Glucoraphanin (GRA) is an aliphatic glucosinolate (GSL), and its hydrolysis product has powerful anticancer activity. ALKENYL HYDROXALKYL PRODUCING 2 (AOP2) gene, encodes a 2-oxoglutarate-dependent dioxyge-nase, which can catalyze GRA to form gluconapin (GNA). However, GRA only present in trace amounts in Chinese kale. To increase the content of GRA in Chinese kale, three copies of BoaAOP2 were isolated and edited using CRISPR/Cas9 system. The content of GRA was 11.71-to 41.29-fold (0.082-0.289 & mu;mol g  1 FW) higher in T1 generation of boaaop2 mutants than in wild-type plants, and this was accompanied by an increase in the GRA/ GNA ratio and reductions in the content of GNA and total aliphatic GSLs. BoaAOP2.1 is an effective gene for the alkenylation of aliphatic GSLs in Chinese kale. Overall, targeted editing of CRISPR/Cas9-mediated BoaAOP2s altered aliphatic GSL side-chain metabolic flux and enhanced the GRA content in Chinese kale, suggesting that metabolic engineering of BoaAOP2s has huge potential in improving nutritional quality of Chinese kale.

Carotenoids are organic pigments that play an important role in both plant coloration and human health; they are a critical subject in molecular breeding due to growing demand for natural molecules in both food and medicine. In this study, we focus upon characterizing BoaCRTISO , the carotenoid isomerase gene before the branch of the carotenoid biosynthetic pathway, which is expressed in all organs and developmental stages of Chinese kale, and BoaCRTISO, which is located in the chloroplast. The expression of BoaCRTISO is induced by strong light, red and blue combined light, and gibberellic acid treatment, but it is suppressed by darkness and abscisic acid treatment. We obtained BoaCRTISO -silenced plants via virus-induced gene silencing technology, and the silence efficiencies ranged from 52 to 77%. The expressions of most carotenoid and chlorophyll biosynthetic genes in BoaCRTISO -silenced plants were downregulated, and the contents of carotenoids and chlorophyll were reduced. Meanwhile, BoaCRTISO -silenced plants exhibited phenotypes of yellowing leaves and inhibited growth. This functional characterization of BoaCRTISO provides insight for the biosynthesis and regulation of carotenoid in Chinese kale.

以白花芥蓝‘四季粗条’为材料克隆了Boa DFR基因,Gen Bank登录号为MT472647,CDS序列长为1 158 bp,编码385个氨基酸,与甘蓝型油菜、甘蓝和白菜等具有高度同源性。Boa DFR表达水平在芥蓝不同发育时期、不同器官中存在显著差异,随植株发育总体呈现先下降后上升的趋势,在幼嫩种子中表达量最高。芥蓝原生质体的亚细胞定位结果显示Boa DFR定位在细胞核上。构建了农杆菌介导的芥蓝瞬时过表达体系,将Boa DFR转入白花芥蓝‘四季粗条’和黄花芥蓝‘福州黄花’中,发现Boa DFR在两种芥蓝中均高水平表达,转基因植株叶片颜色明显变紫,花青素苷显著积累,总含量最高达461.43μg·g-1 FW,而野生型和转空载体芥蓝叶片中几乎检测不到花青素苷。通过HPLC在转基因植株叶片中共检测到8种花青素苷,均为矢车菊花青素苷,其中矢车菊–3–阿魏酰–丙二酰–槐糖苷–5–葡萄糖苷占比最高。

The experiment used the ‘Sijicutiao’ mustard sterile seedlings as the material, firstly constructed the BoaZDS subcellular localization vector with the cloned BoaZDS gene, and extracted the plasmid, then separated the Chinese kale leaf protoplast by enzymatic hydrolysis. After purification, the BoaZDS gene was transiently transformed, and then the position of the BoaZDS gene was observed under a fluorescence microscope. The results showed that the BoaZDS gene was localized in the chloroplast, which was consistent with the predicted results of bioinformatics software. The results provide a basis for studying the function of the ZDS gene in Chinese kale.

The carotenoid isomerase gene (BoaCRTISO) of Chinese kale was targeted and edited using the CRISPR/Cas9 system in the present study. The results showed a high mutation rate (81.25%), and 13 crtiso mutants were obtained. Only two types of mutations, insertions and replacements, were found. Both the total and individual carotenoid and chlorophyll concentrations of the biallelic and homozygous mutants were reduced, and the total levels declined by 11.89-36.33%. The color of the biallelic and homozygous mutants changed from green to yellow, likely reflecting a reduction in the color-masking effect of chlorophyll on carotenoids. The expression levels of most carotenoid and chlorophyll biosynthesis-related genes, including CRTISO, were notably lower in the mutants than in the WT plants. In addition, the functional differences between members of this gene family were discussed. In summary, these findings indicate that CRISPR/Cas9 is a promising technique for the quality improvement of Chinese kale and other Brassica vegetables.

Neoxanthin synthase (NXS) is an important enzyme in carotenoid biosynthesis. Here, the BoaNXS gene was cloned from yellow flower Chinese kale, and preformed for bioinformatics analysis. The BoaNXS gene contains an open reading frame of 699 bp that encodes a 232 - amino acid protein with a calculated molecular mass of 25.52 kD and an isoelectric point (pI) of 9.6. Subcellular localization predicted that the BoaNXS was in the chloroplast. The functional domain of the BoaNXS protein contains DUF4281 Super family. Homology analysis indicates that the NXS protein is apparently conserved during plant evolution, and BoaNXS is most closely related to Brassica napus, and B. oleracea var. oleracea. The findings of the present study provide a theoretical basis for the elucidation of NXS gene function in Chinese kale.

Carotenoid isomerase (CRTISO) is an important enzyme in carotenoid biosynthesis. Here, the Brassica oleracea var. alboglabra CRTISO (BoaCRTISO) gene was cloned from yellow-flower Chinese kale, and preformed for sequence analysis. The BoaCRTISO gene contains an open reading frame of 1,773 bp that encodes a 590-amino acid protein with a calculated molecular mass of 64.88 kD and an isoelectric point (pI) of 6.72. Subcellular localization predicted the BoaCRTISO gene was in the chloroplast. The conserved domain of the BoaCRTISO protein is Rossmann-fold NAD (P) H/NAD (P) (+) binding (NADB) domain. Homology analysis indicates that the CRTISO protein is apparently conserved during plant evolution and is most closely related to B. napus, and B. rapa. The findings of the present study provide a molecular basis for the elucidation of CRTISO gene function in Chinese kale.

以芥蓝(Brassica oleracea var.alboglabra)为材料,以ζ-胡萝卜素脱氢酶(ζ-Carotene desaturase,ZDS)基因为目标基因,建立其CRISPR/Cas9基因组编辑体系.在BoaZDS的编码区近5'端选择靶位点,构建了CRISPR/Cas9表达载体,通过农杆菌介导的遗传转化方法获得了19个芥蓝转基因阳性植株,Sanger测序分析发现其中13株成功突变,CRISPR/Cas9载体在芥蓝上的突变效率为68.42％,且所有突变植株均表现出明显的白化表型.

The CRISPR/Cas9 system has been applied in a variety of plants for targeted gene editing due to its accuracy, high efficiency, and low price. It has been reported that the CRISPR/Cas9 system can lead to mutation in potato, however, the mutation patterns and genotypes of CRISPR/Cas9-induced mutants in tetraploid potato plants have not yet been disclosed in detail. In the present study, the Solanum tuberosum phytoene desaturase (StPDS) gene was selected for inducing targeted mutagenesis. The obtained StPDS knockout mutants exhibited a mutation frequency of 46.67% and clear albino phenotypes. All transgenic plants were tested for the hygromycin resistance gene and a high positive transformation frequency (96.77%) was observed in all tested resistant plants. Genotype analysis of the mutants revealed that heterozygotes accounted for 35.51%, chimeras accounted for 64.29%, and no homozygotes and biallelic mutations were detected. The patterns of the detected mutations consisted mainly of 1 to 2-nucleotide insertions and deletions, followed by 2-bp replacements. In short, the results of this study prove that the CRISPR/Cas9 system is an effective approach for cultivated potato gene engineering.

The hypocotyl of Chinese kale `Cutiaoyusun' was used as explants, the effects of the different concentrations of sucrose and different browning inhibitors (activated carbon, polyvinylpyrrolidone, and ascorbic acid) on the callus proliferation and anti-browning of Chinese kale were studied in this study. The results showed that the proliferation medium with 20 g.L-1 sucrose had the best effects on the proliferation and anti-browning of Chinese kale callus, and its proliferation rate was the highest, reaching 213.5%, and the browning rate was as low as 27.77%. In addition, adding 0.2 g.L-1 ascorbic acid to the culture medium can significantly promote callus proliferation and reduce browning rate. In this treatment, the callus had the lowest browning rate of 19.45% and the highest proliferation rate of 266.98%, and the morphology of the callus was friable. This study lays a foundation for future research in molecular biology and genetic improvement in Chinese kale.

In this study, we report the isolation and purification of protoplasts from Chinese kale (Brassica oleracea var. alboglabra) hypocotyls, and their transient gene expression transformation and subcellular localization of BaMYB75 (Bol042409). The upshot is that the vintage protocol included 5-d hypocotyls that were enzymatically hydrolyzed for 8 h in enzyme solution (3.0% cellulase, 0.5% pectolase, and 0.5 M mannitol), and the protoplasts were purified by precipitation. The total yield of protoplasts was 8 × 105 protoplast g−1 fresh weight, and the protoplasts’ viability was 90%. The maximum transformation efficiency obtained by using green fluorescent protein (GFP) as a detection gene was approximately 45% when the polyethylene glycol (PEG)4000 concentration was 40% and transformation time was 20 min. In addition, BaMYB75 was ultimately localized in the nucleus of Chinese kale hypocotyl protoplasts, verifying the validity and reliability of this transient transformation system. An effective and economical hypocotyl protoplast isolation, purification, and transformation system was established for Chinese kale in this study. This effectively avoided interference of chloroplast autofluorescence compared to using mesophyll cells, laying the foundation for future research in the molecular biology of Brassica vegetables.

Using the cotyledon and hypocotyl of Chinese kale 'Sijicutiao' as explants, the effects of MS medium supplemented with different types and concentrations of growth regulators (2, 4-D, 6-BA, NAA) on the callus induction and regeneration of Chinese kale were studied. The results showed that different hormone combinations of 2,4-D, 6-BA and NAA play a key role in the callus induction and regeneration culture of Chinese kale, and hypocotyl has a better effect than cotyledon with petiole as an explant. MS + 0.1 mg.L-12, 4-D + 0.02 mg.L-1 6-BA was the optimal combination in the process of induction, the hypocotyl explant induction rate was 80%. During the regeneration culture, the best combination was MS + 0.75 mg.L-1 6-BA + 0.03 mg.L-1 NAA, the differentiation rate of adventitious buds was 94%, and the rooting rate was 67%. These results could provide a theoretical reference value for the study of cell suspension culture of Chinese kale.

Endogenous and exogenous plant growth regulators play essential role in the growth and proliferation of calluses. To establish an efficient protocol of callus proliferation, various concentrations of a-naphthaleneacetic acid (NAA) (0.4, 0.5, 0.6 mg.L-1) in combination with 6-benzylaminopurine (6-BA) (1.5, 2.0, 2.5 mg.L-1) on callus proliferation of Chinese kale were evaluated. The results showed that the best callus proliferation was observed on MS medium with 0.4 mg.L-1 NAA and 2.5 mg.L-1 6-BA after 30 d of culture, and the morphology of the callus was yellowish in colour and friable. This study provides a basis for future studies on genetic improvement for Chinese kale.

In this study, the cultivated potato variety Xuanshu 2 was used as plant material, and MS was used as the basic medium, the in vitro regeneration culture of stem segments was carried out to study the optimal combination of phytohormone concentrations for callus induction and bud differentiation. The results indicated that the optimal medium for callus induction in stem segments was MS + 2.0 mg.L-1 6-BA + 0.2 mg.L-1 NAA, with 100% induction rate. The optimal medium for bud differentiation was MS + 2.0 mg.L-1 6-BA + 2.0 mg.L-1 ZT + 0.5 mg.L-1 GA(3), with 88% differentiation rate. In this study, the regeneration system of potato variety Xuanshu 2 was established, laying the foundation for the subsequent genetic transformation research of this variety.

The AOP2.3 gene is an important gene affecting the side chain modification of aliphatic glucosinolate. Here, the Brassica oleracea var. capitata AOP2.3 (BocAOP2.3) gene sequence was obtained from the Brassica database (BRAD) and subjected to sequence analysis. It is indicated that the BocAOP2.3 gene contains a 936 bp open reading frame (ORF) encoding a protein of 311 amino acids with a calculated molecular weight of 33.62 kD, the isoelectric point (pI) is 4.46. The highest number of amino acids is Valine (Val), whereas the lowest number is Tryptophan (Trp). Its prediction formula is C 1455 H 2295 N 397 O 505 S 6 . Its total hydrophobicity index is -0.5, the solution instability index is 29.25, and the fat solubility index is 77.46. Sequence alignment indicated that the BocAOP2.3 protein is more closely related to Brassica napus and Brassica Rapa , those all belonged to the Cruciferous genus. The results of this study provide a molecular basis for elucidating the function of BocAOP2.3 gene in cabbage.

Lycopene β-cyclase (LCYb) is an important enzyme in carotenoid biosynthesis. Here, the Brassica oleracea var. capitata LCYb (BocLCYb) gene sequence was obtained from Brassica database (BRAD), and preformed for bioinformatics analysis. The BocLCYb gene mapped to Scaffold000212, and contains an open reading frame of 1,500 bp that encodes a 499-amino acid protein with a calculated molecular mass of 50.90 kD and an isoelectric point (pI) of 6.77. Subcellular localization predicted the BocLCYb gene was in the cytoplasm and nucleus. The conserved domain of the BocLCYb protein is PLNO2463. The BocLCYb protein is most closely related to Raphanus sativus. The findings of the present study provide a molecular basis for the elucidation of LCYb gene function in cabbage.

CYP79B2 is an important cytochrome P450 monooxygenases in glucosinolate biosynthesis. Here, the Brassica oleracea var. capitata CYP79B2 (BocCYP79B2) gene sequences were obtained from Brassica database (BRAD), and preformed for bioinformatics analysis. The BocCYP79B2.1, BocCYP79B2.2 and BocCYP79B2.3 genes mapped to chromosomes 1, 3 and 7, and contains an open reading frame of 1,623 bp, 1,557 bp and 1,626 bp that encodes a 540, 518, 541 amino acid protein, respectively. Subcellular localization predicted all BocCYP79B2 genes were in the chloroplast. The conserved domain of the BocCYP79B2 protein is PLN02971. Homology analysis indicates that the CYP79B2 protein is apparently conserved during plant evolution. The findings of the present study provide a molecular basis for the elucidation of CYP79B2 gene function in cabbage.

CYP83B1 is an important glucosyltransferase enzyme in glucosinolate biosynthesis. Here, the Brassica oleracea var. capitata CYP83B1 (BocCYP83B1) gene sequence was obtained from Brassica database (BRAD), and preformed for bioinformatics analysis. The BocCYP83B1 gene mapped to chromosomes 8, and contains an open reading frame of 1,473 bp that encodes a 490-amino acid protein with a calculated molecular mass of 55.89 kD and an isoelectric point (pI) of 8.89. Subcellular localization predicted the BocCYP83B1 gene was in the chloroplast. The conserved domain of the BocCYP83B1 protein is belonged the p450 superfamily. The CYP83B1 protein is most closely related to B. rapa. The findings of the present study provide a molecular basis for the elucidation of CYP83B1 gene function in cabbage.

The clustered regulatory interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) system has developed into a powerful gene-editing tool that has been successfully applied to various plant species. However, studies on the application of the CRISPR/Cas9 system to cultivated Brassica vegetables are limited. Here, we reported CRISPR/Cas9-mediated genome editing in Chinese kale (Brassica oleracea var. alboglabra) for the first time. A stretch of homologous genes, namely BaPDS1 and BaPDS2, was selected as the target site. Several stable transgenic lines with different types of mutations were generated via Agrobacterium-mediated transformation, including BaPDS1 and BaPDS2 double mutations and BaPDS1 or BaPDS2 single mutations. The overall mutation rate reached 76.47%, and these mutations involved nucleotide changes of fewer than 10 bp. The clear albino phenotype was observed in all of the mutants, including one that harbored a mutation within an intron region, thereby indicating the importance of the intron. Cleavage in Chinese kale using CRISPR/Cas9 was biased towards AT-rich sequences. Furthermore, no off-target events were observed. Functional differences between BaPDS1 and BaPDS2 were also assessed in terms of the phenotypes of the respective mutants. In combination, these findings showed that CRISPR/Cas9-mediated targeted mutagenesis can simultaneously and efficiently modify homologous gene copies of Chinese kale and provide a convenient approach for studying gene function and improving the yield and quality of cultivated Brassica vegetables.

CYP79B3 is an important cytochrome P450 monooxygenases in glucosinolate biosynthesis.Here, the Brassica oleracea var.capitata CYP79B3 (BocCYP79B3) gene sequence was obtained from Brassica database (BRAD), and preformed for sequence analysis.The BocCYP79B3 gene mapped to Scaffold000053, and contains an open reading frame of 1,632 bp that encodes a 543-amino acid protein with a calculated molecular mass of 61.54 kD and an isoelectric point (pI) of 8.59.Subcellular localization predicted the BocCYP79B3 gene was in the chloroplast.The conserved domain of the BocCYP79B3 protein is PLN02971.The CYP79B3 protein is most closely related to B. napus.The findings of the present study provide a molecular basis for the elucidation of CYP79B3 gene function in cabbage.