Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (20): 3885-3896.doi: 10.3864/j.issn.0578-1752.2022.20.002

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

Identification of the Root-Specific Soybean GmPR1-9 Promoter and Application in Phytophthora Root-Rot Resistance

YAN Qiang(),XUE Dong,HU YaQun,ZHOU YanYan,WEI YaWen,YUAN XingXing,CHEN Xin()   

  1. Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210014
  • Received:2022-05-05 Accepted:2022-06-27 Online:2022-10-16 Published:2022-10-24
  • Contact: Xin CHEN E-mail:yanqiang@jaas.ac.cn;cx@jaas.ac.cn

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Abstract:

【Objective】The objective of this study is to identify the root-specific promotors and the core regulatory sequence of soybean. Then evaluate the potential application of the synthetic promoter in Phytophthora root-rot resistance. 【Method】The genes which specifically expressed in roots with high expression levels were screened based on the transcriptome date of soybean root, stem and leaf tissues in the seedling stage. Based on the distribution of the cis elements, the promoter truncation approach was used to map the minimal promoter controlling root specific expression in soybean hairy roots. The obtained minimal promoter fragment was concatenated with the Phytophthora inducible promoter elements p4XD to construct the synthetic promoter. The synthetic promoter driven over-expression of Phytophthora resistance related gene GmNDR1 in soybean hairy roots, then the resistance level of transgenic tissue to Phytophthora and the expression profiles of GmNDR1 during the interaction had been analyzed. Furthermore, the transgenic Nicotiana benthamiana plants were generated to evaluate the resistance at plant level. 【Result】Though screening, six soybean PR1 homologues with significant root specific expression manner were identified, and GmPR1-9 had the highest promoter activity. Numbers of root specific expression related cis elements were identified in promoter sequence using the online tool PLACE. Truncation analysis of the promoter showed that serial 5’ end deletions L1, L2, L3, L4 and L5 had different GUS activities. The L5 (-166 to -1) fragment had 80% activity of the full-length promoter, and was able to drive GUS expression in roots of transgenic N. benthamiana. GUS enzyme activity was almost undetectable in three 3’ end deletions R1, R2 and R3, and the double terminal deletion mutant M1. When the fusion promoter p4XD-L5 driven GmNDR1 expression in soybean hairy roots, the resistance to P. sojae was significantly enhanced. The disease severity and lesion length were significantly reduced in the over-expression hairy roots when compared with control, and the relative biomass of Phytophthora decreased by 66.5% at 48 h post inoculation. GmNDR1 maintained high expression level in over-expression tissues, with 39.2 times of that in control tissues. The expressions were further up-regulated after inoculation, and reached the highest level at 36 h. In p4XD-L5::NDR1 transgenic N. benthamiana plants, the expression of GmNDR1 was significantly higher in roots than that in stems and leaves. Fifteen days after P. capsica inoculation, the plant height, root length and fresh weight of GmNDR1 over-expression plants were significantly higher, and meanwhile the leaf wilting rate and lesion length were significantly lower. 【Conclusion】This study obtained a soybean root specific promoter and identified the core regulation sequence. The strategy which driven the expression of GmNDR1 by the synthetic promoter p4XD-L5 combined the inducible and tissue-specific promoter core elements can significantly enhance the resistance of transgenic soybean hairy roots and Nicotiana benthamiana plants to Phytophthora pathogens.

Key words: soybean (Glycine max), root specific promotor, GmPR1-9, synthetic promoter, Phytophthora root-rot resistance

Fig. 1

The relative expression of GmPR1-9 in different soybean organs A: Heat map representation of GmPR1-9 and homologous expression (log10FPKM); B: GmPR1-9 expression by RT-PCR analysis"

Fig. 2

Diagram of promoters with root specific expression related cis elements"

Fig. 3

Expression activity analysis of truncated pGmPR1-9 promoters in soybean hairy roots A: Schematic diagrams of truncated pGmPR1-9 promoter constructs. L1-M1: Promoter truncation position information; B: GUS histochemical assays in transgenic soybean hairy roots"

Fig. 4

GUS enzyme activity assays of different truncated pGmPR1-9 promoters Different letters indicate significant differences (P<0.05). The same as below"

Fig. 5

Tissue specific expression analysis of L5 promoter fragment A: Fluorescent screening of transgenic N. benthamiana plants; B-E: Histochemical GUS staining of 10 d old transgenic N. benthamiana whole plants, leaf, stem and root. bar=1 mm"

Fig. 6

Evaluation of Phytophthora resistance in p4XD-L5::NDR1 over-expression soybean hairy roots A: Phenotypes of the soybean hairy roots after P. sojae inoculation; B: Statistics of the lesion length; C: Relative biomass of P. sojae in inoculated hairy roots; D: Relative expression of GmDNR1 in inoculated hairy roots. EV indicates empty vector control. ** indicates significant differences (P<0.01). The same as below"

Fig. 7

The expression levels of GmNDR1 in root, stem and leaf tissues of transgenic N. benthamiana plants"

Fig. 8

Phenotypes of the p4XD-L5::NDR1 over-expression N. benthamiana plants after P. capsici inoculation A-C: Phenotypes of the N. benthamiana plants inoculated with P. capsici zoospores at 0, 5, and 10 dpi; D-E: Stem and root lesion phenotypes of the inoculated N. benthamiana plants at 10 dpi"

Fig. 9

P. capsici resistance of p4XD-L5::NDR1 over-expression N. benthamiana plants A: Plant height; B: Percentage of leaf wilting; C: Lesion length; D: Taproot length; E: Plant fresh weight"

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