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.

组织培养的关键是对外植体灭菌,目前效果最佳的灭菌剂氯化汞属于剧毒试剂,对环境及人体均有危害。为探究更加安全有效的灭菌方式,本研究以绿豆为试验材料,以75%乙醇+30%次氯酸钠联合灭菌、4%过碳酸钠、氯气3种灭菌处理方式搭配灭菌时间共8组处理,统计其萌发及污染情况,并在7d时统计长势相关数据。另外,每组在接种3d时制备外植体转接至分化培养基,观察统计污染及丛生芽状况,根据数据分析结果选出最优灭菌剂及灭菌时间。结果表明, 75%乙醇30s+30%次氯酸钠30min处理下其污染率为4%,丛生芽分化率为82%,生长速率与生长状况良好。因此,该方法最适合于绿豆组织培养的种子灭菌,有利于愈伤组织的诱导。

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.