Brassinazole resistant 1 (BZR1), a brassinosteroid (BR) signaling component, plays a pivotal role in regulating numerous specific developmental processes. Our study demonstrated that exogenous treatment with 2,4-epibrassinolide (EBR) significantly enhanced the accumulation of carotenoids and chlorophylls in Chinese kale (Brassica oleracea var. alboglabra). The underlying mechanism was deciphered through yeast one-hybrid (Y1H) and dual-luciferase (LUC) assays, whereby BoaBZR1.1 directly interacts with the promoters of BoaCRTISO and BoaPSY2, activating their expression. This effect was further validated through overexpression of BoaBZR1.1 in Chinese kale calli and plants, both of which exhibited increased carotenoid accumulation. Additionally, qPCR analysis unveiled upregulation of carotenoid and chlorophyll biosynthetic genes in the T1 generation of BoaBZR1.1-overexpressing plants. These findings underscored the significance of BoaBZR1.1-mediated BR signaling in regulating carotenoid accumulation in Chinese kale and suggested the potential for enhancing the nutritional quality of Chinese kale through genetic engineering of BoaBZR1.1.

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.

The concentrations of chlorophyll and carotenoids were analyzed in two organs (leaves and bolting stems) of 19 varieties of Chinese kale (including four maturity periods and two flower colors). Two chlorophylls and four carotenoids were identified in Chinese kale. The concentrations of chlorophyll and carotenoids varied widely in different organs and varieties. JL-03 was a good candidate for the future breeding programs, since it contained the highest concentrations of chlorophyll and carotenoids (except violaxanthin) in leaves. Lutein was the main component of carotenoids and accounted for approximately 50% of total carotenoids. The concentrations of chlorophyll and carotenoids in leaves significantly exceeded those in bolting stems. Principal component analysis showed that organ was the main source of differences of chlorophyll and carotenoid concentrations in different varieties of Chinese kale, while the maturity and flower color have little effect. Correlation analysis identified a significantly positive correlation between chlorophyll and carotenoids in Chinese kale. These results provided evidence for improving human dietary nutrition and breeding of Chinese kale.

[目的]克隆芥蓝中的BoaBKI1基因,进行生物信息学与表达分析.[方法]采用改良CTAB法提取芥蓝总RNA,并反转录为cDNA;设计引物,克隆BoaBKI1基因;使用生物信息学方法分析BoaBKI1基因序列;用半定量PCR进行BoaBKI1基因的时空表达分析.[结果]成功克隆到BoaBKI1基因,序列分析显示其开放阅读框为1026 bp,编码20种氨基酸;理论分子量为37.30 kD,等电点为9.67,分子式为C1603H2586N476O534S7.同源性序列比对和进化树分析发现芥蓝BoaBKI1与同属植物甘蓝、欧洲油菜的序列一致性高达99％.半定量PCR分析发现,不同发育时期中,BoaBKI1基因在芥蓝真叶期表达量最高,萌动种子期最低;花蕾中花瓣表达水平最高,萼片表达水平最低;开放花朵中雌蕊表达量明显高于其他器官;随花朵开放,BoaBKI1基因在各个花组织中表达水平均出现上调,雌蕊和雄蕊趋势最显著.[结论]上述结果可为研究芥蓝BoaBKI1的功能与调控机制提供理论依据.

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。

Abstract Chinese kale variety ‘Sijicutiao’ was used as the plant material. Chinese kale seedlings were treated by different light qualities, light intensities and phytohormones, and the expression of ζ-carotene desaturase gene (BoaZDS) was analyzed. The results showed that red & blue light significantly promoted the expression of BoaZDS gene, peaked at 6 h and the expression level was 2.33 folds than that of the control. Blue light also induced the expression of BoaZDS gene in the late stage (72 h). Strong light had a significant effect on BoaZDS gene expression, peaked at 24 h and the expression level was 5.64 folds than that of the control, whereas weak light inhibited the BoaZDS gene expression. Abscisic acid and salicylic acid induced the expression of BoaZDS gene, and expression patterns were bimodal. Both methyl jasmonate and gibberellin acid inhibited the expression of the BoaZDS gene. The results provided a basis for further study on the regulation mechanism of BoaZDS gene.

This study presents a systematic analysis of the functional differences between two genes that encode phytoene desaturase (PDS) in Chinese kale. The promoter sequences of both BaPDS1 and BaPDS2 were amplified and cloned, and their lengths were 2005 bp and 2000 bp, respectively. The mining of cis-acting elements in the promoters showed that the two BaPDS genes are mainly associated with light and phytohormone responsiveness. Light quality, light intensity and plant hormone treatments were conducted in seedlings of Chinese kale, and the results indicated that the response of the two genes to different factors differed. Among them, BaPDSs collectively respond to the treatment with salicylic acid and abscisic acid. With regard to response differences, BaPDS1 is sensitive to red and blue light, blue light, and strong light, while BaPDS2 responds to blue light, weak light, darkness, gibberellin and methyl jasmonate. In addition, both BaPDS1 and BaPDS2 are likely targeted to the chloroplast. Furthermore, single and double mutants of BaPDSs were generated via CRISPR/Cas9 technology. Phenotypic analysis showed that the double mutant with edited PDS1 and PDS2 was a pure albino, while the single mutants with edited PDS1 or PDS2 were partly whitened. In summary, BaPDS1 and BaPDS2 genes played different and indispensable roles in Chinese kale, and their functions were partially complementary.

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.