Protein and threonine are the core nutrients of mung bean seeds, which have a critical impact on the nutritional quality of mung beans. Among them, mung bean protein is a high-quality protein source for vegetarians due to its easy digestion, absorption, and rich biological activity, which is of great value to human health. This study aims to explore genetic loci and candidate genes significantly associated with the protein content and relative content of threonine in mung bean seeds, providing a theoretical basis for genetic improvement of protein and threonine content in mung bean seeds.

This study used Kjeldahl nitrogen determination method and liquid chromatography-mass spectrometry to determine the protein content and relative content of threonine in 279 mung bean varieties planted in 2022. The general linear model of Tassel5 software was used for genome-wide association study to explore genetic loci related to protein content and relative content of threonine in mung bean seeds. By combining linkage disequilibrium analysis, transcriptome comparative analysis, and comparative genomics, key candidate genes related to nitrogen metabolism were identified.

Six key loci located on chromosome 3 were identified, which are related to the protein content and relative content of threonine in mung bean seeds, and can explain 6.06% to 12.20% of phenotypic variation. These 6 loci are closely linked and can be mainly divided into two haplotypes, with Hap2 being the dominant haplotype. Then, KASP molecular marker was designed for the key candidate locus SLG03_966208 (A/G), which can successfully classify 80 mung bean varieties into three genotypes: AA, GG, and AG, with a detection accuracy rate of 87.5%. The key candidate gene EVM0000757 was identified within the locus linkage region, and the homologous gene AT5G65750 in Arabidopsis was significantly associated with nitrogen metabolism. Its gene expression was significantly different between high and low protein varieties (P=1.84E-03, |log₂FC|=1.39), making it a reliable candidate gene.

279 mung bean varieties were identified for their grain protein content and relative content of threonine. Six SNP loci were detected within the 939296-1039749 bp region of chromosome 3. A KASP molecular marker for the SLG03_966208 (A/G) locus was developed, which can distinguish the high and low levels of protein and threonine content in mung bean grains. A key candidate gene related to nitrogen metabolism, EVM0000757, was identified.

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.

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.

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.

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.