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。

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.

Flavonoids and ROS Play Opposing Roles in Mediating Pollination in Ornamental Kale （Brassica oleracea var. acephala）

Dear Editor,In most flowering plants, the stigma of the flower represents the barrier that prevents any unrelated or incompatible （genetically similar） pollen from germination and fertilizing the ovule, leading to either defective or genetically unfavorable embryos. In Brassicaceae, self-incompatibility （SI） is a genetic mechanism in the stigmas that can discern compatible versus incompatible mate （pollen）, in turn, rejecting self or incompatible pollen, while allowing non-self or compatible pollen to develop successfully to fertilize the ovules. Thus, stigmas have evolved complex mechanisms and specialized proteins to reject incompatible mates and to recognize and accept compatible pollen （Doucet et al., 2016）. The fact that SI has been shown to exclusively operate in the stigmas temporally before and during anthesis （Kandasamy et al., 1993） indicates that multiple metabolic pathways essential for pollination exist in these stigmatic papillary cells.

The Agronomic Techniques as Determinants of the Phenolic Content and the Biological Antioxidant Effect of Palm-Tree Kale

The content of phytochemicals in vegetables is strongly affected by genetic, agronomic, and environmental factors, and by transportation and storage conditions, potentially affecting the sensory and putative health-promoting properties. In this study the impact of different agricultural techniques on the phenolics content and antioxidant activity of extracts from a traditional Italian kale landrace, called palm-tree kale (Brassica oleracea L., ssp. acephala DC, var. sabellica L.) was evaluated. Furthermore, the antioxidant effects of the different extracts were assessed in a biological system using primary cultures of neonatal rat cardiomyocytes. The final aim was to evidence whether and how the agronomic practices can affect the antioxidant properties of kale not only in vitro but also in cells. Results herein reported underline the influence of the crop production strategies in establishing the potential health benefits of a vegetable. This research approach could be useful for the selection of production techniques that are able to provide edible vegetables with higher functional activity, and evidences the need to study the food domain as a whole with the nutrition domain, and to integrate all results in order to have an overall “foodomics” vision allowing the improvement of health and well-being.

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.

Construction of Plant Antisense Expression Vector with Defective in Anther Dehiscence1 Gene Fragment of Chinese Kale

A pair of primers was designed according to the reported conserved sequence of the defective in anther dehiscencel （DAD1） gene ofArabidopsis thaliana and Brassica rapa. A 558 bp long fragment was amplified from genomic DNA of Chinese kale, showing more than 88% identity with the known DAD1 nucleotide sequence and no intron. The reverse of the amplified fragment was ligated to the downstream of the CaMV35S promoter in the plant expression vector pBIl21. Antisense expression vector pBII21-DAD1F was constructed with DAD1 fragment of Chinese kale, and was transferred into Agrobacterium tumefaciens, which will be used in the transformation to create male sterile materials of Chinese kale.

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.

Molecular Cloning and Expression Analysis of the ζ-Carotene Desaturase Gene in Chinese kale(Brassica oleracea var.alboglabra Bailey)

ζ-Carotene desaturase(ZDS)is an important enzyme in carotenoid biosynthesis.Here,the Brassica oleracea var.alboglabra ZDS(Boa ZDS)gene was cloned from Chinese kale via reverse transcription-polymerase chain reaction(RT-PCR)and deposited in Gen Bank(accession number KY662297).The Boa ZDS gene contains an open reading frame of 1 686 bp that encodes a 561-amino acid protein.Sequence analysis indicates that the ZDS protein is apparently conserved during plant evolution and is most closely related to B.oleracea var.capitata and B.rapa.The promoter sequence of the Boa ZDS gene was predicted to harbor several cis-acting elements that are related to light and phytohormone responses.Semiquantitative RT-PCR analysis showed that Boa ZDS expression varied among different developmental stages and organs.Relative ZDS expression remained stable during germination and seedling stages and rapidly increased at the mature leaf stage.The leaves showed the highest ZDS expression levels compared to the other organs.ZDS expression decreased in all flower tissues during blooming.The fused protein of Boa ZDS was obtained by prokaryotic expression.Heterologous expression of Boa ZDS in Escherichia coli confirmed that Boa ZDS encodes a functionalζ-carotene desaturase that increases β-carotene accumulation in E.coli cells harboring a β-carotene-producing plasmid.The findings of the present study provide a molecular basis for the elucidation of ZDS gene function in Chinese kale.

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。

芥蓝产量性状QTL定位

芥蓝的丰产性状是育种的重要目标性状,由于控制芥蓝的产量性状均为数量性状,常规育种进展缓慢,基因定位及分子标记辅助育种可提高选择效率。本研究利用2个产量差异大的芥蓝自交不亲和系冬强♀(产量高)与Lb07M(产量低)为亲本,构建F2分离群体,F2自交获得F2∶3家系,对产量性状进行了QTL定位分析。对F2∶3家系中单株重、单薹重、株高、薹高、叶长、叶宽、薹粗进行了调查,利用已构建首张芥蓝变种内的SSR和SRAP遗传图谱,结合田间性状调查数据,用QTLNetwork2.0软件通过混合线性复合区间作图法(MCIM)对7个产量性状进行了QTL分析。在10个连锁群中共定位了8个QTL位点,其中控制单薹重、单株重、株高、叶宽的QTL各1个,分别解释表型变异的18.4%,17.8%,19.1%,15.1%;控制主薹高和叶长的QTL各为2个,共解释表型变异的23.8%、22.1%。芥蓝产量性状的QTL定位结果可为分子标记辅助选择高产品种提供参考。

乙酰甲胺磷和NaCl交叉胁迫对花椰菜种子萌发及其幼苗生长的影响

为探究农药和盐交叉胁迫对植物生长影响的机制,试验研究了不同NaCl浓度(0.0%、0.2%、0.4%、0.6%、0.8%)下的不同浓度的乙酰甲胺磷处理液(0.00%、0.03%、0.06%、0.09%、0.12%)对花椰菜种子萌发及其幼苗生长的影响。结果表明:不同浓度乙酰甲胺磷、NaCl及二者的交叉胁迫对花椰菜种子萌发及其幼苗根长、苗高、侧根数目、鲜重和干重的增加均具有显著的抑制作用;乙酰甲胺磷加剧了NaCl对花椰菜的胁迫效应,NaCl也加剧了乙酰甲胺磷对花椰菜的胁迫效应,当乙酰甲胺磷的浓度≥0.09%、NaCl的浓度≥0.8%时,这种相互胁迫效应更加明显;花椰菜对0.2%NaCl处理具有抗性,但对0.03%乙酰甲胺磷处理非常敏感。

Obtaining and Cytological Identification of a Set of Primary Trisomics in Cabbage

Selection of primary trisomics of the cabbage （Brassica oleracea var.capitata L） forms an important basis for gene chromosome mapping and for other genetic studies. The cabbage self-fertilization line - 9601 was used as material, using the root-tip cell chromosome number and pollen mother cell chromosome number identification and karyotype analysis to select the primary trisomics from the progenies of 3x x 2x in the cabbage. Many aneuploid plants with one or two extra chromosomes were obtained and a set of primary trisomics （Tri-1, Tri-2, Tri-3, Tri-4, Tri-5, Tri-6, Tri-7, Tri-8, and Tri-9, in which the Tri-1 and Tri-4 were from 2n＋2 plants and others from 2n＋ 1 plants） was acquired from these plants. Each trisomic exhibited some unique features, such as plant height, plant type, leaf type, size of flower bud, and inflorescence. The triploid crossing by the diploid is a convenient and effective way to select trisomics in the cabbage.

青花菜雄性不育相关基因BoDHAR的克隆与表达分析

以一个与甘蓝显性核不育相关的差异表达片段的序列为信息探针,通过在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、PCR克隆与序列分析,获得了青花菜脱氢抗坏血酸还原酶DHARdehydroascorbatereductase基因的cDNA与DNA全长序列,命名为BoDHAR。并利用双链接头介导PCR的染色体步行技术(genomewalking)克隆了其上游644bp的5′端序列。所获的BoDHAR基因全长1486bp,存在两个内含子,DNA编码区序列633bp,编码210个氨基酸;序列分析表明BoDHAR与同源基因AT1G19570.1cDNA序列有82.3%的一致性,推导的氨基酸序列有79.6%的一致性;编码的水溶性蛋白存在多个磷酸化位点;5′端上游区存在明显的转录调控序列。半定量RT-PCR结果表明BoDHAR在可育系花蕾中的表达量明显高于不育系花蕾,在花药中的表达明显高于其它部位。

羽衣甘蓝游离小孢子培养技术研究及应用

以10个羽衣甘蓝杂交种为试材进行游离小孢子培养，研究胚状体发生和小孢子胚成苗的影响因素，以及小孢子植株倍性鉴定和单倍体加倍方法。结果表明，盛花期为最佳取样时期；培养基中13%的蔗糖较为适宜；继代培养以MS＋6-BA 2 mg·L^-1＋ NAA 0.1mg·L^-1为宜，平均增殖系数为5.06；小孢子再生植株成活率74.6%；小孢子植株自然加倍率为23.33%-37.50%；单倍体试管苗的加倍处理以秋水仙素浓度70mg·L^-1，处理时间9-11d为宜，加倍率达54.55%。

高温胁迫对早熟花椰菜叶片抗氧化系统和叶绿素及其荧光参数的影响

在30～35℃高温胁迫条件下,对早熟花椰菜叶绿素含量、叶绿素荧光参数和叶片抗氧化系统进行研究。结果表明,35℃高温胁迫情况下,叶片叶绿素a、叶绿素b、总叶绿素和类胡萝卜素含量比对照下降25％以上;叶绿素荧光参数,包括原初光能转换效率(Fv/Fm)、光合电子传递量子效率(φPs Ⅱ)、光化学猝灭系数(qP)均低于对照。另一方面,抗氧化酶,包括超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、愈创木酚过氧化物酶(GPX)、过氧化氢酶(CAT)的活性以及抗坏血酸(ASA、DASA)和谷胱甘肽(GSH、GSSG)等抗氧化剂的含量在高温胁迫下明显升高。

结球甘蓝一套初级三体的选育及细胞学鉴定

【目的】创建甘蓝初级三体是其基因染色体定位等遗传研究的基础。【方法】以结球甘蓝自交系-9601为试材,采用根尖细胞、花粉母细胞的染色体计数及核型分析等方法,从其三倍体与二倍体的回交子代中分离和鉴定初级三体。【结果】从回交子代中鉴定分离出了多个2n+1和2n+2等非整倍体植株,经核型分析、染色体联会行为及植株外部形态的观察,初步获得了结球甘蓝一套初级三体(Tr-1～Tr-9),其中从2n+1植株中得到了Tr-2、Tr-3、Tr-5、Tr-6、Tr-7、Tr-8和Tr-9初级三体,从2n+2植株中得到了Tr-1和Tr-4初级三体,各初级三体在株高、株型、叶形、花蕾大小和开花期等特征特性上表现出一定差异。【结论】三倍体与二倍体回交分离结球甘蓝初级三体是方便快捷的有效途径。

高效液相色谱-四极杆飞行时间质谱分析紫甘蓝和羽衣甘蓝中花色苷

应用高效液相色谱-电喷雾/四极杆飞行时间串联质谱联用技术分析了紫甘蓝和羽衣甘蓝中的花色苷成分。选用Agilent TC-C18色谱柱(250 mm×4.6 mm×5 mm),二元线性梯度洗脱,柱后流出液采用电喷雾四极杆飞行时间质谱的正、负离子模式进行检测。根据一级质谱的分子离子和二级质谱碎片离子,获得化合物的准确分子量信息,与相关文献报道进行比较,分别从紫甘蓝和羽衣甘蓝中分离并检测到了16种和17种花色苷,且从紫甘蓝和羽衣甘蓝中发现了锦葵-3-(p-香豆酰基)-芸香苷。本方法快速、准确,无需标准品。

结球甘蓝对根肿病的抗性鉴定与评价

分别利用室内人工接种鉴定、田间人工接种鉴定、田间自然诱发鉴定3种方法,对19份甘蓝材料的抗性进行鉴定,并进行了综合评价,结果表明:‘日本甘蓝’、‘皱叶甘蓝’、‘大楠木’3份材料对根肿病表现为抗病,可以作为抗病育种的材料.3份材料‘天津圆球甘蓝’、‘北黑大平头’、‘小楠木’对根肿病表现为感病.