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

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。

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.

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。

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.

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.

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.

Growth and yield responses of broccoli cultivars to different rates of nitrogen in western Chitwan, Nepal

A field experiment was conducted with the objective to determine the optimum rate of nitrogen (N) fertilizer for effective growth and yield of two varieties of broccoli in southern plain of Nepal. The experiment was laid out with two-factorial completely random block design (RCBD) comprising two varieties of broccoli (Calabrese and Green Sprouting) and five N rates (0, 50, 100, 150 and 200 kg ha-1) with three replication in each treatment combinations. The effects of variety and N rate on total curd yield were significant but the interaction effect was non-significant. Green Sprouting produced 11% higher total curd than Calabrese. Similarly, curd production increased N rate up to 200 kg ha-1 reaching a maximum of 14.47 t ha-1. This indicated that optimum level of N could be beyond the rates tested in this study, which needs further experimentation.

Effect of H_2O_2 on Growth of Collard( Brassis oleracea L. ) Seedlings Under Salt Stress

Collard variety( Brassis oleracea L. var. acephala f. tricolor Hort.) as a research material was treated with exogenous H_2O_2 and H_2O_2 scavenger dimethyl thiourea under 100 mmol/L NaC l stress. Two days later,growth rate,dry weight,fresh weight and relative water content of the plants were determined. After 6h of treatment,the activity and gene expression of three antioxidant enzymes,superoxide dismutase( SOD),catalase( CAT) and ascorbate peroxidase( APX) in plants,were measured. The results showed that the growth rate,dry weight,fresh weight,relative water content,and the activity and gene expression of the three antioxidant enzymes in collard seedlings were higher in the treatment of salt stress with the addition of 0. 05 mmol/L exogenous H_2O_2 than in the simple salt stress treatment; and when endogenous H_2O_2 was removed,the growth rate,dry weight,fresh weight,relative water content,and the activity and gene expression of the three antioxidant enzymes in plant seedlings were lower than those under simple salt stress. It is speculated that under salt stress,H_2O_2 is involved in the regulation of antioxidant defense gene expression,and it might be an important regulator of salt-induced antioxidant system in collard leaves.

Comparison of carotenoid,chlorophyll concentrations and their biosynthetic transcript levels in different coloured cauliflower

Carotenoids and chlorophylls are among the most widely distributed pigments in nature that play essential roles in the photosynthetic apparatus and confer diverse colours in plants.Among all vegetables,cauliflower(Brassica oleracea L.ssp.var.botrytis)is rich in phytochemicals and is an important crop grown all over the world.This study investigates carotenoid and chlorophyll concentrations in differently pigmented cultivars and elucidates the role of transcriptional regulation of carotenoid accumulation including lutein andβ-carotene.Here,we characterised changes in pigments by UHPLC-DAD-ToF-MS and changes in transcript levels of carotenoid metabolic genes by qRT-PCR in florets and leaves of orange(‘Jaffa'and‘Sunset'),purple(‘Di Sicilia Violetto'and‘Graffiti'),green(‘Trevi')and white(‘Clapton')cultivars.Transcript levels of all carotenoid metabolic genes showed different transcript level patterns in the leaves and florets.Compared to the other cultivars,the orange cultivars had the highest levels ofβ-carotene in the florets and lutein in the leaves resulting in changes lutein/β-carotene ratios.In the green cultivar,higher transcript levels were also found,especially for phytoene synthase and phytoene desaturase genes of the core biosynthesis pathway.However,no increased carotenoid concentrations were observed,possibly due to a higher carotenoid turnover induced by the carotenoid cleavage dioxygenase 4 in the green cultivar.In the white(‘Clapton')and purple(‘Di Sicilia Violetto'and‘Graffiti')cultivars the phytoene desaturase transcript levels as well as carotenoid concentrations were low.Chlorophyll concentrations changed in trend comparable to the carotenoid concentrations and were only significantly lower in the leaves of the orange cultivar‘Jaffa'.Also,the chlorophyll a/b ratio changed in‘Jaffa'.In florets the highest chlorophylls concentrations were observed for the green cultivar(‘Trevi')and the purple cultivar(‘Di Sicilia Violetto').Taken together,the study demonstrates the complex source-sink relationship of carotenoid accumulation in different coloured cauliflower.

青花菜快速碱化因子RALF的克隆与序列分析

以一个与甘蓝显性核不育相关的差异表达片段序列为信息探针,在NCBI与TAIR网站数据库中进行同源EST序列搜索,经人工拼接、RT-PCR克隆与序列分析验证,获得了青花菜快速碱化因子RALF(Rapid Alkalinization Factors)基因的cDNA全长序列,命名为BoRALFL1(GenBank序列登录号DQ059310)。该cDNA全长240bp,编码79个氨基酸,与电子克隆获得的序列完全相同。序列分析表明,编码蛋白存在前导信号肽与多个磷酸化位点,与同源基因RALFL8核酸序列在88bp上有82%的一致性,推导的氨基酸序列在74个氨基酸上存在56%的一致性,不同植物间氨基酸序列N-端差异大,C-端具有较高的保守性。

甘蓝自交不亲和信号传导中SRK结合蛋白基因THL1的克隆与序列分析

采用PCR和RT PCR技术 ,从甘蓝基因组和柱头总RNA中扩增获得了 719bp的硫氧还蛋白(THL1)的全长基因序列和 4 30bp的cDNA序列。序列分析首次表明 ,克隆的THL1基因全序列有两个内含子 ,cDNA序列编码

UPLC-MS-MS法快速测定甘蓝和土壤中虱螨脲的残留量

建立了甘蓝（Brassica oleraceaL．）和土壤中虱螨脲残留的超高效液相色谱-三重四极杆串联质谱（UPLC—MS—MS）快速检测的方法。样品用酸化乙腈提取，以QuEChERS法净化，超高效液相色谱一串联质谱电喷雾负离子模式检测。结果表明，虱螨脲在5-500μg/L范围内，浓度和响应峰面积线性关系良好。在0．01、0．02、0．1和0．5mg／kg4个添加浓度下，平均回收率在78．5％～113．6％．相对标准偏差（RSD）为2．5％-8．5％（n=6），虱螨脲在甘蓝和土壤中的定量限（LQD）均为0．01mg／kg。该方法适用于甘蓝和土壤中虱螨脲的检测．

L-精氨酸处理对青花菜贮藏过程中品质的影响

试验用0.2 mmol/L的L-精氨酸(L-Arg)溶液处理青花菜(Brassica oleracea var.italica)20 min,探讨在贮藏过程中对青花菜品质的影响。结果表明,L-Arg处理可有效抑制青花菜的黄化和细胞膜透性的增加,保持可溶性固形物含量,延缓叶绿素的降解和维生素C的流失。L-Arg处理能够有效延缓青花菜的衰老,抑制营养物质的流失。

低温胁迫下结球甘蓝BoNR8 lncRNA过表达对拟南芥种子萌发的影响

长非编码RNA(long no-coding RNA,lncRNA)不含有开放阅读框,且长度大于200 bp,一般不具有编码蛋白质功能。它们在生物体内普遍存在,并发挥着多种生物学作用。植物种子萌发期的特异表达的lncRNA研究较少。BoNR8 lncRNA是结球甘蓝中RNA聚合酶Ⅲ转录的长非编码RNA(约272 bp)。前期研究发现,它在种子萌发期的特异表达;拟南芥中BoNR8 lncRNA过表达抑制了正常条件下种子萌发并降低了萌发种子对ABA的敏感性。本研究对BoNR8 lncRNA序列分析发现,其转录区域内存在冷胁迫基序(ATATAAATAAAT),并且这个基序存在BoNR8 lncRNA二级结构的最大茎环中。生化实验进一步表明,BoNR8 lncRNA响应低温环境,其过表达抑制了低温胁迫下拟南芥种子萌发。BoNR8 lncRNA低温相关功能的发现为植物耐低温研究提供了新材料,丰富了植物耐低温作用机制的研究。

甘蓝晚抽薹基因BoFLC3克隆、序列分析和亚细胞定位

参照甘蓝FLC3基因(GenBank登录号AAP31677)碱基序列设计引物,扩增出甘蓝晚抽薹BoFLC3基因,序列分析结果表明,该基因编码的蛋白质有197个氨基酸,分子量20 990,等电点9. 45。荧光定量PCR分析结果表明:抽薹后BoFLC3基因在甘蓝不同部位表达存在差异,表达量从高到低依次为:叶、茎、花蕾、花瓣和根。亚细胞定位结果表明:BoFLC3蛋白定位在细胞核。

甘蓝LFY基因的克隆及序列分析

以甘蓝ZQ启动抽薹的茎尖为材料,分别提取DNA和RNA,以两对引物扩增并序列拼接得到甘蓝LFYZQ基因DNA序列和cDNA完整编码区,长度分别为2560、1239bp,与花椰菜、拟南芥、芥菜和萝卜同源性分别达到91%、87%、86%、87%。该基因含有3个外显子(452、394、393bp)和2个内含子(514、807bp),共编码412个氨基酸,内含子剪接位点均符合经典GT-AG法则。将LFYZQ与网上公布的12种十字花科植物LFY氨基酸序列按分子进化分成两类:甘蓝LFYZQ与花椰菜以及Jonopsidium属植物Jonopsidium acaule这3个分为一类;其余10种植物分为第二大类。LFYZQ蛋白分子量为46kD,是一个不稳定的疏水蛋白,存在N-十四(烷)酰化位点、蛋白激酶C磷酸化位点、酪蛋白激酶Ⅱ磷酸化位点、酰胺化位点共4种活性位点。