Analysis of Protein Characteristics of Selenite Transporter OsPT2 in Rice

Biaojin ZHANG Qinglong CHEN Tingcan DAI Yaomin ZHOU Yihua WEI Xiangxi ZHANG Linguang LUO

AbstractOsPT2, which is a selenite transporter in rice, belongs to rice phosphate transporter family OsPT. In this study, the amino acid sequence information of OsPT family members was collected using NCBI database. Bioinformatics method was employed to analyze physical and chemical properties and protein structure of OsPT2 and its homologous relationship with other members of the family. The results indicated that OsPT2 was a stable alkaline hydrophobic protein embedded in the cell membrane. The protein contained 11 transmembrane domains, and consisted of 45.08% of alpha helix, 16.48% of extended strand, 10.80% of beta turn and 27.65% of random coil, forming a barrellike threedimensional structure. Members in OsPT family had high homology and could be divided into 5 subgroups. OsPT2 had closer relationship with OsPT1 and OsPT3. This study could provide a basis for further revealing of molecular mechanism of selenite transport in rice.

Key wordsRice (Oryza sativa L.); Selenite; OsPT2; Protein characteristics; Bioinformatics

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Received: May 17, 2018Accepted: August 29, 2018

Supported by Jiangxi Major Research and Development Program (20161BBF60131).

Biaojin ZHANG (1983-), male, P. R. China, research assistant, devoted to research about quality safety and nutritional function of agricultural products.

*Corresponding author. Email: zhangbiaojin@126.com.

Selenium (Se) is a kind of trace nutritional element essential to human body, which has various physiological functions including resisting aging, preventing cancer, resisting oxidation, enhancing immunity of organism, discharging toxic substances and preventing Keshan disease and KaschinBeck disease, and is closely related to human health［1-2］. Se is determined as the third trace nutritional healthcaring elements next to iodine and zinc by World Health Organization (WHO) and Chinese Medical Association (CMA) in the 21st century. However, the intake of Se is insufficient generally all over the world［3］. China is a country lacking Se seriously, where the soil has a Se content only of 0.29 mg/kg averagely, and about 70% of the soil in China is Sedeficient and lowSe areas. In China, the adult takes 26.63 μg of Se per day averagely, which is far less than the safe Se intake 40-240 μg per day recommended in the country and the safe Se intake 50-200 μg per day recommended in America.

Rice is the main food crop in China, which is main source of Se from peoples diet. Therefore, the production of Seenriched rice from soil naturally enriched with Se is an ideal way of supplementing Se through the increase of Se intake from diet. However, expounding the molecular mechanism of Se accumulation in rice is the theoretical basis for production of Seenriched rice. Available Se content in soil is very low, and mainly depends on soluble Se content in soil, which accounts for 1%-3% of total Se in soil, and mainly includes selenite, soluble organic Se and selenate［4］, while selenite is the main existence form of effective Se in paddy soil. Currently, two rice selenite absorption genes, OsNIP2;1［5］ and OsPT2［6］, have been identified. OsPT gene family includes 13 members from OsPT1 to OsPT13［7］. The members in the family play an important role in phosphate absorption and transport in rice, but only OsPT2 has been identified to have selenite absorption function.

In this study, OsPT2 gene information was explored using rice genome database, and the physical and chemical properties and structure of OsPT2 protein and its homologous relationship with other members of the family were analyzed, aiming at providing fundamental basis for revealing of its molecular mechanism of selenite transport in rice.

Materials and Methods

Sources of sequences

The amino acid sequences of OsPT1OsPT13 transporter searched in NCBI database were as follows: AAU84430.1, AAN39049.1, AAN39050.1, AAN39051.1, AAN39052.1, AAN39053.1 and Q7XRH8.2. The amino acid AAU84427.1 of OsPT2 was mainly studied in this study.

Sequence analysis

The physiological and biochemical characteristics of OsPT2 were analyzed online with ProtParam tool (https://web.expasy.org/protparam/) of ExPASy website, including amino acid composition, relative molecular weight, theoretical isoelectric point and stability. The hydrophilcity/hydrophobicity of protein was analyzed with ProtScale (http://web.expasy.org/protscale/). Subcellular localization was performed with PSORT(http://www.psort.org/). Signal peptides of OsPT2 protein was analyzed with SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/). The tansmembrane domains of the protein were analyzed with TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM2.0/). The secondary and tertiary structures of OsPT2 protein were analyzed with SOPMA (https://npsaprabi.ibcp.fr/cgibin/npsa_automat.pl?page=npsa_sopma.html) and SWISSMODEL (https://swissmodel.expasy.org/interactive), respectively. An evolution tree was finally built by NJ (NeighborJoining) method in MEGA7.0［8］.

Results and Analysis

Analysis of physical and chemical properties

The amino acid sequence of rice selenite transporter OsPT2 had a total length of 528 aa. Its physical and chemical properties were analyzed with ProtParam tool, and the results showed that the protein had the relative molecular weight of 57.815 kD, and the theoretical isoelectric point pI was 8.85. The amino acid composition is shown in Table 1. Among the various amino acids, the negativelycharged aspartic acid (Asp) and glutamic acid (Glu) had a total number of 32, accounting for 6.06%, and the positivelycharged arginine (Arg) and lysine had a total number of 38, accounting for 7.20%. The molecular formula of OsPT2 was C2677H4072N672O707S26, consisting of 8 154 atoms. Its instability index was 30.53, lower than 40, suggesting that OsPT2 is a protein with better stability. The fatsoluble index of the protein was 93.22, and the average hydrophilic coefficient was 0.398, so the protein was a hydrophobic protein. The distribution of its hydrophilic/hydrophobic amino acids was further analyzed with ProtScale. As shown in Fig. 1, there were 165 hydrophilic amino acids, accounting for 31.25%, and there were 297 hydrophobic amino acids, accounting for 56.25%.

Analysis of protein structure

PSORT analysis showed that OsPT2 protein was localized in plasma membrane. It was found by SignalP 4.1 that the protein did not contained signal peptide (Fig. 2). TMHMM analysis showed that the protein contained 11 transmembrane domains (FIg. 3). Further analysis of its secondary structure with SOPMA showed that the secondary structure of OsPT2 protein consisted of 45.08% of αhelix, 16.48% of extended strand, 10.80% of β turn and 27.65% of random coil (Fig. 4). Its 3D structure was predicted with SWISSMODEL, to be a structure similar to a barrel (Fig. 5).

Homology analysis of OsPT gene family

Thirteen gene members in OsPT family were aligned with ClustalX2.1, and the results showed that the consistency coefficient reached 64.89%, suggesting that the members in the family have very high homology (Fig. 6). An evolution tree was constructed with MEGA7.0, and it was found that the 13 members could be divided into five subfamilies, and selenite transporter OsPT2 had the closest genetic relationship with OsPT1 and OsPT3 (Fig. 7).

Discussion

Members in OsPT family are mainly phosphate transporter genes, among which OsPT1［9-10］, OsPT4［11］, OsPT6［12-13］, OsPT8［14-15］, OsPT9 and OsPT10［16］ have been identified as efficient phosphate transporter, and OsPT1and OsPT8 are demonstrated to have As accumulation function［17-18］. OsPT2 transporter has very weak affinity to phosphate［19］, but very strong ability of transporting selenite［6］, and it might participate in metabolism of ammonium salt［20］. Furthermore, it is also found to be capable of transporting selenite in tobacco transformed with OsPT8 gene［21］, but whether it has similar transporting capability in rice still needs further demonstration. Overall, few genes related to Se absorption in rice have been explored, and the molecular mechanism of Se metabolism is far from clear, while exploring new Se transportrelated genes by bioinformatics methods is an important way［22］. In this study, the structural characteristics of OsPT2 protein were analyzed by bioinformatics means, and the results showed that it was a barrelshaped channel protein inlaid on plasma membrane, which could provide reference for the discovery of suspected selenite transporters through the exploration of proteins with similar structures by bioinformatics. Meanwhile, the analysis of homology of OsPT2 with other members in OsPT family showed that there was very homology between the genes in this family, which also indicates that other members in this family also might the function of transporting selenite, such as OsPT8.

Selenite is a kind of weak acid, which has the acid dissociation constants pKa1 and pKa2 of 2.57 and 6.60, respectively. It mainly exists in the form of H2SeO3 at the pH lower than 2.5, and in the form of HSeO-3 in the environment with a pH of 2.5-6.6, which is converted to SeO2-3 at the pH higher than 6.6. Therefore, in paddy field environment, selenite could exist in different forms according to different pH values in soil. Researches have been that under different pH conditions, rice root system might have different absorption mechanisms to selenite［23］. Zhao et al.［5］ found that under acidic conditions, rice Si transporter OsNIP2;1 could promote the absorption of selenite, while under alkaline conditions, the transporting capacity was reduced remarkably. Therefore, OsNIP2;1 might mainly transport selenite in the form of H2SeO3. In southern weaklyacidic paddy soil, selenite mainly exists in the form of HSeO-3, which has a structure similar to H2PO-4, and therefore, phosphate transporter might be the main channel protein of HSeO-3. Lazard et al.［24］ found that at the pH

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Fig. 1Distribution of hydrophilic/ hydrophobic amino acids in OsPT2 protein

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Fig. 2Signal peptide analysis of OsPT2 protein

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Fig. 3Transmembrane domain of OsPT2 protein

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:α helix; : extended strand; : β turn; : random coil

Fig. 4Distribution of the secondary structure of OsPT2 protein

value of 6.0, the phosphate transporter protein of yeast has the activity of transporting selenite. Li et al.［25］ also found that wheat could absorb selenite through phosphate transporter. Overall, there might be various selenite absorption pathways, while there has been little understanding on its molecular mechanisms including its absorption, transport and even discharge. Therefore, it is urgent to conduct the exploration of related genes, so as to provide fundamental basis for comprehensively expounding Seenrichment mechanism in plant and breeding Seenriched food crops.

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Fig. 5The tertiary structure of OsPT2 protein

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Fig. 6Alignment of amino acid sequences from members of the OsPT gene family

Biaojin ZHANG et al. Analysis of Protein Characteristics of Selenite Transporter OsPT2 in Rice

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Fig. 7Homologous evolution tree of members in the OsPT gene family

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Editor: Yingzhi GUANGProofreader: Xinxiu ZHU

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