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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (9): 2157-2166.doi: 10.3724/SP.J.1006.2024.44018

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

Cloning and functional analysis of drought-inducible promoter AhMYB44-11- Pro in peanut (Arachis hypogaea L.)

LIU Yong-Hui(), SHEN Yi, SHEN Yue, LIANG Man, SHA Qin, ZHANG Xu-Yao, CHEN Zhi-De*()   

  1. Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China
  • Received:2024-01-29 Accepted:2024-05-21 Online:2024-09-12 Published:2024-06-12
  • Contact: *E-mail: chen701865@aliyun.com
  • Supported by:
    China Agriculture Research System of MOF and MARA(CARS-13);Open Competition Project for Seed Industry of Jiangsu Province(JBGS [2021] 062)

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Abstract:

Drought is a significantly environmental factor that poses a serious threat to peanut yield and quality in China. Our study aims to elucidate the regulatory mechanism of the drought stress response gene AhMYB44-11 and uncover the function of its promoter. We isolated the promoter sequence AhMYB44-11-Pro from the peanut genome and constructed recombinant expression vectors comprising the full-length promoter and several 5'-terminal deletion promoters, each fused with the GUS reporter gene. These vectors were then introduced into plant tissue via Agrobacterium rhizogenes-mediated transformation to assess promoter activity and expression patterns. The results revealed that AhMYB44-11-Pro contains a higher number of drought-responsive cis-regulatory elements, including MBS and Myb-binding sites, compared to the promoter of its homologous gene AhMYB44-01. Furthermore, dehydration treatment significantly enhanced GUS staining and activity in AhMYB44-11-Pro transgenic Arabidopsis, indicating that the promoter’s activity is upregulated under drought stress. These findings confirm that AhMYB44-11-Pro functions as a drought-inducible promoter. Additionally, AhMYB44-11-Pro contains a seed endosperm-specific expression element and gibberellin response elements, demonstrating an increased expression trend during pod development, as evidenced by GUS histochemical staining. This suggests that AhMYB44-11 may play a crucial role in seed development, particularly in dry matter accumulation. This research lays the groundwork for a comprehensive analysis of the biological functions of AhMYB44 and provides a valuable reference for genetically enhancing crop drought resistance.

Key words: peanut, AhMYB44, promoter, drought stress, GUS activity

Table 1

Primers for promoter isolation and vectors construction"

用途
Function		 引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
基因克隆Gene cloning Pro0-F GGCGTGCGGTCTTCTGGGGT
Pro0-R GCTACGATGCCGCTATCCAC
全长以及缺失序列载体构建
Vectors construction of full-length and several deletion sequences		 Pro1-F GTCGACCTGCAGGCATGCAAGCTTGTTCTTGTAACAACTTGAGGC
Pro2-F GTCGACCTGCAGGCATGCAAGCTTAAGTTTGATAGTAAGGGTG
Pro3-F GTCGACCTGCAGGCATGCAAGCTTGTTGATTGGATTCTTTGC
Pro4-F GTCGACCTGCAGGCATGCAAGCTTATGGTCTTGTCTTCGTTA
Pro5-F GTCGACCTGCAGGCATGCAAGCTTCAGAGCAGGGATGACTTG
Pro6-F GTCGACCTGCAGGCATGCAAGCTTATCGTAACGCCCTACCTA
Pro (1-6)-R GAAATTTACCCTCAGATCTACCATGGAGTGCTTAGTTTCTTTTGTTTTG

Fig. 1

Schematic diagram of cis-regulatory elements distribution in the AhMYB44-01/11 promoters"

Fig. 2

Electrophoresis analysis of the full-length promoter and its 5°-deletion fragments M: DL2000 marker; P0: a long fragment containing the full-length promoter; P1-P6: the full-length promoter and its 5°-deletion fragments."

Fig. 3

Vectors diagram expressing GUS in plants driven by different length AhMYB44-11-Pro with 5°-deletion P1-P6: the full-length promoter and its 5°-deletion fragments."

Fig. 4

GUS histochemical detection of different length AhMYB44-11-Pro with 5°-deletion in transgenic tobacco P1-P6: transgenic tobacco leaves transformed with different lengths of AhMYB44-11-Pro series expression vectors respectively."

Fig. 5

GUS activity assay of different length AhMYB44-11-Pro with 5°-deletion in transgenic tobacco P1-P6: transgenic tobacco leaves transformed with different lengths of AhMYB44-11-Pro series expression vectors. Different lower-case letters and upper-case letters above the bars indicate significant differences at P < 0.05 and P < 0.01, respectively."

Fig. 6

GUS histochemical detection of AhMYB44-11-Pro in transgenic Arabidopsis A: seedlings germinated for 2 days; B: seedlings germinated for 3 days; C: seedlings germinated for 7 days; D: stem transection; E: inflorescence; F: stem node; G: silique."

Fig. 7

Drought stress induced expression analysis of AhMYB44-11-Pro transgenic Arabidopsis A: transcriptional level of AhMYB44-11-Pro in transgenic lines Pro-21 and Pro-27 assayed by semi-quantitative reverse transcription PCR; 35S: transgenic plants containing the CaMV35S promoter empty vector; Actin2 was used as an internal control. B, C: GUS staining and activity quantification of transgenic Arabidopsis seedlings under normal growth condition and drought stress, ** above the bars indicates significant differences at P < 0.01."

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