白杨，XP_002300153.3

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

图6 OsPT1的顺式作用元件分析不同颜色的方块代表光响应元件（绿色和红色）、厌氧响应元件（黄色）、MeJA响应元件（粉色）、水杨酸响应元件（蓝色）和疾病、应激响应元件（灰色）

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

图7 OsPT1响应Cd胁迫表达分析 A：地上部分；B：根。*代表在P=0.01水平上差异显著

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

图8 OsPT1对酵母Cd敏感性的影响

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

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