Establishment of a high-efficiency transformation and genome editing method for an essential vegetable and medicine Solanum nigrum.

Solanum nigrum, which belongs to the Solanaceae family, is an essential plant for food and medicine. It has many important secondary compounds, including glycoproteins, glycoalkaloids, polyphenolics, and anthocyanin-rich purple berries, as well as many ideal characteristics such as self-fertilization, a short life cycle and a small genome size that make it a potential model plant for the study of secondary metabolism and fruit development. In this study, we report a highly efficient and convenient tissue culture, transformation and genome editing method for S. nigrum using leaf segments after 8 weeks of tissue culture, with a required period from transformation initiation to harvest of about 3.5 months. Our results also show multi-shoot regeneration per leaf segment and a 100% shoot regeneration efficiency in a shoot regeneration medium. Moreover, over 82% of kanamycin-resistant plants exhibited strong green fluorescence marker protein expression, with genetic integration confirmed by PCR results and green fluorescence protein expression in their T1 progeny. Furthermore, we successfully applied this transformation method to achieve an average of 83% genome editing efficiency of SnMYB1, a gene involved in regulating the anthocyanin biosynthetic pathway of S. nigrum in response to missing nutrients. Taken together, the combination of highly efficient tissue culture, transformation and genome editing systems can provide a powerful platform for supporting fundamental research on the molecular mechanisms of secondary metabolism, fruit development, and production of important compounds by biotechnology.

Cloning and functional analysis of RADIALIS-like 1 gene from Antirrhinum majus.

MYB is one of the largest transcription factor families in plants. Among them, the R3-MYB transcription factor RADIALIS (RAD) plays a very important role in the flowers development in Antirrhinum majus. In this study, a R3-MYB gene similar to RAD was found by analyzing the genome of A. majus, which was named AmRADIALIS-like 1 (AmRADL1). The gene function was predicted through bioinformatics. The relative expression levels in different tissues and organs of wild-type A. majus were analyzed by qRT-PCR. AmRADL1 was overexpressed in A. majus, and the transgenic plants were analyzed by morphological observation and histological staining. The results showed that the open reading frame (ORF) of AmRADL1 gene was 306 bp in length, encoding 101 amino acids. It has typical SANT domain, and the C-terminal contains a CREB motif, which was highly homologous to tomato SlFSM1. The results of qRT-PCR showed that AmRADL1 was expressed in roots, stems, leaves and flowers, and the expression level was higher in flowers. Further analysis of its expression in different floral organs showed that AmRADL1 had the highest expression in carpel. The results of histological staining analysis of the transgenic plants showed that compared with the wild type, although the size of the carpel cells of the transgenic plants did not change significantly, the placenta area in the carpel became smaller and the number of cell decreased. In summary, AmRADL1 may be involved in the regulation of carpel development, but the specific mechanism of action in carpel remains to be further studied.

Functional analysis of flower development related gene SvGLOBOSA from Senecio vulgaris.

The unique capitulum of Asteraceae has important ornamental and research value. Few studies have described the complex molecular mechanism of flower development. In this study, SvGLOBOSA(SvGLO), the MADS-box gene of Senecio vulgaris, was identified by screening the transcriptome data, and its function was examined. The gene structure was analyzed and its function was predicted through bioinformatics. The relative expression levels in different tissues of wild-type S. vulgaris were analyzed by qRT-PCR. SvGLO was overexpressed in Solanum nigrum and morphological observations were made. Histological staining was used in analyzing the histological changes in the ovary of transgenic S. nigrum. The results showed that the open reading frame of SvGLO was 591 bp long, encoding 196 amino acids. It has typical MADS-box and K-box domains and contains a PI motif at the C-terminal. SvGLO belongs to the PI/GLO subfamily of class B MADS-box genes. qRT-PCR results showed that SvGLO was highly expressed in inflorescence tissues but not in vegetative organs. In SvGLO-overexpressed S. nigrum, the sepals showed some characteristics of petals, carpels transformed into stamen-like organs, and fruit development was abnormal. Histological staining revealed that the morphology of ovary wall cells of transgenic S. nigrum was similar to that of anther wall cells of the stamen of wild-type S. vulgaris. Therefore, SvGLO may be involved in the regulation of petal and stamen development in S. vulgaris.

A Stable Agrobacterium rhizogenes-Mediated Transformation of Cotton (Gossypium hirsutum L.) and Plant Regeneration From Transformed Hairy Root via Embryogenesis.

Genetic transformation is a powerful tool to study gene function, secondary metabolism pathways, and molecular breeding in crops. Cotton (Gossypium hirsutum L.) is one of the most important economic crops in the world. Current cotton transformation methods take at least seven to culture and are labor-intensive and limited to some cultivars. In this study, we first time achieved plantlet regeneration of cotton via embryogenesis from transformed hairy roots. We inoculated the cotyledon explants of a commercial cultivar Zhongmian-24 with Agrobacterium rhizogenes strain AR1193, harboring a binary vector pBI-35S::GFP that contained the NPT II (neomycin phosphotransferase) gene and the GFP (green fluorescent protein) gene as a fluorescent marker in the T-DNA region. 82.6% explants produced adventitious roots, of which 53% showed GFP expression after transformation. 82% of transformed hairy roots produced embryonic calli, 12% of which regenerated into stable transformed cotton plants after 7 months of culture. The integration of GFP in the transformed cotton genomes were confirmed by PCR (Polymerase chain reaction) and Southern blot analysis as well as the stable expression of GFP were also detected by semi-quantitative RT-PCR analysis. The resultant transformed plantlets were phenotypically, thus avoiding Ri syndrome. Here we report a stable and reproducible method for A. rhizogenes-mediated transformation of cotton using cotyledon as explants, which provides a useful and reliable platform for gene function analysis of cotton.

[Transcriptome analysis of response to heavy metal Cd stress in soybean root]. / å¤§è±†æ ¹ç³»åº”ç­”é‡é‡‘å±žCd胁迫的转录组分析.

Cadmium (Cd) is a common pollutant not required for life activities, which can have extremely strong toxicity to organisms and affect the growth of crops at a low concentration in soil. To investigate the molecular mechanism of soybean root responding to Cd stress, 7-day old soybean seedlings were stressed by Cd (75 Μmol·L-1) for 0 , 4 , 8, 12 and 48 h. Comparative transcriptome analysis showed 244, 1545, 442 and 1401 of genes responded to the four Cd treatments, respectively, and total 2670 differential expression genes were obtained. GO analysis classified these genes into 56 functional categories and COG analysis classified them into 25 functional categories. KEGG analysis showed that many genes involved in the phenylalanine metabolism, ubiquinone and other terpenoid-quinone biosynthesis and cysteine and methionine metabolism and so on. Further we found that expression of three isoflavones 2'-hydroxylase genes, two isoflavonereductase genes and a chalcone synthase gene were evidently up-regulated in all Cd treatments. The results of RT-PCR analysis of four DEGs were consistent with those of RNA-Seq data, further confirming the reliability of RNA-Seq results.