Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (13): 2295-2308.doi: 10.3864/j.issn.0578-1752.2019.13.009

• HORTICULTURE • Previous Articles     Next Articles

Expression and Structural Analysis of SC MI390-5p and Its Target Genes in Potato Response to Low Temperature

XIE Jie1,WANG Ming1,DING HongYing1,LI Qing1,WANG WanXing2,XIONG XingYao2,QIN YuZhi1()   

  1. 1 College of Horticulture and Landscape, Hunan Agricultural University, Changsha 410128
    2 Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2019-01-03 Accepted:2019-05-13 Online:2019-07-01 Published:2019-07-11
  • Contact: YuZhi QIN E-mail:qyuz@163.com

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Abstract:

The study was carried out to investigate the mechanism of potato leucine-rich repeat receptor-like protein kinase (LRR-RLK) receptor protein kinase SCLRRK1 regulated by miR390 (ScmiR390-5p) in response to abiotic stress. 【Method】 By sequencing and analyzing potato miRNAs in response to low temperature conditions and predicting target genes, we found that ScmiR390-5p responded to low temperatures by regulating a potential LRR-like receptor protein kinase gene. The expression levels of ScmiR390-5p and SCLRRK1 in response to low temperature stress were verified by quantitative real-time PCR (RT-qPCR). The cleavage site of ScLRR390-5p on SCLRRK1 was determined by using RLM-5'RACE. The DNA sequence and CDS sequence of SCLRRK1 were cloned by PCR and the structure and function of SCLRRK1 were predicted by bioinformatics analysis. The expression levels of ScmiR390-5p/SCLRRK1 in various tissues of potato and under various abiotic stress were analyzed by RT-qPCR. 【Result】 The results of RT-qPCR showed that the expression of ScmiR390-5p was induced by low temperature, while the expression of SCLRRK1 was inhibited. There was a negative correlation between the expression of ScmiR390-5p and scrrk1 under low temperature stress. The result of RLM-5′RACE indicated that cleavage site of ScmiR390-5p on SCLRRK1 was ATTCCT// CCTGAGTT, and potato ScmiR390-5p/SCLRRK1 regulatory module was respond to low temperature. The cloning results indicated that the CDS of SCLRRK1 was 2 685 bp in length, encoding 894 amino acids, and the gene sequence was 3 549 bp containing 1 intron, 2 exons, 3' non-coding region and 5' non-coding region. ScmiR390-5p target site was located at SCLRRK1 CDS (960-981 bp, GGAACTATTCCTCCTGAGTTT). Bioinformatics analysis showed that the SCLRRK1 encoded a leucine-richrepeat (LRR)-like receptor protein kinase, belonging transmembrane secreted protein. RT-qPCR analysis of potato tissue expression pattern showed that the expression level of the ScmiR390-5p was highest in the leaves, followed by roots, and relatively lowers in stem (terrestrial stem, tuber and stolon). Differently, the expression level of SCLRRK1 was the lowest in leaves and the highest in stems. The results under various abiotic stresses showed that the expression of ScmiR390-5p and SCLRRK1 was negatively correlated, and ScmiR390-5p was induced by NaCl stress. Compared with the control, the expression of ScmiR390-5p was decreased and then increased slightly after treatment with ABA and 6-BA. The SCLRRK1 level increased continuously under 6-BA treatment, while the expression of SCLRRK1 increased first and then decreased under ABA treatment. The expression of ScmiR390-5p reached the peak after treatment with GA3, PEG and IAA for 8 h. The expression of SCLRRK1 was induced by GA3, PEG and IAA, but the change trend was not correlated to ScmiR390-5p. 【Conclusion】 SCLRRK1 had the amino acid sequence and structural basis of nucleic acid to encode LRR-like receptor protein kinase, which was the target gene of ScmiR390-5p; ScmiR390-5p/SCLRRK1 regulatory module had a significant role in potato tissues; ScmiR390-5p responded to low temperature stress by inhibiting the expression of potato SCLRRK1 at the post-transcriptional level, while ScmiR390-5p/SCLRRK1 regulated module played a role in salt and 6-BA stress response. The ScmiR390-5p/SCLRRK1 regulatory module did not respond to ABA, GA3, IAA and PEG stress, and the ScmiR390-5p and SCLRRK1 were regulated by the above signals at the transcriptional level, respectively.

Key words: potato, ScmiR390-5p, SCLRRK1, quantitative real-time PCR, RLM-5′RACE

Table 1

Primers used in this study"

引物 Primer 序列（5′→3′） Sequence (5′→3′) 用途 Use
SCLRRK1 F ATGACACTTTTGAGGTTTCTGTTAA SCLRRK1 CDS片段克隆
SCLRRK1 R TTAGCTTTCCGTGATTTCTTGA Clone of SCLRRK1 CDS fragments
SCLRRK1 F1 GTTTTCTTCATTTCTTGATATGGC SCLRRK1DNA全长扩增
SCLRRK1 R1 GTTGAATAGTAGCATTCCCTTAATC Amplification of Full-length DNA of SCLRRK1
SCLRRK1 F2 GCTTGAGATCCTAACCACCCG SCLRRK1实时定量PCR
SCLRRK1 R2 TCTTGCAGCATTTCTACCACCTT Quantitative real-time PCR of SCLRRK1
SCLRRK1 outer F3 GCTGATGGCGATGAATGAATGAACACTG RLM-5′RACE 分析作用位点
SCLRRK1 outer R3 CAACAAAGACGTTTCCCGCCAGT Rapid amplification of cDNA 5′ ends
SCLRRK1 inner F4 CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG RLM-5′RACE 分析作用位点
SCLRRK1 inner R4 CCTGAGGCAATTTTCCGTGGAGA Rapid amplification of cDNA 5′ ends
ScmiR390-5p CCAGCGTGAAGCUCAGGAGGG ScmiR390-5p实时荧光定量PCR
ScmiR390-5p CCAGCGTGCGCUAUCCAUCUU Quantitative real-time PCR of ScmiR390-5p

Fig. 1

Comparative analysis of mRNA, miRNA and its target genes with significant differences between different treatments A: TLX2-CLX2; B: TLH1-CLH1"

Table 2

The expression of ScmiR390-5p and its differentially significant target gene between different treatments"

基因编号
ID		 TLX2/CLX2 TLH1/CLH1 基因编号
ID		 基因名
Gene name		 TLX2/CLX2 TLH1/CLH1
上调_下调
Up_down		 上调_下调
Up_down		 上调_下调
Up_down		 上调_下调
Up_down
ScmiR390-5p 上调 Up 上调 Up PGSC0003DMT 400070158 SCLRRK1 Leucine-rich repeat receptor kinase 下调 Down 下调 Down
上调 Up 上调 Up PGSC0003DMT 400010618 Receptor protein kinase CLAVATA1 下调 Down 下调 Down
上调 Up 上调 Up PGSC0003DMT 400034044 Unknown 下调 Down 下调 Down
上调 Up 上调 Up PGSC0003DMT 400009701 Endo-1,3-1,4-beta-d-glucanase 上调 Up 上调 Up

Fig. 2

The analysis of the SCLRRK1 by Real-time PCR and Semi quantitative in low temperature CK: The mixture samples without treatment; TLX1: The mixed samples which acclimated at 5℃ of 1th, 2th and 3th days (control is CLX1); TLX2: The mixed samples of 8th, 9th, and 10th days after 5℃ acclimation (control is CLX2); TLH1: The mixed samples acclimated at 5℃ for 10 days and frozen at -4℃ for the first 1th, 2th and 3th hours (control is CLH1); TLH2: The mixed samples acclimated at 5℃ for 10 days and frozen at -4℃ for the 7th, 8th and 9th hours (control is CLH2)"

Fig. 3

Validation of ScmiR390-5p guided cleavage of SCLRRK1 by RLM-RACE"

Fig. 4

Products of amplification of SCLLRK1 gene from Solanum commersonii-LZ3.4"

Fig. 5

Hydrophobicity/hydrophilicity prediction of SCLRRK1"

Fig. 6

Predicted transmembrane domain of prediction of SCLRRK1"

Fig. 7

Predicted signal peptide of SCLRRK1"

Fig. 8

Nucleotide sequence and amino acid sequence of SCLRRK1 The box is containing Low complexity region; the one underline area is LRR domain; the two underline area is transmembrane helix region; Protein Kinase STYKc domain is shaded area; The ellipse is containing the ScmiR390-5p recognition sites"

Fig. 9

The specific expression of the ScmiR390-5p and SCLRRK1 by Real-time PCR in different tissue form Solanum tuberosum and semi quantitative PCR in different tissue"

Fig. 10

Expression of the SCLRRK1 and ScmiR390-5p by real-time PCR in different abiotic stress"

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