Gene Functional Verification Research Articles

Isolation and Identification of Alkaloid Genes from the Biomass of Fritillaria taipaiensis P.Y. Li

Background/Objectives: Fritillaria taipaiensis P.Y. Li is a valuable traditional Chinese medicinal herb that utilizes bulbs as medicine, which contain multiple alkaloids. Biomass, as a sustainable resource, has promising applications in energy, environmental, and biomedical fields. Recently, the biosynthesis and regulatory mechanisms of the main biomass components of biomass have become a prominent research topic. Methods: In this article, we explored the differences in the heterosteroidal alkaloid components of F. taipaiensis biomass using liquid chromatography–mass spectrometry and high-throughput transcriptome sequencing. Results: The experimental results demonstrated significant differences in the eight types of heterosteroidal alkaloid components among the biomass of F. taipaiensis, including peimisine, imperialine, peimine, peiminine, ebeinone, ebeiedine, ebeiedinone, and forticine. Transcriptomic analysis revealed substantial significant differences in gene expression patterns in the various samples. Three catalytic enzyme-coding genes, 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), and terpene synthase (TPS), were speculated to contribute to the regulation of the differential accumulation of alkaloid synthesis in F. taipaiensis bulbs. A strong positive correlation was observed between the transcriptional level of the TPS gene and the alkaloid content of F. taipaiensis biomass, suggesting that TPS may be a key gene in the biosynthesis pathway of alkaloids. This finding can be used for subsequent gene function verification and molecular regulatory network analysis. Conclusions: This work provides fundamental data and novel insights for the subsequent research on alkaloid biosynthesis in F. taipaiensis.

Dietary supplement of sodium butyrate improves the growth performance and intestinal health by targeting Wnt/β‐catenin signaling pathway in rabbits

AbstractSupplementation of sodium butyrate (SB) has been proved to be beneficial for improving the growth performance and health of animals, but its effects on rabbits have not been well understood. Therefore, this study aimed to investigate the effects of SB supplementation on growth performance, meat quality, and intestinal health of rabbits by evaluating feed intake and efficiency, diarrhea index, blood and cecum metabolites, cecal pH and short‐chain fatty acids (SCFAs), histological staining, nutritional composition of meat, and gene expression profile of cecum. A total of 14 weaned male New Zealand White rabbits (35 ± 2 days, 1.70 ± 0.09 kg) were randomly divided into two groups: rabbits in Control group were fed normally with the basal diet, and rabbits in Butyrate group were supplemented with 0.75 g/kg SB on the basis of the basal diet. After a 1‐week acclimatization period, the formal trial lasted for 10 weeks. Results suggest that dietary SB reduced the feed conversion ratio and diarrhea index of rabbits (p < 0.05). SB treatment significantly increased serum glutamyl transpeptidase, total bile acid, total cholesterol, triglyceride, and glucose levels and decreased tCO2 content relative to the Control group (p < 0.05). Meanwhile, crude ash content in rabbits' meat was significantly increased by SB treatment (p < 0.05). Furthermore, SB treatment significantly increased the integrity of epithelial villi and number of goblet cells in the cecum and decreased cecal pH compared with the Control group. Dietary SB also upregulated levels of Na+‐K+ ATPase, Ca2+‐Mg2+ ATPase, total superoxide dismutase, and glutathione peroxidase in cecum. SCFAs analysis revealed that dietary supplementation with SB increases butyric acid in the cecum contents. Furthermore, SB supplementation is demonstrated to stimulate the proliferation and migration of cecal epithelial cells through activating Wnt/β‐catenin pathways in rabbits both in vitro and in vivo by using transcriptome sequencing and gene function verification technologies. Taken together, our findings provide a theoretical basis for the application of SB as an alternative to antibiotics in livestock production.

Establishment of a Homologous Silencing System with Intact-Plant Infiltration and Minimized Operation for Studying Gene Function in Herbaceous Peonies.

Gene function verification is a crucial step in studying the molecular mechanisms regulating various plant life activities. However, a stable and efficient homologous genetic transgenic system for herbaceous peonies has not been established. In this study, using virus-induced gene silencing technology (VIGS), a highly efficient homologous transient verification system with distinctive advantages was proposed, which not only achieves true "intact-plant" infiltration but also minimizes the operation. One-year-old roots of the representative species, Paeonia lactiflora Pall., were used as the materials; prechilling (4 °C) treatment for 3-5 weeks was applied as a critical precondition for P. lactiflora to acquire a certain chilling accumulation. A dormancy-related gene named HOMEOBOX PROTEIN 31 (PlHB31), believed to negatively regulate bud endodormancy release (BER), was chosen as the target gene in this study. GFP fluorescence was detected in directly infiltrated and newly developed roots and buds; the transgenic plantlets exhibited remarkably earlier budbreak, and PlHB31 was significantly downregulated in silenced plantlets. This study established a homologous transient silencing system featuring intact-plant infiltration and minimized manipulation for gene function research, and also offers technical support and serves as a theoretical basis for gene function discovery in numerous other geophytes.

Association analysis of polymorphisms in SLK, ARHGEF9, WWC2, GAB3, and FSHR genes with reproductive traits in different sheep breeds.

The aim was to investigate the relationship between polymorphisms of gene mutation loci and reproductive traits in local sheep breeds (Duolang Sheep) and introduced sheep breeds (Suffolk, Hu Sheep) in Xinjiang to provide new molecular markers for the selection and breeding of high fecundity sheep. The expression pattern of typing successful genes in sheep tissues was investigated by RT-qPCR technology, providing primary data for subsequent verification of gene function. The 26 mutation loci of WWC2, ARHGEF9, SLK, GAB3, and FSHR genes were typed using KASP. Association analyses were performed using SPSS 25.0, and the typing results showed that five genes with six loci, WWC2 (g.14962207 C>T), ARHGEF9 (g.48271079 C>A), SLK (g.27107842 T>C, g.27108855 G>A), GAB3 (g.86134602 G>A), and FSHR (g.80789180 T>G) were successfully typed. The results of the association analyses showed that WWC2 (g.14962207 C>T), SLK (g.27108855 G>A), ARHGEF9 (g.48271079 C>A), and FSHR (g.80789180 T>G) caused significant or extremely significant effects on the litter size in Duolang, Suffolk and Hu Sheep populations. The expression distribution pattern of the five genes in 12 sheep reproduction-related tissues was examined by RT-qPCR. The results showed that the expression of the SLK gene in the uterus, the FSHR gene in the ovary, and the ARHGEF9 gene in hypothalamic-pituitary-gonadal axis-related tissues were significantly higher than in the tissues of other parts of the sheep. WWC2 and GAB3 genes were highly expressed both in reproductive organs and visceral tissues. In summary, the WWC2 (g.14962207 C>T), SLK (g.27108855 G>A), ARHGEF9 (g.48271079 C>A), and FSHR (g.80789180 T>G) loci can be used as potential molecular markers for detecting differences in reproductive performance in sheep. Due to variations in typing results, the SLK (g.27107842 T>C) and GAB3 (g.86134602 G>A) loci need further validation.

Establishing VIGS and CRISPR/Cas9 techniques to verify RsPDS function in radish

Virus-induced gene silencing (VIGS) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology. However, the investigation of gene silencing and editing in radish remains limited. In this study, a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus (TRV)- and turnip yellow mosaic virus (TYMV)-mediated gene silencing vectors. The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish. The expression level of RsPDS was significantly inhibited using VIGS in ‘NAU-067’ radish leaves. The rootless seedlings of ‘NAU-067’ were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences. Nine adventitious roots were blue with GUS staining, and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing. Furthermore, albino lines were generated with A. tumefaciens-mediated transformation of radish cotyledons. Five base substitutions and three base deletions occurred at target sequence 2 in Line 1, and three base insertions and three base substitutions occurred at target sequence 1 in Line 2. This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish, which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.

Development of a fast and efficient root transgenic system for exploring the function of RsMYB90 involved in the anthocyanin biosynthesis of radish

Radish (Raphanus sativus L.) is recalcitrant to regeneration and genetic transformation, which has severely limited its gene function verification and germplasm innovation. To overcome the limitation, we have developed a rapid and stable regeneration and transformation of hairy roots in radish to explore the potential function of the critical genes involved in the anthocyanin biosynthesis via the optimization of explant selection, bacteria solution concentration, acetosyrinone (AS) concentration and strain types. The single factor experimental designing method was applied and each treatment was conducted for three independent biological replicates. The hairy roots induction rate was more than 90% when using cotyledon with petiole or rootless seedling as explants, while only 53.33% hypocotyls could induce hairy roots. Phenotypic observation and molecular identification indicated that the highest positive transgenic rate of hairy roots could reach approximately 17.51%, when employed optimal optical density (OD600) and AS concentration at 1.0 and 300 μM, respectively, as well as the best strain of MSU440. Subsequently, RsMYB90 was found to play a positive role in the anthocyanin accumulation through the established hairy root transgenic system, and a number of anthocyanin-related genes, such as RsUFGT, RsCHS and RsF3H, were up-regulated by RNA-seq and RT-qPCR analysis. In addition, yeast one-hybrid (Y1H) and dual-luciferase assays (DLA) showed that the RsMYB90 could bind to the promoter of RsUFGT to activate its expression. Taken together, the stable A. rhizogenes-mediated transformation system provides an avenue for gene function assay, genetic engineering and study of secondary metabolisms in radish.

Plastoquinone synthesis inhibition by tetrabromo biphenyldiol as a widespread algicidal mechanism of marine bacteria

Algae and bacteria have complex and intimate interactions in the ocean. Besides mutualism, bacteria have evolved a variety of molecular-based anti-algal strategies. However, limited by the unknown mechanism of synthesis and action of these molecules, these strategies and their global prevalence remain unknown. Here we identify a novel strategy through which a marine representative of the Gammaproteobacteria produced 3,3’,5,5’-tetrabromo-2,2’-biphenyldiol (4-BP), that kills or inhibits diverse phytoplankton by inhibiting plastoquinone synthesis and its effect cascades to many other key metabolic processes of the algae. Through comparative genomic analysis between the 4-BP-producing bacterium and its algicidally inactive mutant, combined with gene function verification, we identified the gene cluster responsible for 4-BP synthesis, which contains genes encoding chorismate lyase, flavin-dependent halogenase and cytochrome P450. We demonstrated that in near in situ simulated algal blooming seawater, even low concentrations of 4-BP can cause changes in overall phytoplankton community structure with a decline in dinoflagellates and diatoms. Further analyses of the gene sequences from the Tara Oceans expeditions and 2750 whole genome sequences confirmed the ubiquitous presence of 4-BP synthetic genes in diverse bacterial members in the global ocean, suggesting that it is a bacterial tool potentially widely used in global oceans to mediate bacteria-algae antagonistic relationships.

In Planta Gene Expression and Gene Editing in Moso Bamboo Leaves.

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and regeneration processes. This method involves Agrobacterium-mediated gene expression via wounding and vacuum for bamboo seedlings. It successfully demonstrated the expression of exogenous genes, such as the RUBY reporter and Cas9 gene, in bamboo leaves. The highest transformation efficiency for the accumulation of betalain in RUBY seedlings was achieved using the GV3101 strain, with a percentage of 85.2% after infection. Although the foreign DNA did not integrate into the bamboo genome, the method was efficient in expressing the exogenous genes. Furthermore, a gene editing system has also been developed with a native reporter using this method, from which an in situ mutant generated by the edited bamboo violaxanthin de-epoxidase gene (PeVDE) in bamboo leaves, with a mutation rate of 17.33%. The mutation of PeVDE resulted in decreased non-photochemical quenching (NPQ) values under high light, which can be accurately detected by a fluorometer. This makes the edited PeVDE a potential native reporter for both exogenous and endogenous genes in bamboo. With the reporter of PeVDE, a cinnamoyl-CoA reductase gene was successfully edited with a mutation rate of 8.3%. This operation avoids the process of tissue culture or callus induction, which is quick and efficient for expressing exogenous genes and endogenous gene editing in bamboo. This method can improve the efficiency of gene function verification and will help reveal the molecular mechanisms of key metabolic pathways in bamboo.

An arginine-to-histidine mutation in flavanone-3-hydroxylase results in pink strawberry fruits.

Fruit color is a very important external commodity factor for consumers. Compared to the most typical red octoploid strawberry (Fragaria × ananassa), the pink strawberry often sells for a more expensive price and has a higher economic benefit due to its outstanding color. However, few studies have examined the molecular basis of pink-colored strawberry fruit. Through an EMS mutagenesis of woodland strawberry (Fragaria vesca), we identified a mutant with pink fruits and green petioles. Bulked-segregant analysis sequencing analysis and gene function verification confirmed that the responsible mutation resides in a gene encoding flavanone-3-hydroxylase (F3H) in the anthocyanin synthesis pathway. This nonsynonymous mutation results in an arginine-to-histidine change at position 130 of F3H. Molecular docking experiments showed that the arginine-to-histidine mutation results in a reduction of intermolecular force-hydrogen bonding between the F3H protein and its substrates. Enzymatic experiments showed a greatly reduced ability of the mutated F3H protein to catalyze the conversion of the substrates and hence a blockage of the anthocyanin synthesis pathway. The discovery of a key residue in the F3H gene controlling anthocyanin synthesis provides a clear target of modification for the molecular breeding of strawberry varieties with pink-colored fruits, which may be of great commercial value.

Advancements and challenges in bamboo breeding for sustainable development.

Bamboo is a highly renewable biomass resource with outstanding ecological, economic and social benefits. However, its lengthy vegetative growth stage and uncertain flowering period have hindered the application of traditional breeding methods. In recent years, significant progress has been made in bamboo breeding. While technical advances in bamboo breeding have been impressive, it is essential to also consider the broader implications we can learn from bamboo's extraordinary features for sustainable development. This review provides an overview of the current status of bamboo breeding technology, including a detailed history of bamboo breeding divided into four eras, a comprehensive map of bamboo germplasm gardens worldwide, with a focus on China, and a summary of available transgenic technologies for gene function verification and genetic improvement. As the demand for bamboo as a sustainable and renewable resource increases continuously, breeding objectives should be focused on enhancing yield, wood properties and adaptability to diverse environmental conditions. In particular, priority should be given to improving fiber length, internode length and wall thickness, as well as regulating lignin and cellulose content for papermaking, substitute for plastic and other applications. Furthermore, we highlight the challenges and opportunities for future research and development in bamboo breeding, including the application of omics technologies, artificial intelligence and the development of new breeding methods. Finally, by integrating the technical advances in bamboo breeding with a discussion of its broader implications for sustainable development, this review provides a comprehensive framework for the development of bamboo industry.

Transcriptome analysis reveals the mechanism of ‘ZhongShan-HongYu’ grape anthocyanin accumulation

During the biosynthesis of anthocyanin, the protein encoding GST transports anthocyanins synthesized in the endoplasmic reticulum to the vesicle, resulting in plant coloration. ‘ZhongShan-HongYu’ (ZS-HY) is a new teinturier germplasm which accumulates anthocyanin in the skin and flesh of the fruit. However, the molecular mechanisms of the flesh coloration in ‘ZS-HY’ remains unclear. We analyzed the transcriptomic profiles of ‘ZS-HY’ and ‘Zico’ flesh (‘ZS-HY’ and ‘Zico’ have similar phenological periods) at 5, 6 and 7 days after anthesis (DAA) using RNA-sequencing technology. A total of 76.27 G of clean data were obtained from our RNA-seq. By analyzing the differentially expressed genes (DEGs) in the flesh, 212 genes potentially involved in anthocyanin biosynthesis were screened. Through further analysis of DEGs, the genes related to anthocyanin accumulation in grape flesh, including VvMYBA1 and VvGST4, were selected for gene function verification. Subcellular localization analysis revealed that VvMYBA1 and VvGST4 were located in cytoplasm and nucleus. The interaction between VvMYBA1 and VvGST4 was analyzed by yeast two-hybrid analysis and BiFC assay. Meanwhile, we found that VvMYBA1 can regulate the activity of VvGST4 promoter by binding with it. Our study provides a basis for the molecular mechanism of anthocyanin transport in grape.

The PcHY5 methylation is associated with anthocyanin biosynthesis and transport in ‘Max Red Bartlett’ and ‘Bartlett’ pears

The red coloring of pear fruits is mainly caused by anthocyanin accumulation. Red sport, represented by the green pear cultivar ‘Bartlett’ (BL) and the red-skinned derivative ‘Max Red Bartlett’ (MRB), is an ideal material for studying the molecular mechanism of anthocyanin accumulation in pear. Genetic analysis has previously revealed a quantitative trait locus (QTL) associated with red skin color in MRB. However, the key gene in the QTL and the associated regulatory mechanism remain unknown. In the present study, transcriptomic and methylomic analyses were performed using pear skin for comparisons between BL and MRB. These analyses revealed differential PcHY5 DNA methylation levels between the two cultivars; MRB had lower PcHY5 methylation than BL during fruit development, and PcHY5 was more highly expressed in MRB than in BL. These results indicated that PcHY5 is involved in the variations in skin color between BL and MRB. We further used dual luciferase assays to verify that PcHY5 activates the promoters of the anthocyanin biosynthesis and transport genes PcUFGT, PcGST, PcMYB10 and PcMYB114, confirming that PcHY5 not only regulates anthocyanin biosynthesis but also anthocyanin transport. Furthermore, we analyzed a key differentially methylated site between MRB and BL, and found that it was located in an intronic region of PcHY5. The lower methylation levels in this PcHY5 intron in MRB were associated with red fruit color during development, whereas the higher methylation levels at the same site in BL were associated with green fruit color. Based on the differential expression and methylation patterns in PcHY5 and gene functional verification, we hypothesize that PcHY5, which is regulated by methylation levels, affects anthocyanin biosynthesis and transport to cause the variations in skin color between BL and MRB.

Transcriptome-Wide Identification and Response Pattern Analysis of the Salix integra NAC Transcription Factor in Response to Pb Stress.

The NAC (NAM-ATAF1/2-CUC) transcription factor family is one of the largest plant-specific transcription factor families, playing an important role in plant growth and development and abiotic stress response. As a short-rotation woody plant, Salix integra (S. integra) has high lead (Pb) phytoremediation potential. To understand the role of NAC in S. integra Pb tolerance, 53 SiNAC transcripts were identified using third-generation and next-generation transcriptomic data from S. integra exposed to Pb stress, and a phylogenetic analysis revealed 11 subfamilies. A sequence alignment showed that multiple subfamilies represented by TIP and ATAF had a gene that produced more than one transcript under Pb stress, and different transcripts had different responses to Pb. By analyzing the expression profiles of SiNACs at 9 Pb stress time points, 41 of 53 SiNACs were found to be significantly responsive to Pb. Short time-series expression miner (STEM) analysis revealed that 41 SiNACs had two significant Pb positive response patterns (early and late), both containing 10 SiNACs. The SiNACs with the most significant Pb response were mainly from the ATAF and NAP subfamilies. Therefore, 4 and 3 SiNACs from the ATAF and NAP subfamilies, respectively, were selected as candidate Pb-responsive SiNACs for further structural and functional analysis. The RT-qPCR results of 7 transcripts also confirmed the different Pb response patterns of the ATAF and NAP subfamilies. SiNAC004 and SiNAC120, which were randomly selected from two subfamilies, were confirmed to be nuclear localization proteins by subcellular localization experiments. Functional prediction analysis of the associated transcripts of seven candidate SiNACs showed that the target pathways of ATAF subfamily SiNACs were "sulfur metabolism" and "glutathione metabolism", and the target pathways of NAP subfamily SiNACs were "ribosome" and "phenylpropanoid biosynthesis". This study not only identified two NAC subfamilies with different Pb response patterns but also identified Pb-responsive SiNACs that could provide a basis for subsequent gene function verification.

Verification of gene function and interaction protein of PhPIF4

In order to investigate the molecular mechanism of PhPIF4 in response to gibberellin-regulated floral transition of Phalaenopsis, qRT-PCR and functional verification experiment of transgenic Arabidopsis thaliana was carried out, and the results showed that PhPIF4 expression was significantly increased in flower buds, leaves and roots after spraying with growth regulator GA3. In PhPIF4 overexpressed Arabidopsis thaliana, hypocotyl elongation, early flowering and increased flowering amount were observed, especially after exogenous GA3 treatment. The content of the endogenous hormone GA3 in the transgenic Arabidopsis thaliana was higher. The interaction between PhDELLA and PhPIF4 was verified by Y2H experiment. The Y1H test demonstrated that PhDELLA could not interact with the PhPIF4 promoter but PhMYB which plays an important role in the regulation of flower development and flowering time could interacte with the PhPIF4 promoter. These findings indicated PhPIF4 participated in the flowering transition and might be directly regulated by GA and the PhPIF4 protein interacts with PhDELLA to go through the GA signaling pathway and the PhPIF4 gene was regulated by PhMYB.

CRISPR/Cas9 editing characteristics of multiple transgenic generations in Fortunella hindsii, an early flowering mini-citrus

The CRISPR/Cas9 editing system is presently regarded as one of the most efficient tools for genome editing and has been implemented in perennial fruit crops. However, the characteristics of transgenic plants and their subsequent generation have rarely been reported in fruit trees. In this study, 15 distinct preserved germplasm resources of Hongkong kumquat (Fortunella hindsii) were utilized to selecting suitable genotypes for genetic transformation. The shoot regeneration efficiency, seed number per fruit, and epicotyl diameter displaid a moderate positive correlation with transformation efficiency, with correlation coefficients of 0.6267, 0.5429, and 0.5388, respectively. The transcription factor RKD family gene FhRWP was knocked out in kumquat, resulting in the production of seven T0 and 34 T1 plants. Editing efficiency of T0 generation ranged from 1.62% to 68.11%, with a significant difference in editing activity among cases. Up to 23 editing types were detected in a single positive transformant, demonstrating that the high degree of citrus editing chimerism. Not all of T1 lines exhibited an increase in editing efficiency relative to T0. The descendants of T0-A2/A3 were all negative, while the other group of T1 lines showed both higher chimera and editing efficiencies of up to 92% than the others. Finally, the CRISPR/Cas9 vector was improved by using the endogenous U6 promoter in F. hindsii. This study provides novel insights into the application of the CRISPR/Cas9 system in asexual plants and serves as a reference for gene function verification in citrus and other species.

Establishment of an efficient root mediated genetic transformation method for gene function verification in citrus

Although several methods of citrus genetic transformation have been studied and published, it is still necessary to further improve the efficiency of genetic transformation and increase the varieties used for citrus genetic transformation. Here, we report a simple, rapid and efficient root transformation technique for citrus to facilitate gene function studies. We found that infestation of Agrobacterium rhizogenes into citrus seedlings of five different species could produce stable transgenic roots in a short period after comparing and optimizing the selection of citrus species, bacterium concentration, cutting time and infection method. PCR and fluorescence observations showed a positive root transformation efficiency of approximately 13%-36% in the five tested citrus species. To demonstrate the utility of this method in the functional verification of citrus genes, a bimolecular fluorescence complementation (BiFC) assay was used to prove the interaction between CrCIPK6/9 and CrCBL5/6 in Citrus reticulata. Additionally, the PCR results showed that CrFT-RFP was transferred from the transgenic positive root to the stem in Citrus reticulata. Finally, overexpression of CjCAD from Citrus junos promoted lignin accumulation in transgenic citrus roots. This method could not only be used to study genes in roots but also various genes involved in plant growth and development. Taken together, we demonstrated that this method allows fast study of gene function in a wide range of citrus species, which would facilitate the understanding of novel genes in citrus.

Virus-Induced Gene Silencing (VIGS) in Chinese Jujube.

Virus-induced gene silencing (VIGS) is a fast and efficient method for assaying gene function in plants. At present, the VIGS system mediated by Tobacco rattle virus (TRV) has been successfully practiced in some species such as cotton and tomato. However, little research of VIGS systems has been reported in woody plants, nor in Chinese jujube. In this study, the TRV-VIGS system of jujube was firstly investigated. The jujube seedlings were grown in a greenhouse with a 16 h light/8 h dark cycle at 23 °C. After the cotyledon was fully unfolded, Agrobacterium mixture containing pTRV1 and pTRV2-ZjCLA with OD600 = 1.5 was injected into cotyledon. After 15 days, the new leaves of jujube seedlings showed obvious photo-bleaching symptoms and significantly decreased expression of ZjCLA, indicating that the TRV-VIGS system had successfully functioned on jujube. Moreover, it found that two injections on jujube cotyledon could induce higher silencing efficiency than once injection. A similar silencing effect was then also verified in another gene, ZjPDS. These results indicate that the TRV-VIGS system in Chinese jujube has been successfully established and can be applied to evaluate gene function, providing a breakthrough in gene function verification methods.

Functional characterization of BoGL5 by an efficient CRISPR/Cas9 genome editing system in broccoli

CRISPR/Cas9 has been widely used for precise and efficient genome editing in a number of organisms, notably in major crops, for gene function characterization and trait improvement. However, the application of this genome editing tool is very limited in Brassica oleracea, especially in broccoli (Brassica oleracea var. italica). Here, we report that the genome of broccoli, a worldwide important Brassicaceae vegetable, can also be precisely edited by a CRISPR/Cas9 system with multiple single-guide RNA-expressing cassettes. The gene BoGL5, previously identified as a candidate for the glossy green mutant, was chosen as the target gene. We generated 14 T0 transgenic plants, among which 14.3% and 50% produced mutations at the expected position in targeted regions 1 and 2, respectively. Two of them with a 100% mutation rate showed a glossy green phenotype and dramatically reduced cuticular wax load. These results indicate that CRISPR/Cas9 can be used as a promising technique for gene functional verification and trait improvement in broccoli.

Transcriptome Analysis Reveals the Central Role of the Transcription Factor MYB in Regulating Anthocyanin Accumulation in Economic Grape Species (Vitis vinifera)

To cultivate different grape varieties according to market needs, it is necessary to study the regulation mechanism of color changes in different development stages of grapes. In this study, RNA-sequencing (RNA-Seq) technology was used to compare and analyze the transcriptome data of four grape varieties at the same development stage. Among the annotated differential genes, the anthocyanin synthesis pathway in the flavonoid pathway was mainly studied. Further RT-qPCR analysis of key enzyme genes, in the flavonoid synthesis pathway of the anthocyanin metabolism pathway, showed that the MYB transcription factor family had binding sites at the start of the four enzyme genes. The relative expression of the MYB transcription factor and enzyme gene in the transcriptome data was verified by reverse transcription polymerase chain reaction. Subcellular localization and gene function verification of the transcription factor MYB2 confirmed its regulatory role in anthocyanins.

Transformation and Detection of Soybean Hairy Roots.

Agrobacterium rhizogenes is a soil bacteria with extensive infectivity, which can infect almost all dicotyledonous plants and a few monocotyledonous plants to induce root nodules. This is caused by the root-inducing plasmid, which contains genes responsible for the autonomous growth of root nodules and crown gall base synthesis. Structurally, it is similar to the tumor-inducing plasmid in that it mainly contains the Vir region, the T-DNA region, and the functional region of crown gall base synthesis. Its T-DNA is integrated into the nuclear genome of the plant with the assistance of Vir genes, causing hairy root disease in the host plant and the formation of hairy roots. The roots produced by Agrobacterium rhizogenes-infested plants are characterized by a fast growth rate, high degree of differentiation, physiological, biochemical, and genetic stability, and ease of manipulation and control. In particular, the hairy root system is an efficient and rapid research tool for plants that have no affinity for transformation by Agrobacterium rhizogenes and low transformation efficiency. The establishment of germinating root culture system for the production of secondary metabolites in the original plants through the genetic transformation of natural plants mediated by root-inducing plasmid in Agrobacterium rhizogenes has become a new technology combining plant genetic engineering and cell engineering. It has been widely used in a variety of plants for different molecular purposes, such as pathological analysis, gene function verification, and secondary metabolite research. Chimeric plants obtained by induction of Agrobacterium rhizogenes that can be expressed instantaneously and contemporarily are more rapidly obtained, compared to tissue culture and stably inheritable transgenic strains. In general, transgenic plants can be obtained in approximately one month.