Mung bean (Vigna radiata) production has been greatly threatened by numerous diseases. Infection with these pathogens causes extensive changes in gene expression and the activation of hormone signal transduction. Quantitative real-time PCR (qRT-PCR) is the most common technique used for gene expression validation. Screening proper reference genes for mung bean under pathogen infection and hormone treatment is a prerequisite for ensuring the accuracy of qRT-PCR data in mung bean disease-resistance research. In this study, six candidate reference genes (Cons4, ACT, TUA, TUB, GAPDH, and EF1α) were selected to evaluate the expression stability under four soil-borne disease pathogens (Pythium myriotylum, Pythium aphanidermatum, Fusarium oxysporum, and Rhizoctonia solani) and five hormone treatments (SA, MeJA, ETH, ABA, and GA3). In the samples from different treatments, the Ct value distribution of the six candidate reference genes was different. Under the condition of hormone treatment, the Ct value ranged from a minimum of 17.87 for EF1α to a maximum of 29.63 for GAPDH. Under the condition of pathogen infection, the Ct value ranged from a minimum of 19.43 for EF1α to a maximum of 31.82 for GAPDH. After primer specificity analysis, it was found that GAPDH was not specific, so the five reference genes Cons4, ACT, TUA, TUB, and EF1α were used in subsequent experiments. The software products GeNorm, NormFinder, BestKeeper and RefFinder were used for qRT-PCR data analysis. In general, the best candidates reference genes were: TUA for SA, ABA, GA3, and Pythium myriotylum treatment; TUB for ETH treatment; ACT for MeJA and Fusarium oxysporum treatment; and EF1α for Pythium aphanidermatum and Rhizoctonia solani treatment. The most stably expressed genes in all samples were TUA, while Cons4 was the least stable reference gene. Finally, the reliability of the reference gene was further validated by analysis of the expression profiles of four mung bean genes (Vradi0146s00260, Vradi0158s00480, Vradi07g23860, and Vradi11g03350) selected from transcriptome data. Our results provide more accurate information for the normalization of qRT-PCR data in mung bean response to pathogen interaction.

Crop yield can be effectively improved by changing root architecture and improving root resistance to stress. Identification of root-specific promoters is prerequisite for research into the regulation of root development and genetic manipulation of root traits. As an important crop species, researches on the genes and promoters specifically expressed in soybean roots is still devoid. In our study, genomic-scale mining of root-specific genes were performed based on the transcriptome date of soybean root, stem and leaf tissues in the seedling stage, among which 105 putative genes were further verified by RT-PCR and 33root-specific genes were identified. Then, 11 promoters（pro1 to pro 11） were cloned from soybean. GUS staining of transgenic soybean hairy roots and Nicotiana benthamiana seedlings showed that the 11 promoters had high root-specific or preferential expression activities. Besides, the GUS enzyme activities driven by pro 1, pro 2, pro 8 and pro 9 were all two folds higher than that of 35S. Our research provided a reference for the identification of tissue specific promoters in soybean and other species. In addition, the promoters identified in this study can be used to drive the specific expression of resistance genes in soybean root tissues, so as to improve the resistance of soybean to root diseases and achieve the purpose of variety improvement.

由于缺乏有效稳定的遗传转化体系,导致基因编辑技术在绿豆中的应用受到极大限制,也使绿豆基因功能研究受到极大阻碍.因此,建立一套基于发根农杆菌的操作简便、快速且高效的绿豆嵌合植株转化技术,可以为绿豆基因功能研究提供技术支撑.首先在 CRISPR/Cas9 载体骨架中插入 1 个绿色荧光蛋白(green fluorescent protein,GFP)表达框用于阳性发状根的快速筛选,然后将含有靶标基因gRNA的CRISPR/Cas9 质粒导入发根农杆菌K599.菌液注射侵染绿豆幼苗植株下胚轴,评价K599 在绿豆中诱导发状根发生及CRISPR/Cas9 系统在绿豆转基因发状根组织中的效率.结果显示,K599 菌液侵染种植7d的绿豆幼苗植株并在保湿条件下培养 3 周可在侵染点诱导发状根产生,此嵌合植株在切除原生根后可在霍格兰培养液中正常生长且培养 1 周后即可用于后续检测.用GFP筛选的结果显示,阳性发状根占比约为(57.8±10.0)％.随机选择 15 个荧光检测阳性的转基因发状根组织,利用测序检测靶位点的编辑效果,结果显示有 10 个靶位点的序列发生了突变,突变比例达到 67％,其中碱基缺失突变 9 个,碱基转换突变 1 个.由本研究结果可知,利用该方法可以在无菌组织培养的条件下于4 周内获得目的基因编辑的转基因组织,极大地便利了绿豆分子水平上的功能研究,也为其他尚缺乏稳定遗传转化体系的作物进行基因功能研究提供了参考.

【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.