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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (4): 896-907.doi: 10.3724/SP.J.1006.2022.14036

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Functional analysis of StMAPK4 in response to low temperature stress in potato

FENG Ya1,2(), ZHU Xi1,2, LUO Hong-Yu1,2, LI Shi-Gui1,2, ZHANG Ning1,2,*(), SI Huai-Jun1,2   

  1. 1College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, Gansu, China
    2Gansu Provincial Key Laboratory of Aridland Crop Science, Lanzhou 730070, Gansu, China
  • Received:2021-02-26 Accepted:2021-06-16 Online:2022-04-12 Published:2021-07-25
  • Contact: ZHANG Ning E-mail:2992919739@qq.com;ningzh@gsau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31960444);Gansu Provincial Key Laboratory of Aridland Crop Science, Gansu Agricultural University(GSCS-2019-Z03)

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Abstract:

Potato is vulnerable to low temperatures resulting in reduced yield production. MAPK gene is widely involved in a variety of environmental stress, and it has been detected to be involved in low temperature regulation. In this present study, to explore StMAPK4 function in response to low temperature stress, potato cultivar ‘Atlantic’ as the experimental material, the expression characteristics of StMAPK4 gene were analyzed in potato root, stem, and leaf at the different time under low temperature (4℃) stress. StMAPK4 gene was analyzed using bioinformatics and its encoded protein subcellular localization was assayed. StMAPK4 overexpression and RNA interference expression vectors were constructed and obtained transgenic potato plants. The activities of superoxide dismutase (SOD) and peroxidase (POD), and the contents of proline (Pro) and malondialdehyde (MDA) in non-transgenic (NT), overexpressed and RNAi interfered transgenic plants were analyzed under 4℃. The results showed that the isoelectric point (pI) of StMAPK4 was 4.97 and it was acidic protein localized in the nucleus and cell membrane. The relative expression levels of StMAPK4 in roots, stems, and leaves significantly increased under low temperature stress. Compared with non-transgenic plants, the activities of SOD and POD and the content of proline in StMAPK4 overexpressed plants were significantly increased, while the content of MDA was significantly decreased. Compared with NT plants, the activities of SOD and POD, and the content of Pro in StMAPK4 overexpression plants were significantly decreased, while the content of MDA was significantly increased. Phenotypic observation revealed that the leaves of non-transgenic and RNAi interfered expression plants wilted seriously, while the leaves of overexpressed plants were less affected. In summary, the overexpression of StMAPK4 gene can enhance the tolerance of low temperature stress in potato plants.

Key words: potato, StMAPK4, low temperature, subcellular localization, genetic transformation

Table 1

Primers of amiR-StMAPK4 for PCR"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')
I GATATACCCTGTGTAAATCGCACTCTCTCTTTTGTATTCC
II GAGTGCGATTTACACAGGGTATATCAAAGAGAATCAATGA
III GAGTACGATTTACACTGGGTATTTCACAGGTCGTGATATG
IV GAAATACCCAGTGTAAATCGTACTCTACATATATATTCCT
A CTGCAAGGCGATTAAGTTGGGTAAC
B GCGGATAACAATTTCACACAGGAAACAG

Table 2

Specific primers for PCR and qRT-PCR"

引物名称
Primer name		 引物编号
Primer ID		 引物序列
Primer sequence (5'-3')
PCR primers for StMAPK4 正向Forward CGGGGGACGAGCTCGGTACCATGGATGCTGAAAACATTGAAAAT
反向Reverse CCATGTCGACTCTAGACTTGGTTGTATCGGGATCAAACT
PCR primers for Ef1a 正向Forward CAGCCAATCCCATCAAGACG
反向Reverse ATCATTCCGGAGCACTCGAT
qRT-PCR primers for StMAPK4 正向Forward ACGCTCTTCACAGGCCCTCACAGAAG
反向Reverse TGGAACTTGAGGCAGCTGCTTGACGT

Fig. 1

Amino acid sequence alignment of StMAPK4 protein in potato and homologous proteins of other species"

Fig. 2

Phylogenetic tree of potato StMAPK4 gene and 11 homologous genes from other species"

Table 3

Analysis of cis-acting elements in StMAPK4 promoter region"

位点名称
Site name		 序列
Sequence		 功能
Function
ABRE ACGTG 脱落酸反应的顺式作用元件
cis-acting element involved in abscisic acid response
ARE AAACCA 厌氧诱导反应的顺式作用调节元件
cis-acting regulatory element essential for the anaerobic induction
CAAT-box CAAAT, CCAAT 启动子和增强子区域顺式作用元件
Common cis-acting element in promoter and enhancer regions
G-Box CACGTT, TAAACGTG 参与光响应的顺式作用调节元件
cis-acting regulatory element involved in light response
LTR CCGAAA 参与低温反应的顺式作用元件
cis-acting element involved in low-temperature response
MBS CAACTG 参与干旱诱导的MYB结合位点
MYB binding site involved in drought-inducibility
P-box CCTTTTG 赤霉素响应元件
Gibberellin-responsive element
TC-rich repeats CACGTT 参与防御和应激反应的顺式作用元件
cis-acting element involved in defense and stress response

Fig. 3

Secondary structure of StMAPK4 protein Blue indicates α-helix (Hh); red is the extension chain (Ee); green represents β-angle (Tt); purple is for random crimp (Cc)."

Fig. 4

Tertiary structure and domain of STMAPK4 protein"

Fig. 5

StMAPK4 protein interaction network"

Fig. 6

StMAPK4 amplification product bands and amir-MAPK4 precursor fragments A: StMAPK4 amplification product bands; B: small fragments of A (primers A and IV), B (primers III and II) and C (primers I and B) amplified using plasmid PRS300 as template; C: d fragment amplified using a mixture of small fragments a, b, and c as a template (primers A and B); M: DL 2000 marker."

Fig. 7

Double digestion of pCE-StMAPK4 (A) and pBI121-miR-StMAPK4 recombinant plasmid (B)"

Fig. 8

Subcellular localization of pCEGFP-StMAPK4 pCEGFP: blank control; pCEGFP-StMAPK4: pCEGFP-StMAPK4 fusion protein; A: pCEGFP fluorescence signal in the dark field: B: autofluorescence of chloroplast; C: cell morphology under bright field; D: combination field."

Fig. 9

Relative expression pattern of StMAPK4 gene in tissue specific under low temperature treatment * and ** mean significant difference at the 0.05 and 0.01 probability levels, respectively, n = 9."

Fig. 10

Relative expression pattern of the transgenic plants by qRT-PCR OE-1, 2, 3, 4, 5, 6: the overexpressed plants; RNAi-1, 2, 3, 4, 5, 6: the interfering expression plants; NT: non-transgenic plants. * and ** mean significant difference at the 0.05 and 0.01 probability levels, respectively, n = 9."

Fig. 11

Phenotype of potato transgenic plants under low temperature stress NT: non-transgenic plants; OE: the overexpression of transgenic potato plants; RNAi: the interfering expression of transgenic potato plants."

Fig. 12

Determination of physiological indexes in the transgenic plants under low temperature stress NT: non-transgenic plants; OE-2, 3, 6: transformed plants with pCE1300-StMAPK4; RNAi-1, 2, 4: transformed plants with pBI121-amiR- StMAPK4. Different lowercase letters above the bars mean significant difference at the 0.05 probability level."

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