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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (3): 590-596.doi: 10.3724/SP.J.1006.2022.14016

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

Cloning and functional analysis of the promoter of purple acid phosphatase gene GmPAP14 in soybean

ZHOU Yue(), ZHAO Zhi-Hua, ZHANG Hong-Ning, KONG You-Bin*()   

  1. North China Key Laboratory for Germplasm Resources of Education Ministry/College of Agronomy, Hebei Agricultural University, Baoding 071001, Hebei, China
  • Received:2021-01-26 Accepted:2021-06-16 Online:2021-07-24 Published:2021-07-24
  • Contact: KONG You-Bin E-mail:1584868048@qq.com;kong_1985@163.com
  • Supported by:
    Natural Science Foundation of Hebei Province(C2018204090);Modern Agricultural Science and Technology Award Post-subsidy Fund Project(17927670H)

Abstract:

The expression of GmPAP14 was induced under low phosphorus condition, and its overexpression could significantly improve the utilization efficiency of organic phosphorus in Arabidopsis. In order to further explore its regulatory mechanism, the promoter sequence of GmPAP14 was cloned according to the sequence of soybean reference genome. The regulatory elements of GmPAP14 promoter were predicted by the database PLACE and PlantCARE, which showed that it contained enhancer regulatory elements, tissue-specific expression elements, root-specific expression elements, and P1BS elements (transcription factor PHR1 binding sites). PGmPAP14-2568-GUS, PGmPAP14-2238-GUS, and PGmPAP14-1635-GUS were constructed and transferred into Arabidopsis thaliana via Floral dip method. The expressional activities of three fragments of GmPAP14 promoter were analyzed through GUS staining and activity measurement. The results revealed that GmPAP14 promoter was mainly expressed in root tip under Pi condition, and GUS staining was extended to the elongation area, mature area, and root hair after low phosphorus treatment. Additionally, Arabidopsis plants with PGmPAP14-2238-GUS had the highest activity among them. These results lay an important foundation for the further study of gene regulation.

Key words: soybean, GmPAP14, promoter, induced expression, low phosphorus

Table 1

Primers used in this study"

引物名称
Primer name
引物序列
Primer sequence (5°-3°)
用途
Function
PGmPAP14-F/R F: AGTCGAAGATGGGGATGT
R: TTTGCTCAAAACCCGTG
GmPAP14启动子克隆
Cloning of GmPAP14 promoter
PGmPAP14-2568-F/R F: AAGCTTAGTCGAAGATGGGGATGT
R: GGATCCTTTGCTCAAAACCCGTG
PGmPAP14-2568克隆
Cloning of PGmPAP14-2568
PGmPAP14-2238-F/R F: AAGCTTTAACAACACAAGTTGTGAATTTC
R: GGATCCTTTGCTCAAAACCCGTG
PGmPAP14-2238克隆
Cloning of PGmPAP14-2238
PGmPAP14-1635-F/R F: AAGCTTCTGAGGAAAGACTACAGTATAC
R: GGATCCTTTGCTCAAAACCCGTG
PGmPAP14-1635克隆
Cloning of PGmPAP14-1635
PGmPAP14-Gus-F/R F: GGATCACTAGAATCACGGGTTTTG
R: ACACTTTTCCCGGCAATAACATAC
转基因植株检测
Detection of transgenic plants

Fig. 1

Cloning of GmPAP14 promoter M: DNA marker DL2000; 1: amplified production of GmPAP14 promoter."

Table 2

Analysis of the cis-elements of GmPAP14 promoter"

调控元件
Regulation element
功能注释
Functional annotation
数目
Number
W box 诱导应答元件 Induced response element 2
P1BS PHR1转录因子结合位点 PHR1-binding site 1
RHERPATEXPA7 根尖特异表达元件 Root hair-specific cis-elemen 2
OSE1ROOTNODULE 组织特异元件 Organ-specific element 5
OSE2ROOTNODULE 组织特异元件 Organ-specific element 8
ROOTMOTIFTAPOX1 组织特异元件 Organ-specific element 20
A/T rich element 增强子元件 Enhancer element 1
ABRELATERD1 脱落酸响应元件 Cis-acting element involved in the abscisic acid responsiveness 1
GAREAT 赤霉素响应元件 GA-responsive element 2
TGACG-motif 茉莉酸甲酯响应元件 Cis-acting regulatory element involved in MeJA response 2
CAAT-box 启动子和增强子区的一般顺式作用元件
Common cis-acting element in promoter and enhancer regions
40
TATA-box 转录起始位点-30核心启动子元件Core promoter element around -30 of transcription start 26

Fig. 2

Amplified fragment in 5° terminal deletion fragments of GmPAP14 promoter A: amplification of 5° terminal deletion fragments. B: the diagram of 5° terminal deletion fragments of GmPAP14 promoter. M: DNA marker DL5000; 1: amplification of PGmPAP14-2568 with Hind III/BamH I; 2: amplification of PGmPAP14-2238 with Hind III/BamH I; 3: amplification of PGmPAP14-1635 with Hind III/BamH I. B: Schematic show of 5° terminal deletion fragments of GmPAP14 promoter."

Fig. 3

Detection of transgenic Arabidopsis plants with GmPAP14 promoter M: DNA marker DL2000; 1: water control; 2: wild type control; 3: plasmid control; 4-6: transgenic Arabidopsis with PGmPAP14-2568-GUS; 7-9: transgenic Arabidopsis with PGmPAP14-2238-GUS; 10-12: transgenic Arabidopsis with PGmPAP14-1635-GUS."

Fig. 4

Chemical staining in different tissues of transgenic Arabidopsis with PGmPAP14-GUS A: root; B: leaf; C: stem; D: flower."

Fig. 5

GUS staining of transgenic Arabidopsis with 5° terminal deletion fragments of GmPAP14 promoter under different P conditions PGmPAP14-2568-GUS, PGmPAP14-2238-GUS, and PGmPAP14-1635-GUS represent transgenic Arabidopsis with 5° terminal deletion fragments of GmPAP14 promoter, respectively. +Pi: KH2PO4; +Po: phytate-P."

Fig. 6

GUS activities of transgenic Arabidopsis with 5° terminal deletion fragments of GmPAP14 promoter under different P condition PGmPAP14-2568-GUS, PGmPAP14-2238-GUS, and PGmPAP14-1635-GUS represent transgenic Arabidopsis with 5° terminal deletion fragments of GmPAP14 promoter, respectively. +Pi: KH2PO4; +Po: phytate-P. Different lowercase letters mean significant difference at the 0.05 probability level."

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