A Genetic Linkage Map of Brassica campestris L.ssp. pekinensis (syn. B. rapa L. ssp. pekinensis)

A molecular genetic map of Chinese cabbage was constructed with a 102 recombinant inbred (RI) population from a cross of two cultivated Chinese cabbage lines 177 and 276, using AFLP and RAPD markers. 352 markers including 265 AFLP markers and 87 RAPD markers were integrated into 17 linkage groups. It covered a total of 2 665. 7 cM with an average interval of 7. 6 cM. AFLP marker is efficient for map construction while it easily forms clusters to cause big gaps in map. A total of 13.92 % abnormal segregation markers distributed in the map. The molecular genetic map is fundamental for gene localization, comparative genomics, and QTL mapping of important agronomic traits.

Effects of Fermented Nori (<i>Pyropia yezoensis</i>) Liquid Fertilizer on Plant Growth Characteristics and Nutrient Content of Komatsuna (<i>Brassica rapa</i>L. var. Wakana Komatsuna) Cultivated in Vermiculite

We conducted plant growth experiments in microbe-free vermiculite to study the effects of four types of fermented seaweed liquid fertilizer (SLF) made from nori (Pyropia yezoensis) seaweed on the germination, plant growth characteristics, SPAD value, and nutrient content and uptake of komatsuna (Brassica rapa L. var. wakana komatsuna). The four types of fermented nori SLF used in this study were prepared by anaerobic fermentation of unwashed nori (SLF1), aerobic fermentation of unwashed nori (SLF2), anaerobic fermentation of washed nori (SLF3), and aerobic fermentation of washed nori (SLF4). Komatsuna seeds treated with 200-, 300-, and 400-fold dilutions of SLFs exhibited improved relative germination ratios (RGRs) at 3 and 4 days after sowing (DAS). At 4 DAS, the RGRs of seeds treated with 10-, 100-, 200-, 300-, and 400-fold SLF dilutions showed no differential effect. Seeds treated with undiluted SLFs did not germinate by 4 DAS. SLF1 may promote komatsuna seed germination. The nitrogen (N), calcium, magnesium, sodium (Na), and iodine (I) contents of plants treated with SLF1 were significantly increased relative to plants treated with the other SLFs. Moreover, the I and Na contents of plants were significantly increased by foliar spray application of different dilutions of SLF1. However, SLF treatment markedly reduced the shoot dry weight compared with 1/2-strength modified Hoagland nutrient (MHN) solution, although the same amounts of N and K were applied. SPAD values of the plants treated with SLFs were significantly higher than those of plants treated with MHN. Foliar treatment with SLFs had no significant effect on plant growth, SPAD value, or uptake of nutrients (except Na) relative to the control, but the I content was increased. Plants treated with SLF1 and SLF2 exhibited the highest Na uptake. Foliar spray treatments with SLF1 resulted in the highest I contents in plants. Based on our results, SLF1 is suitable for use as a liquid fertilizer to promote germination and increase nutrient content in komatsuna. These results need to be followed up in soil experiments in the presence of microbes in the rhizosphere.

Selenium-oxidizing Agrobacterium sp.T3F4 decreases arsenic uptake by Brassica rapa L.under a native polluted soil

Toxic arsenic(As)and trace element selenium(Se)are transformed by microorganisms but their complex interactions in soil-plant systems have not been fully understood.An Asand Se-oxidizing bacterium,Agrobacterium sp.T3F4,was applied to a native seleniferous As-polluted soil to investigate As/Se uptake by the vegetable Brassica rapa L.and As-Se interaction as mediated by strain T3F4.The Se content in the aboveground plants was significantly enhanced by 34.1%,but the As content was significantly decreased by 20.5% in the T3F4-inoculated pot culture compared to the control(P<0.05).Similar result was shown in treatment with additional 5 mg/kg of Se(IV)in soil.In addition,the As contents in roots were significantly decreased by more than 35% under T3F4 or Se(IV)treatments(P<0.05).Analysis of As-Se-bacterium interaction in a soil simulation experiment showed that the bioavailability of Se significantly increased and As was immobilized with the addition of the T3F4strain(P<0.05).Furthermore,an As/Se co-exposure hydroponic experiment demonstrated that As uptake and accumulation in plants was reduced by increasing Se(IV)concentrations.The 50% growth inhibition concentration(IC50)values for As in plants were increased about one-fold and two-fold under co-exposure with 5 and 10μmol/L Se(IV),respectively.In conclusion,strain T3F4 improves Se uptake but decreases As uptake by plants via oxidation of As and Se,resulting in decrease of soil As bioavailability and As/Se competitive absorption by plants.This provides a potential bioremediation strategy for Se biofortification and As immobilization in As-polluted soil.

Differential Expression Analysis of Genie Male Sterility A/B Lines in Chinese Cabbage-Pak-Choi (Brassica campestris ssp. chinensis Makino)

To determine differential expression of genie male sterility A/B lines in Chinese cabbage-pak-choi (Brassica campestris ssp. chinensis Makino var. communis Tsen et Lee), we used the RNA fingerprinting technique, cDNA-AFLP analysis, in different developmental stages and different tissues. While no obvious differential expressions were observed in rosette leaves, florescence leaves, and scapes, some differential expressions were found in alabstrums of A/B lines and among leaves, scapes and alabstrums. We analyzed the al-abstrums collected in different developmental stages with 10 primer combinations. We got a unique band between middle size alabstrums and large alabstrums in B line in one of the ten pair primers, and in another one pair, one band reflecting a higher gene-expression level in A line than that in B line was obtained. No unique bands were found with the other primer combinations. The bands reflecting different gene-expression level were confirmed by Northern hybridization. The results indicated that cDNA-AFLP was a suitable tool for studying differential expression of genie male sterility in plants. SDS-polyacrylamide gel electrophoresis patterns of soluble proteins further verified the difference in A/B lines.

利用InDel标记鉴定典湖1号杂交种纯度

典湖1号是宁波微萌种业有限公司育成的青梗菜新品种,但种子纯度鉴定速度限制了该品种的推广。为实现对该品种杂交种纯度的高通量快速鉴定,在典湖1号父母本(父本EPG-194,母本EPG-168)基因组重测序的基础上开发用于纯度检测的InDel标记。试验以典湖1号及其父母本DNA样品为材料筛选到亲本带型互补、扩增条带清晰和分离速度快的引物D1-7,并用该引物检测田间典湖1号F_(1)群体(EpG-194×EpG-168群体400株,EpG-168×EpG-194群体400株)。试验中,引物D1-7的分子检测结果显示,EpG-194×EpG-168群体和EpG-168×EpG-194群体分别存在非F_(1)杂株146和32株,田间表型鉴定发现EpG-194×EpG-168群体和EpG-168×EpG-194群体分别存在非F_(1)杂株146和32株,对比后二者鉴定的800株单株结果完全一致。结果表明,InDel分子标记引物D1-7准确鉴定了典湖1号群体中混杂的母本自交种,为之后典湖1号杂交种纯度的快速鉴定奠定了基础。

Decreased Pollen Viability and Thicken Pollen Intine in Antisense Silenced Brassica campestris Mutant of BcMF19

Brassica campestris male fertility 19 （BcMF19; GenBank accession number GQ902048.1）, a gene that is specially expressed in tapetum cells and microspores during anther development in B. campestris ssp. chinensis, which is learned from the previous in situ hybridization study. In the present study, we constructed antisense-silenced plants of BcMF19 using B. campestris ssp. chinensis to validate this prediction. The morphology of the pistils, long anthers, and short anthers was significantly affected in 35sbcmf19 compared with the control samples. 4＇-6-Diamidino-2-phenylindole staining revealed that two generative nuclei and one large vegetative nucleus were not affected in the mutant compared with control. Statistical analysis of Alexander＇s staining results showed that 96% of the control pollen grains had vitality, whereas only 86% of the mutant pollen grains did. Under scanning electron microscopy, the mutant demonstrated numerous abnormal pollen grains and resembled dried persimmon. The frequency of normal pollen grains was approximately 18%. Under transmission electron microscopy, the pollen intine during the binucleate and mature pollen stages in 35sbcmf19 exhibited abnormal thickening, especially at the germinal furrows, compared with control. In vitro pollen germination test showed that the tips of the mutant pollen tubes transformed into globular alveoli and stopped growing compared with control. On the other hand, in vivo pollen germination test suggested that BcMF19 affected the pollen tube extension in the pistil. These findings indicate that BcMF19 is essential to the pollen development and pollen tube extension orB. campestris ssp. chinensis.

白菜型油菜IPT基因家族鉴定及表达分析

异戊烯基转移酶(isopentenyltransferase,IPT)是细胞分裂素生物合成的第一限速酶,为分析IPT基因家族在白菜型油菜生长中的功能,利用生物信息学方法从其基因组中鉴定出13个BrIPT基因,它们不均匀地分布在7条染色体上。BrIPTs可分为4个亚族,各家族成员均含有8~10个保守基序和1~2个UTR区。BrIPT基因的启动子区域包含众多响应元件。BrIPT基因家族成员受环境因子、生物激素及防御与应激调控。qRTPCR结果显示,tRNA-IPT基因BrIPT4、BrIPT6、BrIPT9在白菜型油菜体内各个部位均有表达。相比于苗期,成熟期白菜型油菜IPT基因的表达量更高,为后续深入研究IPT基因家族成员的生理生化功能提供了理论依据。

Genome-wide Investigation of micro RNAs and Their Targets in Brassica rapa ssp. pekinensis Root with Plasmodiophora brassicae Infection

Increasing evidence has revealed that micro RNAs play a pivotal role in the post transcriptional regulation of gene expression in response to pathogens in plants. However, there is little information available about the expression patterns of mi RNAs and their targets in Chinese cabbage(Brassica rapa ssp. pekinensis) under Plasmodiophora brassicae stress. In the present study, using deep sequencing and degradome analysis, a genome-wide identification of mi RNAs and their targets during P. brassicae stress was performed. A total of 221 known and 93 potentially novel mi RNAs were successfully identified from two root libraries of one control(635-10CK) and P. brassicae-treated Chinese cabbage samples(635-10T). Of these, 14 known and 10 potentially novel mi RNAs were found to be differentially expressed after P. brassicae treatment. Degradome analysis revealed that the 223 target genes of the 75 mi RNAs could be potentially cleaved. KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis suggested that the putative target genes of the mi RNAs were predominately involved in selenocompound metabolism and plant hormone signal transduction. Then the expression of 12 mi RNAs was validated by quantitative real-time PCR(q RT-PCR). These results provide insights into the mi RNA-mediated regulatory networks underlying the stress response to the plant pathogen P. brassicae.

白菜抽薹性状相关基因的cDNA-AFLP分析

先期抽薹严重制约着我国春大白菜及高原或高海拔地区的春夏白菜栽培,给生产造成重大损失,因而研究白菜作物低温春化途径抽薹和开花的相关基因对白菜育种具有重要意义。文章选取极早抽薹株系DH-54和极晚抽薹株系DH-43为材料,利用256对引物组合进行cDNA-AFLP分析,筛选到与抽薹开花相关的差异表达的转录衍生片段(Transcript-derived fragment,TDF)191条,对其中82个TDF进行克隆测序。结果表明:52个TDF与NCBI或TIGR已有序列同源,功能涉及代谢、胁迫及逆境应答、信号转导、转录调控等等;22个TDF在NCBI或TIGR找到同源序列,但功能未知;有8个TDF在NCBI或TIGR找不到同源序列,可能是一些新基因。

NaCl和Na2SO4胁迫对白菜型冬油菜种子萌发的影响及其耐盐性分析

以6个耐盐性不同的白菜型冬油菜种子为材料,将NaCl和Na_2SO_4按摩尔比1:1混合,设浓度为0(CK)、45、90、135、180、240 mmol·L^(-1)6个处理,测定其对种子萌发和生长参数的影响,研究不同冬油菜种子萌发时的耐盐能力,以期为耐盐冬油菜的筛选提供依据。结果表明:(1)45 mmol·L^(-1)和90 mmol·L^(-1)盐浓度对种子萌发的影响不明显;135 mmol·L^(-1)和180 mmol·L^(-1)盐浓度明显抑制了种子萌发,表现为种子发芽势、发芽率、发芽指数、活力指数、胚根长、胚芽长、鲜重和干重均随盐浓度的增大而明显降低。因此,135~180 mmol·L^(-1)盐浓度可认为是耐盐性鉴定的适宜范围。(2)不同指标对盐胁迫的敏感程度差异较大,发芽指数、活力指数、胚芽长和鲜重与盐浓度均为极显著负相关,对盐胁迫最为敏感。(3)3个耐盐冬油菜ky-1、ky-10-191和ky-NDJ的种子发芽指数、活力指数、胚芽长和鲜重的耐盐临界值显著高于原有品系平油1号、10-191和ky-NDJ。ky-1、ky-10-191、ky-NDJ的耐盐临界值分别为133.895、123.264、107.399 mmol·L^(-1),而平油1号、10-191、NDJ的耐盐临界值分别为117.394、103.947、93.834 mmol·L^(-1)。用隶属函数法综合评价,耐盐性强弱顺序依次为ky-1>ky-10-191>平油1号>kyNDJ>10-191>NDJ。可见,ky-1、ky-10-191和ky-NDJ的耐盐性较平油1号、10-191和NDJ均有所提高。

北方地区白菜型冬油菜与春油菜的SSR和InDel遗传多样性分析

为了合理评价北方地区白菜型油菜种质资源,为抗寒育种及组合配制提供依据,从252对标记中筛选出9对SSR和36对InDel标记对感温性、抗寒性、品质等性状差异明显的19份白菜型油菜品种的遗传多样性和亲缘关系进行了分析.结果表明,共检测到95个等位变异,平均每个标记2.15个;有效等位基因变幅为1.05～3.27个,平均为1.70个.Shannon指数的变幅范围在0.1217～1.2695间,平均值为0.5803;多态性信息量PIC变幅范围为0.0499～0.6377,平均值为0.3081.通过NTSYS计算遗传相似系数GS并按加权配对法（UPGMA）聚类,结果表明19份材料的GS变异在0.52～0.86之间.在GS为0.605水平上可将19份材料按两室与多室性划分为I-1和I-2两大类群,I-1类群在GS为0.655水平上可按冬春性分为II-1、II-2两个亚类群.在GS为0.71水平上,可将冬性材料划分为4个小群.聚类结果表明北方地区白菜型油菜的遗传多样性丰富,冬、春性品种间及春性品种间亲缘关系较远,存在较大遗传差异.

不结球白菜Pol胞质雄性不育系及其保持系的部分生理生化指标分析

对不结球白菜Pol胞质雄性不育系及其保持系的可溶性糖、可溶性蛋白质和脯氨酸含量及其超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性的变化进行研究,结果表明:不育系不同时期的蕾和花中可溶性糖、可溶性蛋白质和脯氨酸含量均低于其保持系;SOD、POD和CAT活性则是不育系高于其保持系;POD同工酶电泳分析发现不育系和保持系也存在差异,这说明上述物质含量和酶活性变化可能与不结球白菜Pol胞质雄性不育有关。

烟区不同类型油菜秸秆腐解及养分释放特性研究

本研究旨在探讨山东和四川烟区适合与烟草轮作的油菜品种,并分析其秸秆腐解及养分释放特性。通过采用尼龙网袋法,在室内模拟田间条件下对2种类型(白菜型和甘蓝型)的4个冬油菜品种进行培养,观察秸秆的腐解过程和养分释放情况。结果显示,培养前10 d,油菜秸秆快速腐解,各养分也快速释放,养分最终释放率表现为K>N>P>C;腐解进程中,秸秆碳氮比有所波动,但大致呈上升趋势;白菜型油菜秸秆的腐解程度、碳和氮的释放率高于甘蓝型油菜,而钾的释放率低于甘蓝型油菜,磷的释放率表现出地区差异;四川烟区油菜秸秆的腐解程度及碳、氮、磷素的释放率高于山东烟区,钾的释放率高于山东白菜型油菜;油菜秸秆腐解可显著提高供试土壤中有机质、氮、磷和钾素的含量,但不同烟区、不同类型油菜秸秆的增效程度有所差异。从秸秆还田利用的角度来看,2烟区中的白菜型油菜品种更适合与烟草轮作种植。

芜菁酸性多糖体内抗肿瘤活性研究

目的:探究芜菁酸性多糖一组分(BRAP-1)对非小细胞肺癌H226细胞荷瘤小鼠的影响。方法:将60只小鼠分为模型组(0.1 mL/10 g)、阳性药物顺铂组[3 mg/(kg·2 d)]、BRAP-1低剂量组[50 mg/(kg·d)]、BRAP-1中剂量组[100 mg/(kg·d)]和BRAP-1高剂量组[200 mg/(kg·d)]。试验期间观察小鼠肿瘤生长情况,计算抑瘤率、脏器指数;酶联免疫法检测各组小鼠血清中的白细胞介素18(IL-18)、IL-1β和肿瘤坏死因子α(TNF-α)含量;实时荧光定量核酸扩增法(q-PCR)、免疫印迹试验检测IL-18、IL-1β、受体相互作用蛋白1(RIP-1)、RIP3、混合谱系激酶结构域样蛋白(MLKL)基因表达情况。结果:与模型组相比较,BRAP-1低、中剂量组抑瘤效果明显(P<0.05),阳性组和BRAP-1高剂量组抑瘤率效果显著(P<0.01);模型组与其他各组相比较,阳性组和BRAP-1高剂量组小鼠血清中IL-1β含量显著增加(P<0.01),IL-18、TNF-ɑ在阳性组中明显降低(P<0.05),BRAP-1各剂量组中显著增加(P<0.01);q-PCR、免疫印迹试验结果显示,与模型组相比IL-1β随给药剂量增加mRNA相对表达量减少(P<0.01),而IL-18、MLKL、RIP1、RIP3的mRNA及蛋白相对表达量增加(P<0.01)。结论:BRAP-1可以通过调节免疫细胞因子水平,上调坏死性凋亡相关蛋白MLKL、RIP1、RIP3抑制肺鳞癌H226细胞体内生长。

白菜Pol胞质雄性不育系和保持系蕾期基因表达差异的SRAP分析

将SRAP技术用于基因差异表达研究,用30对SRAP引物组合对白菜Pol胞质雄性不育系和保持系的cDNA进行扩增,得到两条特异片段BcCMS1、BcCMS2。对特异片段克隆测序分析后发现,BcC-MS1与大白菜细胞生成相关染色体的19个氨基酸有63%的相似性,BcCMS2与芥蓝抑制开花的基因FLC3部分碱基有88%的相似性。这两个序列在GenBank中的登录号分别为DQ914921、DQ914922。此结果显示在胞质雄性不育中细胞质基因改变了部分核基因的表达。

利用DH群体进行白菜株高和开展度的QTL定位分析

应用AFLP、SRAP、RAPD、SSR及同工酶等标记构建的326个标记位点的白菜遗传图谱和81个DH株系群体,采用M apQTL 5软件对白菜的株高和开展度进行QTL定位。结果表明：通过2年试验,在10个连锁群上共检测到27个QTL,主要分布在R 5、R 10连锁群上：其中控制株高的有11个,控制开展度的有16个;获得2年稳定表达的控制株高的QTL 1个,控制开展度的QTL 3个;各QTL加性效应各不相等,其遗传贡献率为13.3%-29.2%;控制株高和控制开展度的QTL位点表现为紧密连锁或相似的位置,主要集中在R 5连锁群上。

铜胁迫下小白菜叶片基因表达谱的cDNA-AFLP分析

本研究以小白菜品种‘上海青’为试验材料，利用Cu2＋质量浓度为10mg·L-1的营养液水培小白菜幼苗，通过cDNA—AFLP技术分析小白菜响应铜胁迫相关基因及其表达情况。试验中利用多态性较好的135对引物进行扩增，共获得5800多条转录衍生片段（TDFs），平均每个5J物组合扩增出30～60条，片段大小主要集中在100～1000bp，根据条带强弱和有无的变化，其表达模式可以分为持续表达、瞬时表达、上调表达和下调表达4种情况。通过筛选获得180条差异表达TDFs，其中上调表达77条（42．8％），下调表达61条（33．9％），瞬时表达42条（23.3％）。选取其中152条差异表达TDFs进行回收测序，最终得到151个有效序列，其中上调表达68条（45％1，下调表达50条（33．1％），瞬时表达33条（21．9％）。Blastx比对结果显示，112条TDFs序列与已知基因具有较高的相似度，涉及能量代谢、物质合成与运输、逆境响应、信号转导等多种途径，16条TDFs序列与功能未知或假定基因的同源性较高，其余23条TDFs序列与已知功能基因的同源性较低或零匹配，可能是一些新的铜胁迫应答基因。从151条TDFs选取4条TDFs，以小白菜actin基因为内参照，对其在不同胁迫处理时间的表达情况进行荧光定量RT—PCR验证，分析结果与cDNA．AFLP结果基本吻合，表明cDNA—AFLP技术是研究小白菜铜胁迫相关基因差异表达的有效方法。小白菜铜胁迫相关基因表达情况较为复杂，涉及多种代谢途径。本研究结果可为探讨小白菜铜胁迫的分子机理和相关基因的克隆提供理论依据。

白菜型油菜春化基因BrFLC序列变异分析

为寻找与冬春性相关的变异,以开发冬春性品种分子辅助标记,采用巢式和半巢式扩增法,对感温性和抗寒性强弱不同的5个白菜型油菜品种的Br FLC基因的第4外显子至第7外显子进行了特异性扩增。除Br FLC5无扩增结果外,其余3个基因均得到扩增。序列比对结果发现,3个Br FLC在第4外显子至第7外显子均存在有不同程度的等位变异,发生的主要等位变异有SNP和Indel两种类型。Br FLC1变异与冬春性相关性不大,与冬春性关系密切的变异主要发生在Br FLC2和Br FLC3上。Br FLC2中检测到1个Indel类型和5个SNP类型变异位点与冬春性有关。Br FLC3中检测到1个Indel类型和16个SNP类型变异位点与冬春性有关。

北方旱寒区冬油菜抗寒性相关指标的QTL定位

以抗寒性不同的白菜型冬油菜品种陇油7号与陇油9号为亲本构建的F2群体为材料,对白菜型冬油菜抗寒性相关的生理指标进行了QTL定位分析。在白菜型油菜的10条染色体上定位了SOD活性、POD活性、CAT活性、MDA含量、游离脯氨酸含量和可溶性蛋白含量共6个性状的24个QTL位点,可解释表型变异为11.1783%~81.1753%;10个显性效应表现为显性正效应,LOD值在3.2787~163.7958之间。染色体2A的Br ID90127-Br ID10421区、Br ID10421-Br ID10709区及Br ID10709-Br ID101165区,5A的BRMS034(R5)-Ra3-H10区、6A的Ra1-F06-Ra2-D04区、8A的Br ID10839-Ra2-E12区和10A的Br ID90115-Ra2-E07区是多个性状QTL共享的标记区间。研究结果对冬油菜抗寒性基因精确定位具有一定价值。

恰玛古异硫氰酸酯的酶解制备及抗宫颈癌活性

优化了恰玛古异硫氰酸酯(Brassica rapa L.isothiocyanate,BRIe)的外源酶解制备工艺,分析、评价了酶解产物的化学成分和抗宫颈癌活性。采用外源黑芥子酶对恰玛古硫代葡萄糖苷进行降解,通过单因素和正交优化实验确定了酶解法的最佳制备工艺:酶量为20μL、酶解温度70℃、pH 6.5、酶解时间2 h、抗坏血酸浓度为0.01 mg/g,在此条件下,异硫氰酸酯的提取率为11.13%;GC-MS鉴定分析所得酶解产物的化学成分有7种,分别为3-氨基-2-恶唑烷酮、2-恶唑烷硫酮、3-丁烯基异硫氰酸酯、2-苯乙基异硫氰酸酯、异硫氰酸异丙酯、异硫氰酸-2-苯乙酯。采用MTT法检测BRIe对宫颈癌Hela、Siha、U14细胞的增殖抑制活性,其中BRIe对Hela细胞的抑制作用最强,72 h IC_(50)为97.48μg/mL;细胞划痕实验显示BRIe抑制了Hela细胞的迁移,PI流式细胞染色显示BRIe将细胞周期阻滞在S期和G2/M期,Hochst 33258、Annexin V-FITC和PI染色表明BRIe诱导了Hela细胞凋亡。研究结果为开发利用恰玛古用于功能食品和抗宫颈癌药物提供了科学依据。