芜菁花青素合成酶基因的克隆、序列分析及表达

目的：克隆津田芜菁和赤丸芜菁花青素合成酶（ANS）基因并研究其表达特性。方法：用UV-A处理津田芜菁和赤丸芜菁块根24h后提取总RNA,通过RT-PCR方法克隆BrANS1和BrANS2基因并进行序列分析,通过Northern杂交检测BrANS1和BrANS2基因的表达。结果：BrANS1和BrANS2的开放读码框为1077bp,编码358个氨基酸残基;BRANS1和BRANS2与甘蓝ANS的同源性达97％,第211-307肽段具有20G-Fe（Ⅱ）加氧酶家族基因的结构域;BrANS1和BRANS2基因具有高度同源性,核苷酸序列在5个位置上存在差异,推导的氨基酸序列完全相同;uv-A可以诱导BrANS1和BrANS2表达,基因的表达量与处理时间相关。结论：克隆了津田芜菁和赤丸芜菁的BRANS1和BrANS2基因,这将为筛选依光型和非依光型花青素生物合成催化酶基因奠定研究基础。     

NbALD1 mediates resistance to turnip mosaic virus by regulating the accumulation of salicylic acid and the ethylene pathway in Nicotiana benthamiana

AGD2-LIKE DEFENCE RESPONSE PROTEIN 1 (ALD1) triggers plant defence against bacterial and fungal pathogens by regulating the salicylic acid (SA) pathway and an unknown SA-independent pathway. We now show that Nicotiana benthamiana ALD1 is involved in defence against a virus and that the ethylene pathway also participates in ALD1-mediated resistance. NbALD1 was up-regulated in plants infected with turnip mosaic virus (TuMV). Silencing of NbALD1 facilitated TuMV infection, while overexpression of NbALD1 or exogenous application of pipecolic acid (Pip), the downstream product of ALD1, enhanced resistance to TuMV. The SA content was lower in NbALD1-silenced plants and higher where NbALD1 was overexpressed or following Pip treatments. SA mediated resistance to TuMV and was required for NbALD1-mediated resistance. However, on NahG plants (in which SA cannot accumulate), Pip treatment still alleviated susceptibility to TuMV, further demonstrating the presence of an SA-independent resistance pathway. The ethylene precursor, 1-aminocyclopropanecarboxylic acid (ACC), accumulated in NbALD1-silenced plants but was reduced in plants overexpressing NbALD1 or treated with Pip. Silencing of ACS1, a key gene in the ethylene pathway, alleviated the susceptibility of NbALD1-silenced plants to TuMV, while exogenous application of ACC compromised the resistance of Pip-treated or NbALD1 transgenic plants. The results indicate that NbALD1 mediates resistance to TuMV by positively regulating the resistant SA pathway and negatively regulating the susceptible ethylene pathway.     

Chemical Constituents from Turnip and Their Effects on <i>α</i>-Glucosidase

Brassica rapa var. rapa (turnip) is an important crop in Qinghai-Tibet Plateau (QTP) with anti-hypoxic effect. Turnip is rich in glucosinolates, isothiocyanates and phenolic compounds with diverse biological activities, involving anti-oxidant, anti-tumor, anti-diabetic, anti-inflammatory, anti-microbial, hypolipidemic, cardioprotective, hepatoprotective, nephroprotective and analgesic properties. In this study, the ethyl acetate (EtOAc) and butanol parts of Brassica rapa were first revealed with inhibitory effects on α-glucosidase, whereas the water part was inactive. Subsequent bioassay-guided isolation on the EtOAc and butanol parts yielded 12 compounds, involving three indole derivatives, indole-3- acetonitrile (1) 4-methoxyindole-3-acetonitrile (2) and indole-3-aldehyde (3) two flavonoids, liquiritin (4) and licochalcone A (5) two phenylpropanoids, sinapic acid (6) and caffeic acid (7) two phenylethanol glycosides, 2-phenylethyl β- glucopyranoside (8) and salidroside (9) and three other compounds, syringic acid (10) adenosine (11) and (3β, 20E)-ergosta-5, 20 (22)-dien-3-ol (12) Licochalcone A (5) and caffeic acid (7) showed α-glucosidase inhibitory activity with IC₅₀ values of 62.4 ± 8.0 μM and 162.6 ± 3.2 μM, comparable to the positive control, acarbose (IC₅₀ = 142 ± 0.02 μM). Docking study suggested that licochalcone A (5) could well align in the active site of α-glucosidase (docking score = -52.88) by forming hydrogen bonds (Gln1372, Asp1420, Gln1372, Arg1510), hydrophobic effects (Tyr1251, Tyr1251, Trp1355, Phe1560, Ile1587, Trp1355, Phe1559, Phe1559) and π-π stacking interaction (Trp1355). This study provides valuable information for turnip as a new resource in searching anti-diabetic candidates.     

Turnip crinkle virus-encoded suppressor of RNA silencing interacts with Arabidopsis SGS3 to enhance virus infection

Most plant viruses encode suppressors of RNA silencing (VSRs) to protect themselves from antiviral RNA silencing in host plants. The capsid protein (CP) of Turnip crinkle virus (TCV) is a well-characterized VSR, whereas SUPPRESSOR OF GENE SILENCING 3 (SGS3) is an important plant-encoded component of the RNA silencing pathways. Whether the VSR activity of TCV CP requires it to engage SGS3 in plant cells has yet to be investigated. Here, we report that TCV CP interacts with SGS3 of Arabidopsis in both yeast and plant cells. The interaction was identified with the yeast two-hybrid system, and corroborated with bimolecular fluorescence complementation and intracellular co-localization assays in Nicotiana benthamiana cells. While multiple partial TCV CP fragments could independently interact with SGS3, its hinge domain connecting the surface and protruding domains appears to be essential for this interaction. Conversely, SGS3 enlists its N-terminal domain and the XS rice gene X and SGS3 (XS) domain as the primary CP-interacting sites. Interestingly, SGS3 appears to stimulate TCV accumulation because viral RNA levels of a TCV mutant with low VSR activities decreased in the sgs3 knockout mutants, but increased in the SGS3-overexpressing transgenic plants. Transgenic Arabidopsis plants overexpressing TCV CP exhibited developmental abnormalities that resembled sgs3 knockout mutants and caused similar defects in the biogenesis of trans-acting small interfering RNAs. Our data suggest that TCV CP interacts with multiple RNA silencing pathway components that include SGS3, as well as previously reported DRB4 (dsRNA-binding protein 4) and AGO2 (ARGONAUTE protein 2), to achieve efficient suppression of RNA silencing-mediated antiviral defence.     

Immunodetection of RNA-Dependent RNA Polymerase from Turnip Yellow Luteovirus Using Monoclonal Antibodies

Monoclonal antibodies (MAbs) to the RNA-dependent RNA polymerase from turnip yellow luteovirus (TYV) were prepared using a recombinant protein as immunogen and were shown to be directed to C-terminal part of the viral replicase. These MAbs were found to interact with a 70-kDa protein found in extracts from TYV-infected plants. Our result is the first successful attempt at detecting the RNA-dependent RNA polymerase of a luteovirus in infected plant extracts. We also found that the protein is not processed further and its accumulation and content in the infected plant obey a definite dynamics during the infection.     

A review of sources of resistance to turnip yellows virus (TuYV) in Brassica species

Turnip yellows virus (TuYV; previously known as beet western yellows virus) causes major diseases of Brassica species worldwide resulting in severe yield-losses in arable and vegetable crops. It has also been shown to reduce the quality of vegetables, particularly cabbage where it causes tip burn. Incidences of 100% have been recorded in commercial crops of winter oilseed rape (Brassica napus) and vegetable crops (particularly Brassica oleracea) in Europe. This review summarises the known sources of resistance to TuYV in B. napus (AACC genome), Brassica rapa (AA genome) and B. oleracea (CC genome). It also proposes names for the quantitative trait loci (QTLs) responsible for the resistances, Tu rnip Y ellows virus R esistance (TuYR), that have been mapped to at least the chromosome level in the different Brassica species. There is currently only one known source of resistance deployed commercially (TuYR1). This resistance is said to have originated in B. rapa and was introgressed into the A genome of oilseed rape via hybridisation with B. oleracea to produce allotetraploid (AACC) plants that were then backcrossed into oilseed rape. It has been utilised in the majority of known TuYV-resistant oilseed rape varieties. This has placed significant selection pressure for resistance-breaking mutations arising in TuYV. Further QTLs for resistance to TuYV (TuYR2-TuYR9) have been mapped in the genomes of B. napus, B. rapa and B. oleracea and are described here. QTLs from the latter two species have been introgressed into allotetraploid plants, providing for the first time, combined resistance from both the A and the C genomes for deployment in oilseed rape. Introgression of these new resistances into commercial oilseed rape and vegetable brassicas can be accelerated using the molecular markers that have been developed. The deployment of these resistances should lessen selection pressure for resistance-breaking isolates of TuYV and thereby prolong the effectiveness of each other and extant resistance.     

Performance of cabbage stem flea beetle larvae (Psylliodes chrysocephala) in brassicaceous plants and the effect of glucosinolate profiles

The cabbage stem flea beetle (CSFB), Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae), is one of the most important pests in European winter oilseed rape production. Adult beetles feed on young leaves whereas larvae mine within the petioles and stems. Larval infestation can cause significant crop damage. In this study, the host quality for CSFB of four oilseed rape (Brassica napus L.) cultivars and seven other brassicaceous species with different glucosinolate (GSL) profiles was assessed under controlled conditions. Larval instar weights and mortality were measured after 14 and 21 days of feeding in the petioles of test plants. To study the impact of GSL on the performance of larvae, the GSL contents in petioles from non-infested and infested plants were analysed before, and 21 days after, the start of larval infestation. Larval performance was not significantly different between the four cultivars of oilseed rape, but differed considerably among the other brassicaceous species tested. In comparison to the weight of larvae in the standard B. napus cv. Robust, the larval weight was higher in turnip rape (Brassica rapa L. var. silvestris) and significantly reduced in white mustard (Sinapis alba L.), oil radish (Raphanus sativa L. var. oleiformis), and cabbage (Brassica oleracea L. convar. capitata var. alba). The duration of larval development increased in white mustard and oilseed radish. The GSL profiles of the petioles showed little difference between non-infested and infested plants of oilseed rape whereas the content of aliphatic GSL increased in the infested turnip rape plants. In contrast, the aliphatic and benzenic GSL decreased in infested Indian rape (B. rapa subsp. dichotoma Roxb.). Larval weight was not correlated with the total GSL content of plants, neither before infestation nor 21 days after. Larval weight was positively correlated with progoitrin and 4-hydroxyglucobrassicin. White mustard, which provides inferior host quality for larval development, has the potential to introduce insect resistance into high-yielding oilseed rape cultivars in breeding programmes.     

Replication of the cauliflower mosaic virus: role and stability of the cloned delta 3 discontinuity sequence

A fragment of cauliflower mosaic virus (CaMV) DNA, containing delta 3, one of the three discontinuity sequences, was cloned in various ways into CaMV DNA deleted for the delta 3 sequence. The series of constructions was monitored for the appearance of the typical single-strand (ss) discontinuity after hybrid CaMV replication in plants. The delta 3 discontinuity was observed only if the orientation of inserted DNA sequence was the same as in the wild-type virus. Long polylinker sequences used for insertion of the fragment into cloned viral DNA, affected the stability of the insert in progeny viral DNA in plants by acting as recombination targets.     

Epithiospecifier protein in turnip and changes in products of autolysis during ontogeny

An epithiospecifier protein present in turnip tissue gives rise to 1-cyano-epithioalkanes during autolysis. Volatile hydrolysis products are produced from glucosinolates during autolysis of seeds, seedlings and plant tissue more than 6 weeks after sowing.