Investigation of Polymorphisms in Coding Region of OsHKT1 in Relation to Salinity in Rice

doi:10.1016/j.rsci.2016.05.004

Abstract

Abstract:

Rice (Oryza sativa) is sensitive to salinity, but the salt tolerance level differs among cultivars, which might result from natural variations in the genes that are responsible for salt tolerance. High-affinity potassium transporter (HKTs) has been proven to be involved in salt tolerance in plants. Therefore, we screened for natural nucleotide polymorphism in the coding sequence of OsHKT1, which encodes the HKT protein in eight Vietnamese rice cultivars differing in salt tolerance level. In total, seven nucleotide substitutions in coding sequence of OsHKT1 were found, including two non-synonymous and five synonymous substitutions. Further analysis revealed that these two non-synonymous nucleotide substitutions (G50T and T1209A) caused changes in amino acids (Gly17Val and Asp403Glu) at signal peptide and the loop of the sixth transmembrane domain, respectively. To assess the potential effect of these substitutions on the protein function, the 3D structure of HKT protein variants was modelled by using PHYRE2 webserver. The results showed that no difference was observed when compared those predicted 3D structure of HKT protein variants with each other. In addition, the codon bias of synonymous substitutions cannot clearly show correlation with salt tolerance level. It might be interesting to further investigate the functional roles of detected non-synonymous substitutions as it might correlate to salt tolerance in rice.

Key words: polymorphism, salt stress, OsHKT1 gene, rice

Quynh-Hoa Pham, Xuan-An Tran, Thi-Nha-Trang Nguyen, Thi-Thuy-Anh Tran, Hai-Yen Hoang, Thi-Hong-Van Nguyen, Thi-Hanh Tang, Thi-Phuc Do. Investigation of Polymorphisms in Coding Region of OsHKT1 in Relation to Salinity in Rice[J]. Rice Science, 2016, 23(6): 334-338.

Figures/Tables 4

Fig. 1. PCR products using four pairs of primers. A, PCR product of 499 bp using primer pair 1 (F1); B, PCR product of 828 bp using primer pair 2 (F2); C, PCR product of 603 bp using primer pair 3 (F3); D, PCR product of 668 bp using primer pair 4 (F4). M, 1 kb ladder marker; Lanes 1 to 9, Nipponbare, Nep Non Tre, Chiem Cu, Re Nuoc, Hom Rau, Nep Oc, Ngoi, Dau An Do and Nep Deo Dang, respectively.

Fig. 2. Polymorphism in OsHKT1 sequence. A, Schematic representation of polymorphism locations in the protein. The position of changes in amino acid are indicated with a star; MPM, transmembrane-pore loop-transmembrane domain. B, Nucleotide sequence of OsHKT1 was compared among the cultivars with the Nipponbare sequence. Non-synonymous substitutions are indicated in dark grey, and the encoded amino acids are listed above. Synonymous substitutions are indicated in light grey. G17V means that glycine is replaced by valine; D403E means that aspartic acid is replaced by glutamic acid.

Fig. 3. 3D ribbon model of OsHKT1 protein visualized by Discovery studio 4.5 visualizer. A, 3D ribbon model of Ni-OsHKT1 protein visualized from the top that shows the pore. Four residues in Ser-Gly-Gly-Gly ion selection motif are labeled and Na+ ion is indicated as a black round circle at the center of the pore. B, Visualization of Ho-OsHKT1 transporter from the side that shows the zoom in D403E variance position (green) (G17V substitution belongs to the signal peptide so they are not modeled in 3D structure of OsHKT1 transporter).

Table 1 Changes in codon usage bias by nucleotide substitutions in nine rice cultivars

Position of nucleotide variance	Former codon	Usage frequency of former codon (‰)	Substituted codon	Usage frequency of substituted codon (‰)	Amino acid	Cultivar
360	CUA	7.7	CUG	21.0	Leu	Nep Non Tre, Dau An Do, Nep Oc, Chiem Cu
645	UCA	12.4	UCG	12.3	Leu	Chiem Cu
708	ACA	11.6	ACG	11.4	Thr	Nep Non Tre, Dau An Do, Nep Oc, Chiem Cu
744	ACG	11.4	ACA	11.6	Thr	Nep Non Tre, Dau An Do, Nep Oc, Chiem Cu
1440	AAG	32.3	CAA	13.5	Lys	Nep Non Tre
Values in bold meant that the substituted nucleotide caused an increase in codon usage frequence.

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28	(Managing Editor: Wang Caihong)

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