Expression of mouse metallothionein-I gene confers cadmium resistance in transgenic tobacco plants

Transgenic tobacco plants containing a mouse metallothionein-I (MT-I) gene fused to the cauliflower mosaic virus 35S (CaMV 35S) promoter and nopaline synthase (nos) polyadenylation site were obtained by transforming tobacco leaf discs with an Agrobacterium tumefaciens strain carrying the chimaeric gene. Transformants were directly selected and rooted on medium containing cadmium and kanamycin. A total of 49 individual transgenic tobacco plants were regenerated. Among them 20% showed a very high expression level and their growth was unaffected by up to 200 M cadmium, whereas the growth of control plants was severely affected leaf chlorosis occurred on medium containing only 10 M cadmium. The concentration of MT-I in leaves of control and transgenic tobacco was determined with Cd/haemoglobin saturation assay, a polarographic method and western blotting. In addition, seeds from self-fertilized transgenic plants were germinated on medium containing toxic levels of cadmium and scored for tolerance/susceptibility to this heavy metal. The ratio of tolerant to susceptible plants was 3:1 indicating that the metallothionein gene is inherited as a single locus.     

金属螯合亲和层析纯化金属硫蛋白

将二价铜离子螯合在Chelating Sepharose Fast Flow凝胶上制成亲和层析柱,锌诱导兔肝和镉诱导小鼠肝经匀浆、乙醇处理后上柱,用pH4.0的醋酸盐缓冲液平衡,再用pH5.2不同浓度的醋酸盐缓冲液分别洗脱,可得两个金属硫蛋白(MT)洗脱峰,经确定先后为MT-2和MT-1.分离方法比传统的凝胶过滤-离子交换法简单、省时,适于实验室规模分离纯化.     

α-Domain of human metallothionein IA can bind to metals in transgenic tobacco plants

We have introduced a genetically marked Dissociation transposable element (Ds ^HPT ) into tomato (Lycopersicon esculentum) by Agrobacterium tumefaciens-mediated transformation. Probes for the flanking regions of the T-DNA and transposed Ds ^HPT elements were obtained with the inverse polymerase chain reaction (IPCR) technique and used in RFLP linkage analyses. The RFLP map location of 11 T-DNAs carrying Ds ^HPT was determined. The T-DNAs are distributed on 7 of the 12 tomato chromosomes. To explore the feasibility of gene tagging strategies in tomato using Ds ^HPT , we examined the genomic distribution of Ds ^HPT receptor sites relative to the location of two different, but very closely linked, T-DNA insertion sites. After crosses with plants expressing Ac transposase, the hygromycin phosphotransferase (HPT) marker on the Ds element and the excision markers β-glucuronidase (GUS) and Basta resistance (BAR) facilitated the identification of plants bearing germinally transposed Ds ^HPT elements. RFLP mapping of 21 transposed Ds ^HPT elements originating from the two different T-DNA insertions revealed distinct patterns of reintegration sites.     

通过和玉米杂交诱导硬粒小麦单倍体

硬粒小麦（Ｔｒｉｔｉｃｕｍ ｄｕｒｕｍ Ｄｅｓｆ．）ＤＲ１４７授以超甜玉米（Ｚｅａ ｍ ａｙｓ Ｌ．） ｓｓ ７７００的花粉后,在８３．４％ 的柱头上观察到花粉萌发及花粉管长入胚囊,有９．９％ 的子房发生了卵细胞的单受精,１．９％ 的子房发生了中央细胞的单受精,３２．７％ 的子房发生了双受精。尽管双受精后可同时形成胚和胚乳,但胚乳往往发育迟缓,甚至败育。硬粒小麦×玉米形成的杂合子核型高度不稳定,在最初的几次细胞分裂中,来自父本玉米的染色体逐步被排除,最后形成硬粒小麦单倍体胚。在授以玉米花粉４ ｈ 后用１００ ｐｐｍ ２,４－Ｄ溶液浸蘸硬粒小麦穗部,可以有效地促进幼胚在缺乏胚乳或胚乳败育情况下的生长和发育。授粉９—１３ ｄ 后由５３３个硬粒小麦子房解剖出２５个胚,得胚率为４．７％ 。通过幼胚拯救获得１１棵正常植株,植株获得率为２．１％ 。根尖细胞染色体计数表明,它们为单倍体（２ｎ＝ ２ｘ＝ １４）。     

Identification of cancer hallmark‐associated gene and lncRNA cooperative regulation pairs and dictate lncRNA roles in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common malignant tumour in the oral and maxillofacial region. Numerous cancers share ten common traits (“hallmarks”) that govern the transformation of normal cells into cancer cells. Long non‐coding RNAs (lncRNAs) are important factors that contribute to tumorigenesis. However, very little is known about the cooperative relationships between lncRNAs and cancer hallmark‐associated genes in OSCC. Through integrative analysis of cancer hallmarks, somatic mutations, copy number variants (CNVs) and expression, some OSCC‐specific cancer hallmark‐associated genes and lncRNAs are identified. A computational framework to identify gene and lncRNA cooperative regulation pairs (GLCRPs) associated with different cancer hallmarks is developed based on the co‐expression and co‐occurrence of mutations. The distinct and common features of ten cancer hallmarks based on GLCRPs are characterized in OSCC. Cancer hallmark insensitivity to antigrowth signals and self‐sufficiency in growth signals are shared by most GLCRPs in OSCC. Some key GLCRPs participate in many cancer hallmarks in OSCC. Cancer hallmark‐associated GLCRP networks have complex patterns and specific functions in OSCC. Specially, some key GLCRPs are associated with the prognosis of OSCC patients. In summary, we generate a comprehensive landscape of cancer hallmark‐associated GLCRPs that can act as a starting point for future functional explorations, the identification of biomarkers and lncRNA‐based targeted therapy in OSCC.     

Comparative Transcriptomic Analysis of Storage Roots in Cassava During Postharvest Physiological Deterioration

Plant Molecular Biology Reporter - Cassava is an important starchy and food crop; however, the commercial value of cassava is seriously constrained by postharvest physiological deterioration (PPD)....     

Drought stress and plant ecotype drive microbiome recruitment in switchgrass rhizosheath

The rhizosheath, a layer of soil grains that adheres firmly to roots, is beneficial for plant growth and adaptation to drought environments. Switchgrass is a perennial C4 grass which can form contact rhizosheath under drought conditions. In this study, we characterized the microbiomes of four different rhizocompartments of two switchgrass ecotypes (Alamo and Kanlow) grown under drought or well-watered conditions via 16S ribosomal RNA amplicon sequencing. These four rhizocompartments, the bulk soil, rhizosheath soil, rhizoplane, and root endosphere, harbored both distinct and overlapping microbial communities. The root compartments (rhizoplane and root endosphere) displayed low-complexity communities dominated by Proteobacteria and Firmicutes. Compared to bulk soil, Cyanobacteria and Bacteroidetes were selectively enriched, while Proteobacteria and Firmicutes were selectively depleted, in rhizosheath soil. Taxa from Proteobacteria or Firmicutes were specifically selected in Alamo or Kanlow rhizosheath soil. Following drought stress, Citrobacter and Acinetobacter were further enriched in rhizosheath soil, suggesting that rhizosheath microbiome assembly is driven by drought stress. Additionally, the ecotype-specific recruitment of rhizosheath microbiome reveals their differences in drought stress responses. Collectively, these results shed light on rhizosheath microbiome recruitment in switchgrass and lay the foundation for the improvement of drought tolerance in switchgrass by regulating the rhizosheath microbiome.     

The ScCas9++ variant expands the CRISPR toolbox for genome editing in plants

The development of clustered regularly interspaced palindromic repeats (CRISPR)-associated protein (Cas) variants with a broader recognition scope is critical for further improvement of CRISPR/Cas systems. The original Cas9 protein from Streptococcus canis (ScCas9) can recognize simple NNG-protospacer adjacent motif (PAM) targets, and therefore possesses a broader range relative to current CRISPR/Cas systems, but its editing efficiency is low in plants. Evolved ScCas9⁺ and ScCas9⁺⁺ variants have been shown to possess higher editing efficiencies in human cells, but their activities in plants are currently unknown. Here, we utilized codon-optimized ScCas9, ScCas9⁺ and ScCas9⁺⁺ and a nickase variant ScCas9n⁺⁺ to systematically investigate genome cleavage activity and cytidine base editing efficiency in rice (Oryza sativa L.). This analysis revealed that ScCas9⁺⁺ has higher editing efficiency than ScCas9 and ScCas9⁺ in rice. Furthermore, we fused the evolved cytidine deaminase PmCDA1 with ScCas9n⁺⁺ to generate a new evoBE4max-type cytidine base editor, termed PevoCDA1-ScCas9n⁺⁺. This base editor achieved stable and efficient multiplex-site base editing at NNG-PAM sites with wider editing windows (C₋₁–C₁₇) and without target sequence context preference. Multiplex-site base editing of the rice genes OsWx (three targets) and OsEui1 (two targets) achieved simultaneous editing and produced new rice germplasm. Taken together, these results demonstrate that ScCas9⁺⁺ represents a crucial new tool for improving plant editing.