Crops ›› 2018, Vol. 34 ›› Issue (4): 53-61.doi: 10.16035/j.issn.1001-7283.2018.04.010

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Cloning and Expression of ScHAK10 Gene in Sugarcane

Luo Haibin1,Jiang Shengli1,Huang Chengmei1,Cao Huiqing1,Deng Zhinian2,Wu Kaichao2,Xu Lin2,Lu Zhen1,Wei Yuanwen1   

  1. 1 Guangxi Crop Genetic Improvement and Biotechnology Laboratory, Nanning 530007, Guangxi, China
    2 Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences/Key Laboratory of Sugarcane Biotechnology and Genetic Improvement of Guangxi, Ministry of Agriculture and Rural Affairs, Nanning 530007, Guangxi, China
  • Received:2018-01-25 Revised:2018-06-15 Online:2018-08-15 Published:2018-08-23

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Abstract:

To explore the function of ScHAK10 in Sugarcane HAK gene family and molecular response mechanism under drought stress, the gene ScHAK10 coding sequence (CDS) from the ROC22 was cloned by homologous cloning. Sequencing and bioinformatics analysis indicated that ScHAK10 sequence obtained was 2 433bp in length, coding 810 amino acids, the molecular weight of ScHAK10 was 89.83kD, isoelectric point (pI) was 8.46, hydrophobic basic protein was prediction. Sequence comparison with maize (Zea mays L.), sorghum [Sorghum bicolor (L.) Moench] and other crops showed that ScHAK10 gene was highly homologous with that of other species. ScHAK10 protein contains multiple trans-membrane domains so that it is predicted to be located on the cytoplasmic membrane. Secondary structure analysis showed that it had alpha helix (38.15%), extended long chain (19.26%) and irregular curling (37.16%). Prediction of protein function showed that it may be involved in biological reactions such as transcription, signal transduction, and immune responses. Real-time PCR result indicated that ScHAK10 expressed in different tissues, the highest expression in leaf, followed by stem, and the lowest in root. ScHAK10 gene expression was up-regulated by drought stress. According to the deepening of the stress level, the expression of ScHAK10 rose initially, and then dropped, and decreased after rewaterring. These results indicated that contributes were made for further illustrating its function and its molecular mechanism in the regulation of sugarcane drought.

Key words: Sugarcane, Potassium transporter gene (HAK), Gene cloning, Bioinformatics analysis, Gene expression

Fig.1

RT-PCR amplification electrophoresis results of ScHAK10 gene M:DL2000 DNA Marker;1:ScHAK10;2:ScHAK10"

Fig.2

Nucleotide sequences in coding regions of ScHAK10 gene and their deduced amino acid sequences"

Table 1

Homology analysis between ScHAK10 gene and HAK gene from other species"

登录号Accession No. 物种Species 同源性Homology (%) 基因Gene
XM_021449505.1 高粱 95 potassium transporter 10
KF815907.1 玉米 94 high-affinity potassium transporter10
XM_004965768.3 小米 90 potassium transporter 10
XM_015788218.1 水稻 87 potassium transporter 10
XM_020332943.1 山羊草 86 potassium transporter 10
XM_003563544.3 二穗短柄草 85 potassium transporter 10
XM_010943891.2 油棕 80 potassium transporter 10

Fig.3

Prediction for hydrophily hydrophobicity of ScHAK10 protein||| Horizontal axis indicate amino acid position; the hydrophobic position and hydrophilic position are plotted above and below the ordinate, respectively"

Fig.4

ScHAK10 protein transmembrane region analyzed by TMHMM software"

Fig.5

ScHAK10 signal peptide analyzed by SignalP 4.1 Server"

Fig.6

Predicted secondary structure of ScHAK10 protein"

Fig.7

Predicted tertiary structure of ScHAK10 protein"

Table 2

Functional category for ScHAK10 protein"

功能分类Functional category 概率Probability 比值
Odds		 基因本体分类
Gene ontology category		 概率
Probability		 比值
Odds
运输和结合Transport and binding 0.773 1.885 电压门控性离子通道Voltage-gated ion channel 0.279 12.682
嘌呤和嘧啶Purines and pyrimidines 0.331 1.362 转录Transcription 0.219 1.711
辅酶合成Biosynthesis of cofactors 0.210 2.917 信号传导Signal transducer 0.205 0.958
脂肪酸代谢Fatty acid metabolism 0.017 1.308 离子通道Ion channel 0.169 2.965
运输Translation 0.071 1.614 阳离子通道Cation channel 0.146 3.174

Fig.8

Phylogenetic tree based on HAK gene The tree is constructed using the neighbor-joining method with bootstrap values calculated from 500 resamplings. Numbers at the nodes indicate the bootstrap values, The scale bar represents 0.05 substitution per nucleotide position"

Fig.9

Multiple amino-acid alignment of ScHAK10 with other HAK proteins"

Fig.10

Relative expression of ScHAK10 gene in different tissues of sugarcane Error bars represent the standard error of each treating group (n=3). The same below"

Fig.11

Expression profiles of ScHAK10 under drought stress"

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