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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (9): 2462-2471.doi: 10.3724/SP.J.1006.2023.24236

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

Function analysis of potato StCYP85A3 in promoting germination and root elongation

LIU Jie1,2(), CAI Cheng-Cheng1,2, LIU Shi-Feng1,2, DENG Meng-Sheng3, WANG Xue-Feng2, WEN He1,2, LI Luo-Pin1,2, YAN Feng-Jun1,2, WANG Xi-Yao1,2,*()   

  1. 1State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
    2Potato Research and Development Center, College of Agriculture, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
    3College of Biological Engineering, Sichuan University of Light Chemical Industry, Yibin 644000, Sichuan, China
  • Received:2022-10-19 Accepted:2023-02-21 Online:2023-09-12 Published:2023-03-10
  • Supported by:
    Molecular Mechanism and Application of Seed Potato Vigor Regulation(SKL-ZY202203);Sichuan potato Innovation Team Project of National Modern Agricultural Industry Technology System(sccxtd- 2023-09)

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Abstract:

Brassinolide (BR), a kind of phytosterol hormone, plays the significant role in regulating plant growth and development and stress response. The function of StCYP85A3 as the encoding gene of BR synthease in potato remains to be further explored. In this study, the gene was cloned from potato ‘Chuanyu 10’. Bioinformatics analysis showed that this protein had a typical aerobic binding domain for catalyzing BR synthesis and belonged to the CYP85As family. Tissue expression analysis indicated that the relative expression level of this gene was the highest in the bud eye of the tuber and the root, and the expression amount in the bud eye increased with the extension of storage time, and the expression increased rapidly when the dormancy of the tuber bud eye was released. At the same time, the exogenous BR could induce the relative expression level of StCYP85A3 in potato bud eye and root, which significantly promoted the germination of seed potato and the elongation of seedling root. The overexpression of StCYP85A3 in Arabidopsis wild-type and mutant cyp85a2 demonstrated that the seed germination and root elongation of the overexpression strains were earlier than wild type, and the defects of mutant seed germination and plant root growth retardation were remedied by supplementing strains. In addition, exogenous BR did not significantly promote seed germination and root elongation of over expressed strains, but significantly promoted seed germination and root elongation of wild type, mutant, and complementary strains. The above potato and Arabidopsis thaliana experiments showed that StCYP85A3 had the function of promoting germination and root elongation.

Key words: potato, StCYP85A3, BR synthetase, root system, germination

Table 1

Primers used in the study"

基因Gene name 编号Gene ID 引物Primer sequence (5°-3°)
EF1αL Soltu.DM.06G005580 F: CTTGTACACCACGCTAAGGAG
R: GTCAATGCAAACCATTCCTTG
qCYP85A3 Soltu.DM.02G009130.1 F: CCACATACTTGGTTGCCCTAC
R: TGCTCCTCTAATGTGCTTGTG
CYP85A3 Soltu.DM.02G009130.1 F: ATGGCCATTTTCTTGATTGTTTT
R: CTAGTAAGTTGAAACCTTAATC

Fig. 1

Prediction of subcellular location, conservative alignment of protein sequences, and evolutionary tree analysis A: the prediction of subcellular localization; B: protein sequence conservative alignment; C: the analysis of the evolutionary tree; D: gene structure analysis. The stack height is determined by the deviation of the position’s letter emission frequencies from the background frequencies of the letters. As a new feature, the stack width now visualizes both the probability of reaching the state and the expected number of letters the state emits during a pass through the model."

Table 2

Some important cis-acting regulatory elements in the promoters of StCYP85A3"

调控元件
Cis-element		 核心序列
Core sequence		 元件功能描述
Functional description of cis-element		 位置
Position
ABRE CACGTG ABA响应元件
Cis-acting element involved in the abscisic acid responsiveness		 -6, -574
ARE AAACCA 顺式作用调节元件对无氧诱导至关重要
Cis-acting regulatory element essential for the anaerobic induction		 -1467
TGA-element AACGAC 生长素响应元件
Auxin-responsive element		 -205, -438
Box4 ATTAAT 参与光响应的保守DNA模块的一部分
Part of a conserved DNA module involved in light responsiveness		 -12, -112, -818, -1082, -1442
GT1-motif GGTTAA 光响应元件
Cis-acting element for light responsive		 -91
G-box TACGTG 光响应元件
Cis-acting element for light responsive		 -6, -574
CAT-box GCCACT 与分生组织表达相关的顺式作用调节元件
Cis-acting regulatory element related to meristem expression		 -245

Fig. 2

Analysis of CYP85A3 tissue expression and expression of CYP85A3 in the eye bud of C10 during storage A: CYP85A3 tissue expression; B: CYP85A3 expression during C10 storage. The error line represents the standard deviation. Student’s t-test is used for statistical analysis. Different lowercase letters represent significant differences between tissues and after storage at P < 0.05."

Fig. 3

Effect of BR exogenous application on potato tuber germination and root length A: the effects of different concentrations of BR on potato tuber germination rate; B: the effect of different concentrations of BR; C: the effect of BR treatment on the expression of StCYP85A3 in potato bud eyes; D: the effect of BR treatment on the expression of StCYP85A3 in potato seedling roots. The error line represents the standard deviation. Student’s t-test is used for statistical analysis. Different lowercase letters represent significant differences at P < 0.05."

Fig. 4

Overexpression, mutant, and supplementary germination rate A: the statistics of germination rate of different strains of Arabidopsis thaliana on MS medium; B: the statistics of germination rate of different lines of Arabidopsis thaliana on MS medium containing 50 nmol L-1 BR; C: the germination of Arabidopsis thaliana of different strains on MS medium after 3 days of planting; D: germination of Arabidopsis thaliana of different strains on MS medium containing 50 nmol L-1 BR after 60 hours of planting. The error line represents the standard deviation. The 60 hours data in Fig. A and Fig. B are jointly analyzed for significance. Student’s t-test is used for statistical analysis. Different lowercase letters indicate significant differences in germination of different strains at P < 0.05."

Fig. 5

Overexpression, mutant, and root length A: the statistics of root length of different lines of Arabidopsis thaliana on MS and MS medium containing 50 nmol L-1 BR; B: root length phenotypes of different Arabidopsis lines on MS medium; C: changes of StCYP85A3 expression in Arabidopsis thaliana roots treated with 50 nmol L-1 BR over time; D: root length phenotype of Arabidopsis thaliana lines treated with 50 nmol L-1 BR after 10 days of germination. The error line represents the standard deviation. Student’s t-test is used for statistical analysis. Different lowercase letters indicate significant differences in root length and the relative expression level at P < 0.05."

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