Helianthus annuus L. (oil sunflower) is ideal candidate for phytoremediation of cadmium (Cd)-contaminated soil. To understand the relationship between the Cd accumulation in oil sunflower and its associated rhizosphere bacteria and root endophytic bacteria, this paper discussed the rhizosphere microecological characteristics and Cd accumulation of oil sunflower in various low-moderate Cd-contaminated farmlands. Low-molecular-weight organic acids in rhizosphere soil synergistically promoted Cd absorption by roots: tartaric acid (acidic soils > 2 mg kg-1, alkaline soil < 1 mg kg-1) played a direct role, while oxalic, lactic, and succinic acids indirectly affected Cd absorption through regulating available P (increase rate: 4.73-31.63 %). Organic acids and available P also affect rhizosphere bacteria (e.g., norank Acidobacteria Gp3) and root endophytic bacteria (e.g., norank Candidatus Saccharibacteria), further enhancing Cd absorption. Soil pH had a greater impact on root endophytic bacterial communities than Cd concentration. Although Cd concentrations (all > 2 mg kg-1) and bioaccumulation factors (BCF > 3) in roots, stems, and leaves were significantly higher in acidic soils than in alkaline soil (Cd < 1 mg kg-1, BCF < 1), the Cd concentration in oil remained below food safety standards. Thus, oil sunflower is more suitable for the phytoremediation of low-moderate Cd-contaminated acidic soils.

Engineering anthocyanin biosynthesis in herbs could provide health-promoting foods for improving human health. Rehmannia glutinosa is a popular medicinal herb in Asia, and was a health food for the emperors of the Han Dynasty (59 B.C.). In this study, we revealed the differences in anthocyanin composition and content between three Rehmannia species. On the 250, 235 and 206 identified MYBs in the respective species, six could regulate anthocyanin biosynthesis by activating the ANTHOCYANIDIN SYNTHASE (ANS) gene expression. Permanent overexpression of the Rehmannia MYB genes in tobacco strongly promoted anthocyanin content and expression levels of NtANS and other genes. A red appearance of leaves and tuberous/roots was observed, and the total anthocyanin content and the cyanidin-3-O-glucoside content were significantly higher in the lines overexpressing RgMYB41, RgMYB42, and RgMYB43 from R. glutinosa, as well as RcMYB1 and RcMYB3 in R. chingii and RhMYB1 from R. henryi plants. Knocking out of RcMYB3 by CRISPR/Cas9 gene editing resulted in the discoloration of the R. chingii corolla lobes, and decreased the content of anthocyanin. R. glutinosa overexpressing RcMYB3 displayed a distinct purple color in the whole plants, and the antioxidant activity of the transgenic plants was significantly enhanced compared to WT. These results indicate that Rehmannia MYBs can be used to engineer anthocyanin biosynthesis in herbs to improve their additional value, such as increased antioxidant contents.

为了研究CRISPR/Cas9技术在天目地黄基因编辑中的应用,克隆了天目地黄(Rehmannia chingii)的八氢番茄红素脱氢酶基因(RcPDSI),利用PCR方法扩增其cDNA序列和基因组DNA序列.通过构建单靶点CRISPR/Cas9载体,利用根癌农杆菌介导的遗传转化方法侵染天目地黄无菌苗叶盘,通过TA克隆测序法分析基因编辑的类型.结果显示,克隆获得了 1个天目地黄RcPDS1的全长cDNA序列,其具有1个长度为1 743 bp的开放阅读框,编码580个氨基酸残基,基因组DNA序列长度8 041 bp,包含14个内含子和15个外显子.通过遗传转化共获得57个转基因再生株系,其中具明显白化表型的株系有20个(35.09％).TA克隆测序结果显示,RcPDS1靶位点突变类型主要包括碱基缺失、替换和插入.利用CRISPR/Cas9基因编辑技术在天目地黄中成功实现了对RcPDS1基因的靶向敲除.

MYB转录因子是植物最大的转录因子家族之一,广泛参与植物的生长发育、逆境胁迫和次生代谢产物积累.该研究通过同源比对和功能注释,在地黄(Rehmannia glutinosa)转录组中筛选出MYB的转录本,设计特异性引物对MYB基因的cDNA序列进行PCR扩增,用水杨酸(SA)、Ag+、茉莉酸甲酯(MeJA)和腐胺(Put)这4种诱导子处理地黄毛状根,并通过实时荧光定量PCR(qRT-PCR)检测候选MYB基因的表达.结果显示:(1)成功克隆到1个地黄MYB基因,命名为RgMYB10;该基因编码247个氨基酸残基,蛋白质相对分子质量28.48 kD,等电点为5.14,属于R2R3-MYB转录因子.(2)qRT-PCR结果显示,RgMYB10在须根中表达量最高,其次为茎,块根中的表达量最低.(3)RgMYB10在MeJA处理后的毛状根中显著上调表达,为特异响应MeJA诱导的基因,推测Rg-MYB10基因可能是响应MeJA参与地黄毛蕊花糖苷生物合成的关键转录因子.研究表明,地黄MYB10基因可能参与地黄毛蕊花糖苷的生物合成,为进一步研究MYB10基因在地黄毛蕊花糖苷合成中的功能奠定了基础.

WRKYs play important roles in plant metabolism, but their regulation mechanism in Rehmannia glutinosa remains elusive. In this study, 37 putative WRKY transcription factors (TFs) with complete WRKY domain from R. glutinosa transcriptome sequence data were identified. Based on their conserved domains and zinc finger motif, the R. glutinosa WRKY TFs were divided into five groups. Structural feature analysis shows that the 37 RgWRKY proteins contain WRKYGQK/GKK domains and a C2H2/C2HC-type zinc finger structure. To identify the function of RgWRKY members involved in acteoside biosynthesis, transcriptional profiles of 37 RgWRKYs in hairy roots under salicylic acid (SA), methyl jasmonate (MeJA), and hydrogen peroxide (H2O2) treatments were systematically established using RNA-seq analysis. Based on the correlationship between the expression levels of RgWRKY genes and acteoside content, RgWRKY7, RgWRKY23, RgWRKY34, RgWRKY35, and RgWRKY37 were suggested to be involved in acteoside biosynthesis in R. glutinosa, and RgWRKY37 was selected for gene functional research. Overexpression of RgWRKY37 increased the content of acteoside and total phenylethanoid glycosides (PhGs) in hairy roots and enhanced the transcript abundance of seven enzyme genes involved in the acteoside biosynthesis pathway. These results strongly suggest the involvement of the WRKY transcription factor in the regulation of acteoside biosynthesis.

Here, we cloned a phytoene desaturase (PDS) gene from Rehmannia glutinosa, and realized RgPDS1 knock out in R. glutinosa resulted in the generation of albino plants. Rehmannia glutinosa is a highly important traditional Chinese medicine (TCM) with specific pharmacology and economic value. R. glutinosa is a tetraploid plant, to date, no report has been published on gene editing of R. glutinosa. In this study, we combined the transcriptome database of R. glutinosa and the reported phytoene desaturase (PDS) gene sequences to obtain the PDS gene of R. glutinosa. Then, the PDS gene was used as a marker gene to verify the applicability and gene editing efficiency of the CRISPR/Cas9 system in R. glutinosa. The constructed CRISPR/Cas9 system was mediated by Agrobacterium to genetically transform into R. glutinosa, and successfully regenerated fully albino and chimeric albino plants. The next-generation sequencing (NGS) confirmed that the albino phenotype was indeed caused by RgPDS gene target site editing, and it was found that base deletion was more common than insertion or replacement. Our results revealed that zCas9 has a high editing efficiency on the R. glutinosa genome. This research lays a foundation for further use of gene editing technology to study the molecular functions of genes, create excellent germplasm, accelerate domestication, and improve the yield and quality of R. glutinosa.

The present study analyzed the effects of planting density on the development, quality, and gene transcription characte-ristics of Rehmannia glutinosa using 85-5 and J9 as materials with three planting densities of 5 000, 25 000, and 50 000 plants/Mu(1 Mu≈667 m~2). The agronomic characteristics of leaves and tuberous roots, the content of catalpol and acteoside, and the changes of gene expression were determined. The results showed that the leaf size, the diameter of tuberous root, leaf biomass, tuberous root number, and tuberous root biomass per plant at low density were significantly higher than those of medium and high densities. The content of catalpol and acteoside in leaves was higher at high density. The content of catalpol in tuberous roots was higher at low density, and the change trend was similar to that in leaves, while the content of acteoside in tuberous roots was higher at high density. Transcriptome analysis found that about 1/2 of the expansin genes could change regularly in response to density treatment, which was rela-ted to the development of tuberous roots. The change trend of the gene expression of multiple catalytic enzymes involved in the biosynthesis of catalpol and acteoside was consistent with that of their content, which was presumedly involved in the accumulation and regulation of density-responsive medicinal components. Based on the analysis of the development, medicinal components, and gene expression characteristics of R. glutinosa at different densities, this study is expected to provide an important basis for regulating the quality and yield of medicinal materials of R. glutinosa by managing the planting density.

目的 克隆地黄Rehmannia glutinosa毛蕊花糖苷合酶基因(RgAcS1),分析其亚细胞定位和表达模式.方法 在地黄的转录组数据库中通过注释和比对,获得地黄RgAcS1的cDNA序列,利用聚合酶链式反应(PCR)方法进行分子克隆.构建绿色荧光蛋白(GFP)融合表达载体,以农杆菌瞬时表达法观测RgAcS1的亚细胞定位.利用实时荧光定量PCR(qRT-PCR)检测RgAcS1基因在地黄块根不同部位的表达模式.结果 获得地黄1个莽草酸-O-羟基肉桂酰基转移酶的全长编码序列,cDNA长度为1 659 bp,包含1个1 296bp的开放阅读框,编码431个氨基酸残基,蛋白质相对分子质量为475 900,具有莽草酸-O-羟基肉桂酰基转移酶的典型结构域,命名为RgAcS1.亚细胞定位结果显示RgAcS1主要分布在细胞质中,在细胞核中也有分布.qRT-PCR分析表明,RgAcS1在地黄的周皮和根毛中表达量较高,在木质部和韧皮部表达量较低.RgAcS1基因在地黄品种北京1号、QH1和85-5中非菊花心中表达量均高于菊花心中的表达量,且差异达极显著水平.结论 获得地黄RgAcS1的cDNA序列,明确了RgAcS1的亚细胞定位和时空表达模式,为进一步研究RgAcS1基因在毛蕊花糖苷合成过程中的作用奠定基础.

基因编辑是一项能够在生物体基因组水平上实现对DNA序列精确定向修饰的新技术.该技术主要利用序列特异性核酸酶靶向识别切割基因组上目标位点,造成DNA双链断裂(DSBs),进一步诱发非同源末端连接(NHEJ)和同源性重组(HR)2种修复机制对断裂的DNA双链进行修复,实现修复位点碱基的插入、缺失和替换,从而达到对靶基因精确编辑的目的.对基因编辑技术的类型、作用原理及该技术在植物领域中的应用进行综述,并对基因编辑技术在药用植物功能基因组学和遗传改良中的应用前景进行展望.