Improved genome annotation of Brassica oleracea highlights the importance of alternative splicing

Brassica oleracea has been developed into many important crops,including cabbage,kale,cauliflower,broccoli and so on.The genome and gene annotation of cabbage(cultivar JZS),a representative morphotype of B.oleracea,has been widely used as a common reference in biological research.Although its genome assembly has been updated twice,the current gene annotation still lacks information on untranslated regions(UTRs)and alternative splicing(AS).Here,we constructed a high-quality gene annotation(JZSv3)using a full-length transcriptome acquired by nanopore sequencing,yielding a total of 59452 genes and 75684 transcripts.Additionally,we re-analyzed the previously reported transcriptome data related to the development of different tissues and cold response using JZSv3 as a reference,and found that 3843 out of 11908 differentially expressed genes(DEGs)underwent AS during the development of different tissues and 309 out of 903 cold-related genes underwent AS in response to cold stress.Meanwhile,we also identified many AS genes,including BolLHCB5 and BolHSP70,that displayed distinct expression patterns within variant transcripts of the same gene,highlighting the importance of JZSv3 as a pivotal reference for AS analysis.Overall,JZSv3 provides a valuable resource for exploring gene function,especially for obtaining a deeper understanding of AS regulation mechanisms.

Molecular Cloning and Sequence Analysis ofBoPGIP2 Gene from Brassica oleracea L. var. alboglabra

PGIP gene was obtained from Brassica oleracea L. var. alboglabra, named BoPGIP2. The full length of BoPGIP2 gene is 1 102 bp and the exon is 993 bp which encodes a protein of 330 amino acids with a predicted molecular mass of 37.1 kDa, interrupted by one intron of 95 bp in, length. Sequence analysis revealed that it has five potential N-giycosylation sites, two protein kinase C phosphrylation sites, five casin kinase Ⅱ phosphrylation sites and four N-myristoylation sites. The amino acids sequences alignment confirmed that ^145 LRR stucture was highly conserved in all aligned PGIP sequences.

与花椰菜(Brassica oleracea ssp.botrytis)抗黑腐病基因连锁的RAPD标记

利用 RAPD技术 ,在花椰菜 ( Brassica oleracea ssp.botrytis)抗感黑腐病的近等位基因系之间进行 1 0碱基随机引物的筛选 .34 5条 RAPD引物扩增的产物表明 ,有 7条引物在近等基因系中呈多态性 .进一步的重复实验表明 ,只有引物 OP2 2 4能够产生稳定的多态性 .为了确定扩增产物 OP2 2 4/1 6 0 0与抗黑腐病基因的连锁程度 ,利用 F2分离群体进行检测 ,结果表明 :花椰菜种质 C71 2抗黑腐病生理种 BJ的抗性由一对显性基因控制 .RAPD标记OP2 2 4/1 6 0 0与抗病基因连锁 ,其遗传距离为 ( 4 .5± 1 .37) c

花椰菜(Brassica oleracea var.botrytis)抗黑腐病差异表达cDNA片段的克隆及功能的初步研究

采用花椰菜抗黑腐病近等基因系C731（感病系）和C712（抗病系）作为材料，利用cDNA—AFLP技术，研究了花椰菜黑腐病抗性在黑腐病菌侵染和非侵染条件下基因表达的情况．获得了两个阳性克隆M2和M6．Northern杂交和点杂交进一步证明M2是组成型表达的cDNA片段，只是在病菌侵染与非侵染条件下表达丰度不同．而M6是差异表达的cDNA片段．Southern杂交表明M6 cDNA片段在花椰菜基因组中是单拷贝序列．随后的序列分析发现M6cDNA片段与拟南芥1号染色体的BACF19P19中66665～66813bp有84％同源性，该片段编码拟南芥的2A6蛋白的部分序列．推测的氨基酸序列与拟南芥的2A6蛋白部分序列有91％同源性．用H2O2作为外源分子胁迫处理的初步功能分析发现：M6cDNA片段受H2O2诱导，在诱导早期16～24h高度表达，其在叶片中的积累呈逐渐上升趋势．根据序列分析和初步的功能分析的结果推测：M6cDNA片段可能就是编码花椰菜中类2A6蛋白的部分基因片段．是参与花椰菜抗病反应信号途径的相关调控基因片段．

花椰菜(Brassica oleracea var.botrytis)黑腐病抗性基因同源序列分离及克隆的研究

通过从 NBS 保守序列设计简并引物 PCR 的方法,以花椰菜(Brassica oleracea vat.botrytis)抗、感黑腐病的近等基因系为材料,分离得到 NBS-LRR 型抗性基因同源序列,并获得1个克隆,命名为 RGA330-7.Southern 杂交表明,该片段在近等基因系中存在明显的多态性,且该片段在抗黑腐病基因位点至少存在3个以上类似 RGA330-7的同源拷贝.序列分析结果认为该克隆与 NBS-LRR 型抗性基因的部分 CDSs 有很高的同源性,说明该片段属于 NBS-LRR 型.系统进化分析该序列与甘蓝型油菜的2个抗病同源序列归为一类,很可能这3个不同来源的抗性基因同源序列同属于一种抗性基因家族.因此推测该序列与花椰菜抗黑腐病基因紧密相关,为进一步克隆花椰菜抗黑腐病基因提供了可靠的候选基因,对分子标记辅助抗性育种具有重要意义。

根癌农杆菌介导白细胞介素-4基因转化甘蓝(Brassica oleracea L.)研究

利用根癌农杆菌介导法，以白细胞介素-4（interleukin-4，IL-4）基因转化中甘11号甘蓝。本研究对可能影响il-4转化率的外植体类型、外植体的预培养时间、农杆菌的侵染时间以及外植体与农杆菌的共培养时间进行了优化。试验结果表明：il-4基因对带1-2mm子叶柄的子叶的转化率显著高于下胚轴；带柄子叶在预培养1d、侵染3min、共培养1d时，转化效果最好，转化率达23．3%;下胚轴在预培养1-3d，侵染3～5min，共培养1-2d时，转化效果较好，转化率最高可达13．3％。经PCR检测及PCR-Southem杂交，初步证明目的基因il-4已经转入甘蓝的再生植株。转il-4基因甘蓝的PCR阳性再生植株已经开花并产生了后代。

Transformation of insect-resistant gene into cauliflower (Brassica oleracea L.var. botrytis)

Cowpea Trypsin Inhibitor (CpTI) gene was transferred into the cotyle dons and hypocotyls of cauliflower by Agrobacterium-mediated transformation met hod. The best selective concentration of kanamycin (kan) was 15 mg L-1. The con centration of carbencillin (carb) was 500 mg L-1. 14 transgenic cauliflower pla nts were obtained. The putative transformants were assayed by PCR and Southern b lotting analysis. The results indicated that CpTI gene was transferred into caul iflower successfully.

Study on the Relationship Between the Ploidy Level of Microspore-Derived Plants and the Number of Chloroplast in Stomatal Guard Cells in Brassica oleracea

The relationship between the ploidy level of microspore-derived plants and chloroplast number in stomatal guard cells was studied in cabbage, broccoli, and Chinese kale. In the experiment, distribution statistics analysis and t-test were used to perform statistical analysis on chloroplast number of different ploidy level in those stomatal guard cells mentioned above, and morphology identifying and chromosome counting were used to test accuracy of counting chloroplast number in stomatal guard cells. The chloroplast average number in stomatal guard cells was very similar among the different leaf positions on the same plant and among significantly among the different ploidy the different locations in the same stoma in the same variety. All the leaf, while the chloroplast number varied distributions of the chloroplast number in different ploidy stoma were normal distribution fitted. A correlation has been established between ploidy and chloroplast number in the stomatal guard cells. In every single stoma of microspore-derived plants, the chloroplast number for a haploid should not be more than 10, diploids 11 to 15, and polyploids more than 15. The accuracy of this method for identification of different ploidy plants was 93.93%. Furthermore, the accuracy of this method was reliable and did not vary with the plants growth conditions. Therefore, the chromosome ploidy of plants derived from microspore culture in cabbage, broccoli, and Chinese kale can be identified by simply counting the chloroplast number in stomatal guard cells.

Plant Male Sterility Induced by Anti-Gene CYP86MF in Brassica oleracea var. italica

An anti-gene CYP86MF was introduced into hypocotyls of broccoli （Brassica oleracea L.var. italica Plenck） with Agrobacterium tumefaciens, and the transgenic plants were obtained by kanamycin selection. The results of PCR, Southern blot and Northern blot indicated that the anti-CYP86MF has been integrated into chromosome of the transgenic plant. And also, plants with hypogenetic stamina or ungerminated pollen were observed. The transgenic male sterility plant could fructify via artificial pollination with normal pollen. Thus it was proved that the pistil of male sterility plant was normally developed, and the sterility originated from anti-CYP86MF.

Nuclear and chloroplast genome diversity revealed by low-coverage whole-genome shotgun sequence in 44 Brassica oleracea breeding lines

Whole-genome shogun sequence(WGS)data generated by next-generation sequencing(NGS)platforms are a valuable resource for crop improvement.We produced 5–6×WGS coverage of 44 Brassica oleracea breeding lines representing seven subspecies/morphotypes:cabbage,broccoli,cauliflower,kailan,kale,Brussels sprout,and kohlrabi to systematically evaluate the nuclear and chloroplast(Cp)diversity in the 44 B.oleracea breeding lines.We then exploited the impact of low-coverage NGS by evaluating nuclear genome diversity and assembly,annotation of complete chloroplast(Cp)genomes and 45 S nuclear ribosomal DNA(45S nrDNA)sequences,and copy number variation for major repeats.Nuclear genome diversity analysis has revealed a total of 496463 SNPs and 37493 indels in the nuclear genome across the 44 accessions.Interestingly,some SNPs showed subspecies enrichment at certain chromosomal regions.The assembly of complete Cp genomes contained 153361–153372 bp with 37 variants including SNPs and indels.The 45S nrDNA transcription unit was 5802 bp long with a total of 31 SNPs from the 44 lines.The phylogenetic tree inferred from the nuclear and Cp genomes coincided and clustered broccoli,cauliflower,and kailan in one group and cabbage,Brussels sprout,kale,and kohlrabi in another group.The morphotypes diverged during the last 0.17 million years.The Cp genome diversity reflected the unique cytoplasm of each subspecies,and revealed that the cytoplasm of many breeding lines was replaced and intermingled via inter-subspecies crosses during the breeding process instead.The polymorphic Cp markers provide a classification system for the cytoplasm types in B.oleracea.Furthermore,copy numbers of major transposable elements(TEs)showed high diversity among the 44 accessions,indicating that many TEs have become active recently.Overall,we demonstrated a comprehensive utilization of low-coverage NGS data and might shed light on the genetic diversity and evolution of diverse B.oleracea morphotypes.

Genome-wide identification and analysis of cytokinin dehydrogenase/oxidase(CKX) family genes in Brassica oleracea L.reveals their involvement in response to Plasmodiophora brassicae infections

Cytokinins are a class of phytohormones that promote cell division and differentiation and are thought to affect plant immunity to multiple pathogens.However,a comprehensive analysis of cytokinin dehydrogenase/oxidase(CKX)family genes in cabbage has not been reported.In this study,a total of 36 CKX genes were identified using a genome-wide search method.Phylogenetic analysis classified these genes into three groups.They were distributed unevenly across nine chromosomes in B.oleracea,and 15 of them did not contain any introns.The results of colinearity analysis showed that 36 CKX gene in Arabidopsis was present in several copies in the Brassica oleracea genome.An analysis of cisacting elements indicated that all genes possessed at least one stress or hormone responsive cis-acting element.A heatmap of CKX gene expression showed the patterns of expression of these genes in various tissues and organs.Three genes(Bol028363,Bol031036 and Bol018140)were relatively highly expressed in all of the investigated tissues under normal conditions,showing the expression profile of housekeeping genes.Generally,the expression patterns of CKX genes in Jingfeng 1 and Xiangan 336 were quite different under the same treatment.Notably,three genes(Bol020547,Bol028392 and Bol045724)were significantly down-regulated and up-regulated in the susceptible and resistant material,respectively,after inoculation,which may indicate their crucial roles in resistance to clubroot disease.The results provide insights for better understanding the roles of CKX genes in the B.oleracea–P.brassicae interaction.

Genome-wide identification,evolutionary selection,and genetic variation of DNA methylation-related genes in Brassica rapa and Brassica oleracea

DNA methylation plays an important role in plant growth and development,and in regulating the activity of transposable elements(TEs).Research on DNA methylation-related(DMR)genes has been reported in Arabidopsis,but little research on DMR genes has been reported in Brassica rapa and Brassica oleracea,the genomes of which exhibit significant differences in TE content.In this study,we identified 78 and 77 DMR genes in Brassica rapa and Brassica oleracea,respectively.Detailed analysis revealed that the numbers of DMR genes in different DMR pathways varied in B.rapa and B.oleracea.The evolutionary selection pressure of DMR genes in B.rapa and B.oleracea was compared,and the DMR genes showed differential evolution between these two species.The nucleotide diversity(π)and selective sweep(Tajima’s D)revealed footprints of selection in the B.rapa and B.oleracea populations.Transcriptome analysis showed that most DMR genes exhibited similar expression characteristics in B.rapa and B.oleracea.This study dissects the evolutionary differences and genetic variations of the DMR genes in B.rapa and B.oleracea,and will provide valuable resources for future research on the divergent evolution of DNA methylation between B.rapa and B.oleracea.

A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var.capitata L.)

The dominant genic male sterility （DGMS） gene CDMs399-3 derived from a spontaneous mutation in the line 79-399-3 of spring cabbage （Brassica oleracea var. capitata L.）, has been successfully applied in hybrid seed production of several cabbage cultivars in China. During the development of dominant male sterility lines in cabbage, the conventional identification of homozygous male-sterile plants （CDMs399-3/CDMs399-3） is a laborious and time-consuming process. For marker-assisted selection （MAS） of the gene CDMs399-3 transferred into key spring cabbage line 397, expressed sequence tag-simple sequence repeats （EST-SSR） and SSR technology were used to identify markers that were linked to CDMs399-3 based on method of bulked segregant analysis （BSA）. By screening a set of 978 EST-SSRs and 395 SSRs, a marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the genetic background of cabbage line 397 were identified. 7 homozygons male-sterile plants in population P1170 with 20 plants were obtained finally via MAS of BoE332. Thus, BoE332 will greatly facilitate the transferring of the gene CDMs399-3 into the key spring cabbage line 397 and improve the application of DGMS in cabbage hybrid breeding.

Photosynthetic Excitation Pressure Causes Violaxanthin De-epoxidation in Aging Cabbage （Brassica Oleracea L.） Leaves

The purpose of the present studies was analysis of the age induced changes in photochemical efficiency and xanthophyils cycle pigments of the primary cabbage （Brassica oleracea L. cv. Capitata f. alba） leaves. Photochemical efficiency of photosystem Ⅱ （PS Ⅱ） was studied by a pulse amplitude modulated chlorophyll fluorescence apparatus, chlorophyll concentration was analysis spectrophotometrically and xanthophyll cycle pigments were estimated by high-pressure liquid chromatography （HPLC）. Leaf senescence was accompanied with a decrease both in chlorophylls concentration, the photochemical efficiency and rate constant for PS Ⅱ photochemistry whereas non-photochemical parameters increased. Excitation pressure （1-qP） which is a measure of relative lumen acidification increased by 1.2x in aging leaves. The maximum quantum yield of PS Ⅱ showed no significant change. The level of de-epoxidised xanthophylls increased but the concentration of mono- and di-epoxy xanthophylls decreased in aging leaves. A linear relationship between the excitation pressure and the depoxidation state of the xanthophyll cycle pigments and lutein, during the onset of senescence suggests that excitation pressure can be used as a sensor for monitoring the onset of senescence as well for the de-epoxidation state of the xanthophylls responsible for non-photochemical quenching in stressed leaves.

Genetic diversity analysis of Brassica oleracea L.by SSR

SSR analysis on genetic diversity of 30 samples was carried out. Five primers selected from 36 primers were used to amplify 30 samples in this experiment, PCR products were separated by 6% polyacrylamide gel electrophoresis, silver staining and photographed. The results of SSR were analyzed by UPGMA clustering. The results showed that a total of 21 gene alleles were detected by 5 SSR primers. The number of alleles ranged from 2 to 5 with an average of 4.2.PIC range was 0.257-0.92 1, with an average of 0.543. The average coefficient of genetic similarity of SSR markers among materials was 0.432. Some of cabbage cultivars in the experiment were divided into four groups except cultivars which come from Japan.

Review of Research Situation of Brassica oleracea var.italica

Broccoli(Brassica oleracea var.italica),also called green broccoli,green cauliflower,and calabrese,belongs to genus Brassica in family Cruciferae,and is annual or biennial herbaceous plant.Both broccoli and cauliflower are varieties of Brassica oleracea L.Broccoli is rich in nutrients.Its protein,amino acids and vitamins are higher than cauliflower,and broccoli is easy to grow,the supply period is long,and it has a good market and economic value.This paper introduced broccoli-related information from broccoli's nutritional value,cultivation techniques,existing problems and prospects.In addition,on the basis of existing studies,it discussed the future development prospects of broccoli,in order to promote the production research of broccoli in China.

花椰菜(Brassica oleracea var.botrytis L.)凝集素的细胞凝集和糖抑制作用的研究

花椰莱(Brassica oleracea var. botrytis L.)凝集素能凝集兔、大鼠和小鼠的红细胞,其中对兔的红细胞的凝集活性最高,最低凝集浓度为0.68μg/ml。但不凝集我们所测试的其它16种红细胞。浓度为0.1mg/ml时,花椰菜凝集素也能凝集小鼠艾氏腹水瘤细胞,S_(180)肉瘤细胞,大鼠W_(256)肿瘤细胞、人的MGC_(80-3)胃癌细胞、小鼠和大鼠的脾脏淋巴细胞、骨髓细胞以及牛精子细胞,而不凝集人的Hela细胞。该凝集素对免的红细胞的凝集活性可被L—鼠李糖和D—树胶醛糖所抑制,最低抑制浓度分别为33.3m mol/L和16.7m mol/L。

Effect of Avocado （Persea Americana）, Cabbage （Brassica Oleracea） and Ginger （Zingiber Officinale） on Rat Liver and Thyroid Injuries Induced by CCI4 （Carbon Tetrachloride）

Avocado, Cabbage, and Ginger are a part of a regular human diet and have antioxidant, and antitumor effects. The effect of AVOE （avocado）, GE （Ginger） and CE （Cabbage） extracts separately on liver NO （nitric oxide）, MDA （malondialdehyde）, as well as serum AST （aspartate aminotransferase）, ALT （alanine aminotransferase）, total bilirubin, TC （total cholesterol）, T.G （triglyceride）, HDL cholesterol （high-density lipoprotein）, LDL cholesterol （low-density lipoprotein）, TSH （thyroid-stimulating hormone）, T3 （Triiodothyronine）, T4 （Thyroxine） in rats treated and untreated with CC14 （carbon tetrachloride） was studied. The levels of NO, MDA, as well as serum AST, ALT, total bilirubin, TC, T.G, LDL, and TSH, showed an elevation while, HDL, T3 and T4 showed the decline in rats treated with CC14 as compared to control. Treatment of rats with AVOE and GE pre, during, and post CC14 administration improve NO, MDA, as well as serum AST, ALT, total bilirubin, TC, T.G, HDL, LDL, TSH, T3, T4 as compared to CC14. Treatment of rats with CE pre, during, and post CC14 administration did not improve in the thyroid hormones and lipid profile levels as compared to CC14. These findings suggest that avocado and ginger treatment exerts a protective effect on metabolic disorders by decreasing oxidative stress.

Effects of Brassica oleracea extract on impaired glucose and lipid homeostasis in highfat diet-induced obese mice

Objective: To examine the effect of Brassica oleracea extract(BO) on impaired glucose and lipid homeostasis in high-fat diet(HFD)-induced obese mice. Methods: Obesity of ICR mice was induced by feeding a HFD(45 kcal% lard fat) for 16 weeks. During the last 8 weeks of study period, obese mice were additionally administered with BO(100 and 200 mg/kg/day). The metabolic parameters were determined. The gene expressions of hepatic lipogenesis were also studied. Results: After 8 weeks of treatment, BO(100 and 200 mg/kg) significantly reduced hyperglycemia and improved insulin sensitivity(P < 0.05). The serum lipid(total cholesterol, triglyceride, and non-esterified fatty acid) and hepatic triglyceride and nonesterified fatty acid were decreased(P < 0.05). The levels of insulin and leptin in serum were also decreased(P < 0.05). Moreover, the expressions of hepatic lipogenic genes including sterol regulatory element-binding protein 1 c, fatty acid synthase, and acetyl-CoA carboxylase were decreased by BO treatment(P < 0.05). Conclusions: These results suggest that BO is a new therapeutic agent for improving the homeostasis of glucose and lipid in HFD-induced obese mice probably by suppression of lipogenic genes in liver tissue.

The protoplasts isolation,culture and shoots regeneration of broccoli(Brassica oleracea var.italica)

Eight F1-hybrid cultivars of broccoli were studied.We obtained cell division,celled colonies and p-calli in 5 cultivars,roots and shoots regeneration in one cultivar.The leavesof propagated plantlets in vitro were cut into 1—2mm pieces,isolated with an enzyme solutioncontaining 2% cellulase and 1%macerase on a rotary shaker（50 rpm,21℃,3h,2500 lux light）,and purified with a 0.5M sucrose solution.The purified protoplasts were placed on a drop of 1%agarose.2—3 ml liquid medium was added around the agarose drops,and all of the cultures wereincubated at 25℃ under light（4000 lux）for 16 hours.3—5 days after isolation the cell divisionwas found.About 7 days after incubation 4 multicellular colonies were formed.After 3—5 wksome p-calli were developed.When the p-calli were 2—3 mm in diameter it was transferred to asolidified medium.Once they were developed to 1 cm in diameter they were transferred on a re-generation medium.About 5 months after incubation some roots and shoots grown from the calliwere