Molecular mechanism of flowering time regulation in Brassica rapa:similarities and differences with Arabidopsis

Properly regulated flowering time is pivotal for successful plant reproduction.The floral transition from vegetative growth to reproductive growth is regulated by a complex gene regulatory network that integrates environmental signals and internal conditions to ensure that flowering takes place under favorable conditions.Brassica rapa is a diploid Cruciferae species that includes several varieties that are cultivated as vegetable or oil crops.Flowering time is one of the most important agricultural traits of B.rapa crops because of its influence on yield and quality.The transition to flowering in B.rapa is regulated by several environmental and developmental cues,which are perceived by several signaling pathways,including the vernalization pathway,the autonomous pathway,the circadian clock,the thermosensory pathway,and gibberellin(GA)signaling.These signals are integrated to control the expression of floral integrators BrFTs and BrSOC1s to regulate flowering.In this review,we summarized current research advances on the molecular mechanisms that govern flowering time regulation in B.rapa and compare this to what is known in Arabidopsis.

Genome-wide identification and characterization of the lateral organ boundaries domain gene family in Brassica rapa var.rapa

The Lateral Organ Boundaries Domain(LBD)genes encode highly conserved plant-specific LOB domain proteins which regulate growth and development in various species.However,members of the LBD gene family have yet to be identified in Brassica rapa var.rapa.In the present study,fifty-nine LBD genes were identified and distributed on 10 chromosomes.The BrrLBD proteins are predicted to encode hydrophobic polypeptides between 118 and 394 amino acids in length and with molecular weights ranging from 13.31 to 44.24 kDa;the theoretical pi for these proteins varies from 4.83 to 9.68.There were 17 paralogous gene pairs in the BrrLBD family,suggesting that the amplification of the BrrLBD gene family involved largescale gene duplication events.Members of the BrrLBD family were divided into 7 subclades(class I a to e,class II a and b).Analysis of gene structure and conserved domains revealed that most BrrLBD genes of the same subclade had similar gene structures and protein motifs.The expression profiles of 59 BrrLBD genes were determined through Quantitative Real-time fluorescent PCR(qRT-PCR).Most BrrLBD genes in the same subclade had similar gene expression profiles.However,the expression patterns of 7 genes differed from their duplicates,indicating that although the gene function of most BrrLBD genes has been conserved,some BrrLBD genes may have undergone evolutionary change.

番茄植株中生物碱类对菜青虫(Pieris rapae L.)活性组分的分离和提纯

为寻找和开发不污染环境、不伤害天敌而对菜青虫有活性的行为物质,本研究对番茄植株中的生物碱类进行提取、分离和部分纯化,得到了极性和非极性生物碱类以及番茄苷(tomatine)纯品,与标样对照薄层试验吻合。将提取物对5龄菜青虫进行试验表明:生物碱类及其中的番茄苷对菜青虫有明显拒食作用;番茄苷对菜青虫有抑制生长发育的作用。

Comparison Between a Tetraploid Turnip and Its Diploid Progenitor(Brassica rapa L.):The Adaptation to Salinity Stress

Polyploidy is pursued in plant breeding programs due mainly to its ability to yield larger vegetative or reproductive organs. In controlled growth chamber experiments, a tetraploid turnip （cv. Aijiaohuang, 4n） and its diploid progenitor （cv. Aijiaohuang, 2n） were evaluated for their tolerance to salinity stress via investigations on a group of physiological parameters. The results indicate that the tetraploid turnip exhibit better adaptation to a high concentration salt medium （200 mmol L-1）, as evidenced by a less-affected germination rate and a healthier morphological appearance at the seedling stage. Furthermore, an extension of salinity stress up to a certain period of time at the 5-7-leaf stage shows differences between the tetraploid turnip and its diploid progenitor. The former had a higher K＋/Na＋ ratio in the roots, higher glutathione concentration and antioxidant activities in the leaves, and smaller reductions in photosynthetic capacity in terms of leaf chlorophyll content. Studies on the differences between an autopolyploid and its respective relative, from which the autopolyploid originated, in terms of their tolerance to salinity and/or other abiotic stresses, have remained rather limited. The comparison is interesting due to a homogenous genetic background.

OS-mhscFv联合RAPA对骨肉瘤细胞凋亡及HIF-1α、VEGF、MMP-2表达的影响

目的:探讨抗人骨肉瘤人源化单链抗体(OS-mhscFv)联合雷帕霉素(RAPA)对人骨肉瘤MG-63细胞凋亡及HIF-1α、VEGF、MMP-2表达的影响及其作用机制。方法:将人骨肉瘤MG-63细胞分为生理盐水组、OS-mhscFv组、RAPA组和OSmhscFv+RAPA联合组,应用流式细胞仪检测OS-mhscFv联合RAPA对MG-63细胞凋亡的影响;应用Real time PCR法检测4组细胞中HIF-1α、VEGF、MMP-2的mRNA表达量。结果:与生理盐水组和单独给药组比较,OS-mhscFv+RAPA联合组能诱导MG-63细胞的凋亡,差异有统计学意义(P<0.01)。OS-mhscFv+RAPA联合组MG-63细胞中HIF-1α、VEGF、MMP-2的mRNA相对表达量均显著低于单独用药组和生理盐水组,差异有统计学意义(P<0.01)。结论:OS-mhscFv联合RAPA能够有效诱导MG-63细胞凋亡,其诱导机制可能是通过下调HIF-1α、VEGF、MMP-2的mRNA表达量完成。

Molecular cloning and expression analysis of turnip (Brassica rapa var. rapa) sucrose transporter gene family

In higher plants, sugars(mainly sucrose) are produced by photosynthetically assimilated carbon in mesophyll cells of leaves and translocated to heterotrophic organs to ensure plant growth and development. Sucrose transporters, or sucrose carriers(SUCs), play an important role in the long-distance transportation of sucrose from source organs to sink organs, thereby affecting crop yield and quality.The identification, characterization, and molecular function analysis of sucrose transporter genes have been reported for monocot and dicot plants. However, no relevant study has been reported on sucrose transporter genes in Brassica rapa var. rapa, a cruciferous root crop used mainly as vegetables and fodder.We identified and cloned 12 sucrose transporter genes from turnips, named BrrSUC1.1 to BrrSUC6.2according to the SUC gene sequences of B. rapa pekinensis. We constructed a phylogenetic tree and analyzed conserved motifs for all 12 sucrose transporter genes identified. Real-time quantitative polymerase chain reaction was conducted to understand the expression levels of SUC genes in different tissues and developmental phases of the turnip. These findings add to our understanding of the genetics and physiology of sugar transport during taproot formation in turnips.

番茄植株中黄酮类对菜青虫(Pieris rapae L.)的活性组分及其对截形叶螨(Tetranychus truncalus Ehara)的毒效

本研究对番茄(Lycopersicon esculentum)植株中黄酮类进行了提取、分离和部分纯化,从中得到一种黄酮醇,经红外光谱、紫外光谱、质谱、核磁共振等分析,并与标准图谱对照,确定其分子结构为芦丁。生物测定结果表明,黄酮类对菜青虫有一定拒食作用,但未达到显著标准;黄酮类及芦丁有抑制菜青虫生长发育的作用;芦丁对截形叶螨有毒效,LC_(50)为94.00 μg·g^(-1)。

家蚕微孢子虫(Nosema bombycis)感染菜粉蝶(Pieris rapae)幼虫初探

采用不同浓度的家蚕微孢子虫感染三峡库区菜粉蝶幼虫—菜青虫,结果表明:家蚕微孢子虫对菜青虫感染性强,致死率较高,其半致死浓度(LC50)为:1.46×104/mL。当微孢子虫浓度为1.0×107/mL时,菜青虫死亡率高达88.9%,校正死亡率也高达87.2%,显著体现了家蚕微孢子虫对菜青虫幼虫的致病能力,由此探索了家蚕微孢子虫做为生物农药的可行性。

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.

Development and Characterization of Microsatellite Markers in Brassica rapa ssp.chinensis and Transferability Among Related Species

Simple sequence repeat （SSR） or microsatellite marker is a valuable tool for several purposes, such as mapping, fingerprinting, and breeding. In the present study, an intersimple sequence repeat （ISSR）-PCR technique was applied for developing SSR markers in non-heading Chinese cabbage （Brassica rapa）. A total of 190 SSRs were obtained. Among these, AG or CT （54.7%） was the most frequent repeat, followed by AC or GT （31.6%） of the microsatellites. The average number of the SSRs length array was 16 and 10 times, respectively. Based on the determined SSR sequences, 143 SSR primer pairs were designed to evaluate their transferabilities among the related species of Brassica. The number of alleles produced per marker averaged 2.91, and the polymorphism information content （PIC） value ranged from 0 to 0.863 with an average of 0.540. Monomorphism was observed in 16 primer pairs. The transferability percentage in CC genome was higher than in BB genome. More loci occurred in the BBCC genome. This result supported the hypothesis that BB genome was divergent from A and C genomes, and AA and CC genomes were relatively close. The polymorphic primers can be exploited for further evolution, fingerprinting, and variety identification.

Transformation of Chinese Cabbage(Brassica rapa L. ssp. pekinensis)by Agrobacterium Micro-Injection into Flower Bud

We obtained two lines of Chinese head cabbage(Brassica rapa L. ssp. pekinensis)selfed progenies containing both an anti-sense gene of BcpLH and a gene for resistance to kanamycin by micro-injecting buds of their primary transformants(T0)with Agrobacterium tumefaciens strain LBA4404. 31 positive plants resistant to kanamycien were recovered. Southern blot analysis confirmed the presence of T-DNA in two transgenic plants. One(DHZ-13-1)exhibits the characteristics of out-toward rosette and cauline leaves, and nested flower model in which secondary complete flower developed from the base of the primary ovary and the third flower from the ovary in the secondary flower, and so on, while another(DHZ-6-1)has no phenotype change. ABA and IAA affected the root growth of progeny of DHZ-13-1, but 6-BA was insensitive to hypocotyl growth during its seedling development.

Genetic Diversity of European and Chinese Oilseed Brassica rapa Cultivars from Different Breeding Periods

The Brassica oilseed crops went through two major breeding bottlenecks during the introgression of genes for zero erucic acid and low glucosinolate content, respectively, which may lead to reduced genetic biodiversity of the crop. This study investigates the impact of these bottlenecks on the genetic diversity within and across European and Chinese winter B. rapa cultivars. We compared eight cultivars from Europe and China, representing three different seed qualities from three different breeding periods： （1） high erucic acid, high glucosinolates （＋＋）; （2） zero erucic acid, high glucosinolates（0＋）; （3） zero erucic acid, low glucosonolates （00, canola quality）. Diversity was estimated on 32 plants per cultivar, with 16 simple sequence repeat （SSR） markers covering each of the B. rapa linkage groups. The analysis of molecular variance （AMOVA） showed that genetic variations within cultivars, across cultivars and across regions （Europe and China） were significant, with about 60% of the total variation within cultivars. There was a slight, but non-significant loss in genetic diversity within cultivars when comparing the three breeding periods as indicated by effective number of alleles （2.39, 2.23, and 1.99 for breeding periods 1, 2, and 3, respectively）, Shannon information index （0.93, 0.90, 0.75）, and expected heterozygosity （0.51, 0.49, 0.42）. By cluster analysis （UPGMA dendrogram） and principal coordinate analysis, Chinese and European cultivars were clearly divided into two distinct groups. In conclusion, quality improvement did not significantly reduce the genetic diversity of European and Chinese B. rapa cultivars.

Control Efficiency of Four Biological Pesticides against Pieris rapae Linne and Their Impacts on Yield and Benefit of Chinese Cabbage

[ Objective] The paper was to screen out ideal biological pesticides, in order to provide guidance for pollution-free control against Pieris rapae Linne. [Method] Taking Chinese cabbage variety Taiyuan Erqing as the test material, the field control efficiencies of four pesticides including PrGV · Bt WP 1 000 times dilution, NPV · Bt SC 750 times dilution, 0.5% azadirachtin EC 750 times dilution, and 0.3% matrine AS 500 times dilution against P. rapae were studied, and their impacts on yield and planting benefits of Chinese cabbage were also determined. Using foliar spray method, the pesticides were sprayed for the first time when the second or third instar larvae of P. rapae first occurred in fields, and sprayed for the second time with the interval of 15 d. The fields sprayed with beta-cypermethrin EC （organic chemical pesticide） and water were set as control. [ Result] NPV · Bt SC 750 times dilution had the best effect after spraying for two times： the control efficiency against P. rapae at 15 d after spraying was 90.11% ; the damage rate of Chinese cabbage was only 0.21%, while the commodity rate reached 100% ; compared with chemical pesticide spraying, the commodity yield （177 262.5 kg/hm2 ） and the income after deducting spraying cost （48 858.5 yuan/hm2 ） were increased by 14.7% and 13.75%, respectively. [ Conclusion] Although biological pesticides are more expensive, they have long persistence and good control effect, resulting in green and safe Chinese cabbage with high commodity rate and yield, and higher eventual economic benefit after deducting spraying cost.

Development of InDel Markers for Brassica rapa Based on a High-resolution Melting Curve

Brassica rapa is one of the most important leafy vegetable crops with large cultivated area in China.To increase the availability of DNA markers in B.rapa,we developed insertion-deletion(InDel)markers utilizing high-resolution melting(HRM)curve analysis.We designed primers for 252 InDels(≥3 bp)evenly distributed in the genome and tested gene polymorphisms with eight accessions.In total,208 markers were specifically amplified,and 148 InDels with polymorphism were genotyped successfully using HRM.We further analyzed the correlation with InDel size,GC number,and predicted the difference in Tm values(Tm)using 208 markers with specific amplification.We found that the success rate of InDel markers was correlated with the GC number of InDel and the predicted-Tm,but not clearly correlated with the length of InDel.When the GC number within InDel was≥8,the successful rate exceeded 90.0%.When the predicted-Tm reached 0.5°C,the success rate was greater than 90.0%,and when it was≥0.6°C,the rate climbed to 100.0%,indicating their role as the optimal parameter for successful development of an applicable InDel marker.The polymorphic InDel markers can be easily genotyped using HRM.They are of great value in genetic analysis,construction of linkage map,and molecular marker-assisted selection in B.rapa.

菜粉蝶Pieris rapae(L)的人工饲养及幼虫取食量的测定与防治适期

通过对菜粉蝶的人工饲养，测定各龄期在甘蓝上的取食量，累计取食量，观察其生物学特性，结果显示其取食量从１—５龄逐步递增，在菜青虫卵孵化后８天左右，累计取食量进入激增，１５天左右达到高逢。此为制订菜青虫化防适期提供科学依据。

A Genetic Linkage Map of Brassica rapa Based on AFLP Markers

A F2 mapping population was developed by crossing a Chinese cabbage-pe-tsai variety CC156 and an oil type Rapid cycling RC144 which were different from each other in morphology, maturity, self-compatibility, plant height, etc. Using 244 AFLP markers a map was constructed containing 10 main linkage groups covering a total distance of 857 cM, corresponding to 3.5 cM per marker. Length of linkage groups varied from 43 to 125 cM and the number of AFLP markers linkage to each group ranged from 7 to 41.

菜粉蝶(Pieris rapae L.)种群抽样技术研究

在研究菜粉蝶种群幼虫、蛹空间分布型的基础上,利用分布型参数Kc值;Iwao回归式中的α,β;Taglor幂律中的a,b以及Morisita的I_δ求得理论抽样模型,从而导出在不同置信度t,不同的平均虫口密度X(头/株)和不同允许误差下D的理论抽样数。利用Kuno(1969),Iwao(1975)和willson(1983)提出的序贯抽样法,得到菜粉蝶幼虫的5个序贯抽模型,并对这些模型进行比较。结合Kuno和Iwao的模型,获得菜粉蝶幼虫的Kuno-Iwao复合序贯抽样法,从而在某些情况下大大减少了抽样数。

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.

Developing Stable Cultivar through Microspore Mutagenesis in ×<i>Brassicoraphanus koranhort</i>, Inter-Generic Allopolyploid between <i>Brassica rapa</i>and <i>Raphanus sativus</i>

A stable progeny was developed through induced mutation, using microspore culture, of the hybrid (F1F1) produced by crossing a newly synthesized, unstable allopolyploid (F1) and a stable cultivar, BB#1(F1) in xBrassicoraphanus. An F1F1 plant was subjected to the induced mutation system established during production of BB#1. Morphological characteristics of the progeny such as color, and leaf number and length, differed from those of BB#1. The bolting time of the progeny in spring cropping was very late compared to BB#1, allowing it to be grown to an adult plant in spring. Genomic in situ hybridization analysis of pollen mother cells at prophase I identified 19 bivalents, 10 from Brassica rapa and 9 from Raphanus sativus. The glucoraphenin content was almost identical to that of BB#1. Two cultivars are available in the baemoochae crop now. These results indicate that induced mutation using microspore culture is a viable method of stabilizing intergeneric allopolyploids between B. rapa and R. sativus.

An optimized protocol for detecting guard cell specific gene expression by in situ RT-PCR in Brassica rapa

It is important to detect specific genes expressed in the guard cells,which control gas exchange and play key roles in response to drought and salt stresses.Due to the genetic transformation of Chinese cabbage(Brassica rapa)has not been well developed,in situ RT-PCR is a valuable option for detecting guard cell specific genes.We reported an optimized protocol of in situ RT-PCR by using an FAMA homologous gene Bra001929 in Brassica rapa.FAMA in Arabidopsis has been verified to be specially expressed in guard cells.We designed specific RT-PCR primers and optimized the protocol in terms of the(a)reverse transcription time,(b)blocking time,(c)antigen-antibody incubation time,and(d)washing temperature.Our approach provides a sensitive and effective in situ RT-PCR method for locating expression in the guard cells in Brassica rapa.Moreover,we proved the guard cell specific expression of Bra001929 in the epidermis indicating its’applicability as a marker gene for guard cells of Brassica rapa.