Effects of Heavy Metals and Saline-alkali on Growth,Physiology and Biochemistry of Orychophragmus violaceus

[Objective] The aim was to study on effects of heavy metals and saline-alkali on growth, physiology and biochemistry of Orychophragmus violaceus. [Method] Taken Orychophragmus violaceus as materials, growth, physiology and biochemistry were explored under stress of saline-alkali and heavy metals (light, moderate and severe saline-alkali, Pb, Pb + Cd, light saline-alkali + Pb, moderate saline-alkali + Pb, severe saline-alkali + Pb, light saline-alkali + Pb + Cd, moderate saline-alkali + Pb + Cd and severe saline-alkali + Pb + Cd) with control group set. [Result] Light stress of saline-alkali had little effect on membrane permeability, as follows: MDA contents in leaves and root systems declined by 25.6% and 9.0% compared with control group; Pb (500 mg/L) stress promoted synthetization of photosynthetic pigments, as follows: chlorophyll a and b and carotenoid increased by 0.86%, 0.69% and 6.25% than those of control group; combined stresses of Pb and Cd destroyed synthetization of photosynthetic pigments, among which carotenoid was more sensitive; under combined stresses of saline-alkali, Pb and Cd, POD and SOD activities, soluble saccharides and Pro content all increased and activities of POD and SOD in root system were both higher than those in leaves. [Conclusion] Orychophragmus violaceus is with resistance against light combined stresses of saline-alkali and Pb (500 mg/L).

诸葛菜(Orychophragmus violaceus)Toc33基因编码区的克隆及序列分析

以诸葛菜(Orychophragmusviolaceus)新鲜嫩叶为材料提取总DNA,并以其为模板,根据已报道的拟南芥(Arabidopsisthaliana)Toc33基因的编码序列,设计一对PCR引物,扩增诸葛菜叶绿体外膜蛋白转运器构件蛋白基因Toc33,得到两条扩增带,将这两条带分别割胶回收,与T载体连接转化E.coli,筛选重组子并测序,结果表明克隆到的这两个片段分别长1475bp,1573bp,通过同源性比较发现,它们之间的同源性达到88%.这两个片段与拟南芥Toc33基因有较高的同源性,分别命名为OvToc33 1,OvToc33 2.参考拟南芥Toc33外显子和氨基酸序列,分别确定了所克隆的两个诸葛菜Toc33基因片段的7个外显子和6个内含子,得到了诸葛菜Toc33基因的全编码区序列,并推导了其氨基酸序列.这两个DNA序列与拟南芥Toc33基因编码区的同源性分别高达91.8%和92.4%,说明这一蛋白是高度保守的.

油菜品质改良的重要天然遗传资源——诸葛菜(ORYCHOPHRAGMUS VIOLACEUS)利用前景展望

Orychophragmus Violaceus L.O.E.Schulz属,中国种,诸葛菜,其种子含有特高含油率和特高亚油酸,且具有十分理想的脂肪酸组成的天然植物资源。从现资料看,属世界上的首次发现,也是我国第一个低芥酸源,就油苯脂肪酸组成改良育种的前景看,本文已论证,这里是一个极其宝贵的天然资源。

诸葛菜(Orychophragmus violaceus)查尔酮合成酶基因OvCHS的克隆与序列分析

根据GenBank发布的基因序列,设计PCR引物,分别从诸葛菜(Orychophragmus violaceus)的花瓣基因组DNA和cDNA克隆到查尔酮合成酶基因,并定名为OvCHS,序列已上传至NCBI数据库,登陆号为EF408918。序列分析表明,OvCHS基因的基因组全长为1263 bp,具一个75 bp的内含子,编码区全长为1188 bp,编码395个氨基酸。与模式植物拟南芥(Arabidopsis thaliana)查尔酮合成酶基因AtCHS比较发现,两基因编码区有135个碱基不同,相似性为88.64%,氨基酸序列中仅16个氨基酸残基的差异,相似性达95.95%。

Study on the Flower Development of Testtube Plantletsof Orychophragmus violaceus

In this study, testtube plantlets of O.Violaceus were cultured on MS media and treated with low temperature, phytohormones, etc. The following results were obtained: 1. The cold treatment of 5～7℃ for more than 7 days was needed for flower initiation of the testtube plantlets originated from germinated seeds. 2. When cultured on the MS media supplemented with 2mg/L gibberellin (GA3), the testtube plantlets could initiate flowers without cold treatment. 3. When the MS media was supplemented with a reduced amount of NH4NO3, flower initiation of the testtube plantlets could be accelerated. The results of the study on flower development of O.Violaceus may be useful for the production of testtube flowers of this ornamental plant species.

Preliminary Studies on the Intergeneric Hybridization Between Brassica napus with OguCMS and Orychophragmus violaceus

In the cross between B. napus with OguCMSOrychophragmus violaceus, pre-fertilizatiocompatibility was observed. An intergeneric hybrid was successfully obtained by usinovary culture. The hybrid status was confirmed by cytology, morphology, fertilityisozyme and miosis analysis. The seed setting rate was 0.0072%. An amphidiploid of thhybrid was also produced by the use of chromosome-doubling method with 0.1% colchicinetreatment. Obtaining this hybrid and its amphidiploid has important theoretical anutilization value.

Does bicarbonate affect the nitrate utilization and photosynthesis of Orychophragmus violaceus?

The effect of bicarbonate(HCO_3^-) on the growth and development of plants varies by species. To better understand inorganic carbon and nitrogen assimilation changes of karst-adaptable plants under different HCO_3^- treatments, we conducted experiments on seedlings and in vitro plantlets of Orychophragmus violaceus(Ov).We found that the vital photosynthesis potential(as measured by net photosynthetic rate, actual photochemical efficiency of photosystem-II, photochemical quenching coefficient, and the instantaneous carbon isotope ratio of3-phosphoglycerate) was consistent under different HCO_3^- treatments of Ov. Bicarbonate's lack of effect on carbon assimilation of Ov may be related to carbonic anhydrase in Ov converting HCO_3^- to H2 O and CO2. In this way, Ov could prevent HCO_3^- ion toxicity and high pH from harming its growth and development under HCO_3^- stress.This study also found that high HCO_3^- concentrations could promote nitrogen assimilation and utilization of Ov through changes in related indexes(foliar nitrogen isotope fractionation ratio, stable nitrogen isotope assimilation ratio, foliar stable nitrogen isotope fractionation, nitrate nitrogen utilization efficiency, and nitrate utilization share)under different HCO_3^- treatments. Bicarbonate has different effects on photosynthesis and on inorganic nitrogen assimilation of Ov, which may be connected tophotosynthesis providing electrons for nitrate/nitrite reduction through the photosynthetic chain.

Transformation of Orychophragmus violaceus Using Agrobacterium tumefaciens And Regeneration of Transgenic Plantsa

In this paper, we reported firstly the transgenic plants of Orychophragmus violateus in the world. Excised cotyledon and hypocotyls of Orychophragmus violaceus were used as explants for genetic transformation. After 2-3 days of co-cultivation with Agrobacterium tumefaciens strain A208SE(PROA93),the hypocotyls and cotyledon were transferred onto selection medium containing 25 mg/L Km and 250 mg/L Ap. 8 weeks later, shoots emerged,then the shoots were excised and transferred onto root medium containing 25 mg/L Km and 100 mg/L Cef. The roots were formed within 4-5 weeks.The whole plants were transplanted into pots and grew well. The frequency of plant regeneration of hypocotyls was about 30%,and that of cotyledon was 51%.The regenerated plants showed high enzymatic activities ofglucuronidase and neomycin phosphotransferase II. Southern blot analysis confirmed that NPT II gene had been stably integrated into the chromosomal genome of Orychophragmus violaceus .the transformation frequency of hypocotyls was 10%,and that of cotyledon was 5.5%.

PEG－mediated Gene Transfer into Orychophragmus Violaceus Cotyledon Protoplast and Regeneration of Transgenic Plants

Transgenic plants were obtained by PEG-mediated tranfer of foreign gene into cotyledon protoplasts of Orychophragums violaceus. Systematic study was carricd out on PEG-mediatcd transformation of cotyledon protoplast using transient expression system, which showed 25-30 μg of pasmid, 15% PEG and a pH value of 8.0 as the optimal parameters contributing to the highest expression level. Using these parameters, cotyledon protoplasts were isolated, treated with bacterial plasmid DNA (pBI222 with HPT as selective marker) and PEG, and cultured at a density of 5×10 4/ml.After 10-15 days,they were selected by adding 25 μg/ml hygromycine. One month later, a few calli were observed, which were then transferred onto a solid medium with 50-100 μg/ml hygromycine for proliferation. Later they were transferred successively onto differentiation and rooting media and finally hygromycineresistant whole plants were obtaincd. The plants grew well in pots and a regeneration rate of 5 ×10(-5) was achieved. Then,excised leaves of the transgenic plants were used as explants for Southern blot analysis, which confirmed the stable integration of HPT gene into the chromosomal genome of Orychophragmus violaceus The transformation frequency was 10-5.

Morphological and Cytogenetic Analysis on the Advanced Generations of Generic Hybrids between Brassica napus and Orychophragmus violaceu

[Objective] This study aimed to reveal the genetic changes of advanced generation hybrids between Brassica napus and Orychophragmus violaceus. [Method] The morphological characteristics such as the plant shape, branching sites, leaf shape, leaf color, primary branches and secondary branches, as well as the cytological characteristics of the advanced generation hybrids (F8-F10) between B. napus and O. violaceus were observed. [Result] The morphology analysis revealed that the hybrid progeny was more like B. rapa in leaf shape, leaf color, plant shape and early flowering phenotype, whereas more like B. napus in number of secondary branches, silique length and 1 000-seed weight. Analysis on the cytogenetics characteristics showed that these advanced inbred progenies were hypoploids with less than 38 chromosomes; moreover, all the chromosomes from O. violaceus had been lost. Chromosome pairings at meiotic diakinesis of hybrids between the advanced inbred progenies and B. rapa revealed that chromosomes lost in hypoploids possibly belonged to the C genome of B. oleracea. With generations developing, chromosomes number of plants from two populations gradually increased and developed into the number of B. napus (2n=38). [Conclusion] This study will provide reference to reveal the source of chromosome lost in hypoploids and the morphological change of hybrids.

诸葛菜Orychophragmus violaceu(sL.)O.E.Schulz.

诸葛菜是十字花科诸葛菜属一年或二年生草本植物,高可达50 cm,无毛;茎直立,基生叶及下部茎生叶大头羽状全裂,顶裂片近圆形或短卵形,侧裂片卵形或三角状卵形,叶柄疏生细柔毛;花紫色、浅红色或褪成白色,花萼筒状,紫色,花瓣宽倒卵形,密生细脉纹,长角果线形,种子卵形至长圆形,黑棕色,4~5月开花,5~6月结果。

外源GA_3对NaCl胁迫下二月兰种子萌发的影响

为研究外源GA3处理对NaCl胁迫下二月兰种子萌发的影响,设定了0、0.5%、1.0%和2.0%4个NaCl胁迫浓度,分别用0、50、100、200、300mg/L和500mg/L外源GA3溶液处理,测定二月兰种子的发芽势、发芽率、发芽指数、活力指数和α-淀粉酶活性。结果表明:在较低浓度NaCl胁迫下,GA3处理对二月兰种子萌发具有促进作用,其发芽势、发芽率、发芽指数、活力指数和α-淀粉酶活性均显著提高,且随着GA3浓度的增加,其促进作用增强,200 mg/L时达到最高。随着GA3浓度的继续增加,其促进作用逐渐下降,说明适宜浓度的GA3处理可以缓解NaCl胁迫对二月兰种子萌发的抑制作用;而在较高浓度NaCl胁迫下,GA3处理对二月兰种子的萌发的促进作用不明显。

常压室温等离子体对NaCl胁迫下二月兰种子萌发和幼苗生长的影响

以二月兰种子为试验材料,采用培养皿纸上发芽法研究不同质量浓度NaCl胁迫下常压室温等离子体(ARTP)处理对二月兰种子萌发和幼苗生长的影响。结果表明:在清水处理条件下,ARTP处理降低了幼苗地上部和地下部鲜质量,处理10 min时根长显著增加,出苗率显著降低;在5 g/L NaCl处理条件下,ARTP处理对二月兰种子萌发与幼苗生长没有显著影响;在10 g/L NaCl处理条件下,ARTP处理对二月兰幼苗株高无显著影响,但显著降低了出苗率,处理10 min时发芽率显著降低,但幼苗地上部鲜质量显著增加。

外源BABA对NaCl胁迫下二月兰幼苗生长和生理特性的影响

以二月兰为试材,研究不同β-氨基丁酸(BABA)浓度(0.25、0.50、1.00、2.00 mmol·L^-1)对NaCl胁迫下二月兰幼苗生长指标、质膜透性、脯氨酸含量、抗氧化酶活性以及叶绿素含量的影响。结果表明:NaCl胁迫下,随着BABA处理浓度的增加,二月兰幼苗的相对电导率呈现先下降后上升的趋势,生物量、根系生长指标、脯氨酸含量、抗氧化酶活性以及叶绿素含量均呈现先上升后下降的趋势。相对电导率在0.25 mmol·L^-1时降幅达最大,脯氨酸含量和叶绿素含量均在0.50 mmol·L^-1时增幅达最大。过氧化物酶(POD)活性和过氧化氢酶(CAT)活性均在0.25 mmol·L^-1变化最显著,达到最大值,超氧化物歧化酶(SOD)活性在0.50 mmol·L^-1时达到最大值。表明0.25、0.50 mmol·L^-1浓度的BABA处理能够有效缓解NaCl胁迫对二月兰幼苗的伤害。

Foliar modifications induced by inhibition of polar transport of auxin

The effects of auxin polar transport inhibitors, 9-hydroxy-fluorene-9-carboxylic acid (HFCA); 2, 3, 5-triiodobenzoic acid (TIBA) and trans-cinnamic acid (CA)on leaf pattern formation were investigated with shoots formed from cultured leaf explants of tobacco and cultured pedicel explants of Orychophragmus violaceus, and the seedlings of tobacco and Brassica chinensis. Although the effective concentration varies with the inhibitors used, all of the inhibitors induced the formation of trumpet-shaped and/or fused leaves. The frequency of trumpet-shaped leaf formation was related to the concentration of inhibitors in the medium.Histological observation of tobacco seedlings showed that there was only one main vascular bundle and several minor vascular bundles in normal leaves of the control, but there were several vascular bundles of more or less the same size in the trumpet-shaped leaves of treated ones.These results indicated that auxin polar transport played an important role on bilateral symmetry of leaf growth.

花生与绿肥轮作对土壤含水量和土壤肥力的影响

探讨在冬春裸地上种植绿肥对后茬作物春花生养分含量、土壤含水量和土壤肥力的影响,为山东地区春花生等冬闲田建立一种合理的轮作方式提供理论依据。在田间试验条件下,设置了春花生—冬闲田、夏花生—冬小麦、春花生—二月兰和春花生—毛叶苕子4种轮作处理。结果表明:种植二月兰和毛叶苕子绿肥作物,可促进后茬作物花生氮磷钾养分增加,二月兰增加程度高于毛叶苕子。种植二月兰和毛叶苕子绿肥作物,可提高后茬作物花生土壤含水量和土壤肥力,在0~20 cm土层中,2021年,与春花生—冬闲田相比,春花生—二月兰、春花生—毛叶苕子土壤相对含水量分别升高了4.70%、6.14%,在20~40、40~60 cm土层中也表现出相似趋势,2022年与2021年相比,表现出相似规律;与春花生—冬闲田相比,春花生—二月兰处理有机质、碱解氮、有效磷和速效钾含量平均增加了5.71%、8.98%、7.22%和7.72%,春花生—毛叶苕子处理有机质、碱解氮含量平均增加了4.54%、14.91%。可以利用春花生种植期间的冬春裸地种植一季冬绿肥,促进后茬作物养分积累量增加,改善土壤肥力。

盐胁迫对二月兰(Orychophragmus violaceus)种子萌发的影响

为了了解盐渍化土壤条件对二月兰(Orychophragmus violaceus)种子萌发的影响,及种子萌发及萌发过程中可溶性糖、蛋白质、淀粉酶活性的影响,本研究分别用浓度为0.3%、0.6%、0.9%、1.2%、1.5%的NaCl、Na_2SO_4和二者质量比为1:1的混合盐处理二月兰种子,并观测溶性糖、蛋白质、淀粉酶活性。结果表明,二月兰种子的发芽率、发芽势、发芽指数均随着盐浓度增加而下降,且盐浓度愈高对种子萌发抑制作用愈强。二月兰种子耐盐适宜范围为NaCl 0.29%、Na_2SO_4 0.30%、混合盐0.24%,耐盐半致死浓度为NaCl 0.63%、Na_2SO_4 0.65%、混合盐0.60%,耐盐极限浓度为NaCl 1.18%、Na_2SO_41.21%、混合盐1.18%。说明二月兰种子萌发中蛋白质的分解受到一定程度的抑制,较高浓度盐处理强烈抑制了淀粉酶活性,盐处理使可溶性糖含量增加。为以后二月兰在盐渍化条件下的应用提供理论依据。

Genome assembly of the Brassicaceae diploid Orychophragmus violaceus reveals complex whole-genome duplication and evolution of dihydroxy fatty acid metabolism

Orychophragmus violaceus is a Brassicaceae species widely cultivated in China,particularly as a winter cover crop in northern China because of its low-temperature tolerance and low water demand.Recently,O.violaceus has also been cultivated as a potential industrial oilseed crop because of its abundant 24-carbon dihydroxy fatty acids(diOH-FAs),which contribute to superior high-temperature lubricant properties.In this study,we performed de novo assembly of the O.violaceus genome.Whole-genome synteny analysis of the genomes of its relatives demonstrated that O.violaceus is a diploid that has undergone an extrawhole-genome duplication(WGD)after the Brassicaceae-specific a-WGD event,with a basic chromosome number of x=12.Formation of diOH-FAs is hypothesized to have occurred after the WGD event.Based on the genome and the transcriptome data from multiple stages of seed development,we predicted that OvDGAT1-1 and OvDGAT1-2 are candidate genes for the regulation of diOH-FA storage in O.violaceus seeds.These results may greatly facilitate the development of heat-tolerant and eco-friendly plant-based lubricants using O.violaceus seed oil and improve our understanding of the genomic evolution of Brassicaceae.

Nuclear reconstitution of demembranated Orychophragmus violaceus sperm in Xenopus laevis egg extracts

The cell-free extracts from animal Xenopus laevis egg could induce chromatin decon- densation and pronuclear formation from demembranated plant (Orychophragmus violaceus)sperm. The demembranated Orychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.

The genome of Orychophragmus violaceus provides genomic insights into the evolution of Brassicaceaepolyploidizationandits distinct traits

Orychophragmus violaceus,referred to as‘‘eryuelan’’(February orchid)in China,is an early-flowering ornamental plant.The high oil content and abundance of unsaturated fatty acids in O.violaceus seeds make it a potential high-quality oilseed crop.Here,we generated a whole-genome assembly for O.violaceus using Nanopore and Hi-C sequencing technologies.The assembled genome of O.violaceus was~1.3 Gb in size,with 12 pairs of chromosomes.Through investigation of ancestral genome evolution,we determined that the genome of O.violaceus experienced a tetraploidization event from a diploid progenitor with the translocated proto-Calepineae karyotype.Comparisons between the reconstructed subgenomes of O.violaceus identified indicators of subgenome dominance,indicating that subgenomes likely originated via allotetraploidy.O.violaceus was phylogenetically close to the Brassica genus,and tetraploidy in O.violaceus occurred approximately 8.57 million years ago,close in time to the whole-genome triplication of Brassica that likely arose via an intermediate tetraploid lineage.However,the tetraploidization in Orychophragmus was independent of the hexaploidization in Brassica,as evidenced by the results from detailed phylogenetic analyses and comparisons of the break and fusion points of ancestral genomic blocks.Moreover,identification of multi-copy genes regulating the production of high-quality oil highlighted the contributions of both tetraploidization and tandem duplication to functional innovation in O.violaceus.These findings provide novel insights into the polyploidization evolution of plant species and will promote both functional genomic studies and domestication/breeding efforts in O.violaceus.