The molecular mechanism of WRINKLED1 transcription factor regulating oil accumulation in developing seeds of castor bean

The transcription factor WRINKLED1(WRI1),a member of AP2 gene family that contain typical AP2 domains,has been considered as a master regulator regulating oil biosynthesis in oilseeds.However,the regulatory mechanism of RcWRI1 in regulating oil accumulation during seed development has not been clearly addressed.Castor bean(Ricinus communis)is one of the most important non-edible oil crops and its seed oils are rich in hydroxy fatty acids,widely applied in industry.In this study,based on castor bean reference genome,three RcWRIs genes(RcWRI1,RcWRI2 and RcWRI3)were identified and the expressed association of RcWRI1 with oil accumulation were determined.Heterologous transformation of RcWRI1 significantly increased oil content in tobacco leaf,confirming that RcWRI1 activate lipid biosynthesis pathway.Using DNA Affinity Purification sequencing(DAP-seq)technology,we confirmed RcWRI1 binding with Transcription Start Site of genes and identified 7961 WRI1-binding candidate genes.Functionally,these identified genes were mainly involved in diverse metabolism pathways(including lipid biosynthesis).Three cis-elements AW-box([CnTnG](n)7[CG])and AW-boxes like([GnAnC](n)6[GC]/[GnAnC](n)7[G])bound with RcWRI1 were identified.Co-expression network analysis of RcWRI1 further found that RcWRI1 might be widely involved in biosynthesis of storage materials during seed development.In particular,yeast one hybrid experiments found that both AP2 domains within RcWRI1 were required in binding targeted genes.These results not only provide new evidence to understand the regulatory mechanism of RcWRI1 in regulation of oil accumulation during castor bean seed development,but also give candidate gene resource for subsequent genetic improvement toward increasing oil content in oilseed crops.

Proteomic Study of Differentially Expressed Proteins in Seeds between Parents and Offspring of Castor Bean(Ricinus communis L.)

Castor bean(Ricinus communis L.),is one of the top 10 oilseed crops in the world and,therefore,of high economic value.Hybridization is one of the most effective ways to breed new varieties with high yield,high oil content,and better stress resistance.Therefore,prediction of desired traits in castor hybrid offspring is particularly important.In this study,proteomic analysis was performed to identify differentially expressed proteins(DEPs)in seeds between castor hybrid offspring and their female(Lm female line aLmAB2)and male parents(CSR·181).Among the DEPs upregulated in the seeds of hybrid offspring,the majority were related to seed yield and stress tolerance,while some were related to oil synthesis and fatty acid synthesis and metabolism in seeds.In other words,the hybrid offspring showed heterosis for seed yield,stress tolerance,oil synthesis,and fatty acid synthesis and metabolism when compared with their parents.Further,real-time quantitative polymerase chain reaction assays were performed on 12 genes encoding DEPs involved in oil synthesis,pollen abortion,yield,and stress tolerance of seeds.The results showed that the expression levels of the 12 genes were consistent with those of the DEPs.

Genome-wide analysis of the B3 transcription factors reveals that RcABI3/VP1 subfamily plays important roles in seed development and oil storage in castor bean(Ricinus communis)

The B3 transcription factors(TFs)in plants play vital roles in numerous biological processes.Although B3 genes have been broadly identified in many plants,little is known about their potential functions in mediating seed development and material accumulation.Castor bean(Ricinus communis)is a non-edible oilseed crop considered an ideal model system for seed biology research.Here,we identified a total of 61 B3 genes in the castor bean genome,which can be classified into five subfamilies,including ABI3/VP1,HSI,ARF,RAV and REM.The expression profiles revealed that RcABI3/VP1 subfamily genes are significantly up-regulated in the middle and later stages of seed development,indicating that these genes may be associated with the accumulation of storage oils.Furthermore,through yeast one-hybrid and tobacco transient expression assays,we detected that ABI3/VP1 subfamily member RcLEC2 directly regulates the transcription of RcOleosin2,which encodes an oil-body structural protein.This finding suggests that RcLEC2,as a seed-specific TF,may be involved in the regulation of storage materials accumulation.This study provides novel insights into the potential roles and molecular basis of B3 family proteins in seed development and material accumulation.

Genomic characterization and expression profiles of stress-associated proteins(SAPs)in castor bean(Ricinus communis)

Stress-associated proteins(SAPs)are known as response factors to multiple abiotic and biotic stresses in plants.However,the potential physiological and molecular functions of SAPs remain largely unclear.Castor bean(Ricinus communis L.)is one of the most economically valuable non-edible woody oilseed crops,able to be widely cultivated in marginal lands worldwide because of its broad adaptive capacity to soil and climate conditions.Whether SAPs in castor bean plays a key role in adapting diverse soil conditions and stresses remains unknown.In this study,we used the castor bean genome to identify and characterize nine castor bean SAP genes(RcSAP).Structural analysis showed that castor bean SAP gene structures and functional domain types vary greatly,differing in intron number,protein sequence,and functional domain type.Notably,the AN1-C2H2eC2H2 zinc finger domain within RcSAP9 has not been often observed in other plant families.High throughput RNA-seq data showed that castor bean SAP gene profiles varied among different tissues.In addition,castor bean SAP gene expression varied in response to different stresses,including salt,drought,heat,cold and ABA and MeJA,suggesting that the transcriptional regulation of castor bean SAP genes might operate independently of each other,and at least partially independent from ABA and MeJA signal pathways.Cis-element analyses for each castor bean SAP gene showed that no common cis-elements are shared across the nine castor bean SAP genes.Castor bean SAPs were localized to different regions of cells,including the cytoplasm,nucleus,and cytomembrane.This study provides a comprehensive profile of castor bean SAP genes that advances our understanding of their potential physiological and molecular functions in regulating growth and development and their responses to different abiotic stresses.

Study on Free Amino Acid and Short Peptide Fertilizer Production by Solid State Fermented Castor Bean Meal

Using microbial fermentation to increase the content of free amino acids and short peptides in the organic fertilizer for castor bean meal can effectively promote plant growth and improve fruit quality.Using free amino acid and short peptide content as an indicator,through single factor and response surface optimization experiments,the process parameters(moisture content,fermentation time and inoculum quantity)of castor meal solid-state fermentation were optimized.The best process parameters for the solid-state fermentation were:the moisture content 62%,the fermentation time 20 d,and the inoculum quantity 0.23%.The moisture content had the greatest impact on the conversion rate of free amino acids and short peptides,and the protein conversion rate reached 65.6%.The scale-up experiment under the optimal conditions showed that the solid-state fermentation using the inoculum had a significant beneficial effect compared with other fermentation methods.The fermentation of castor cake fertilizer provides a theoretical and practical basis for production feasibility,and has important guiding significance for the effective utilization of castor bean meal.

Characterization and functional divergence of genes encoding sucrose transporters in oilseeds castor bean

Castor bean(Ricinus communis L.)is an economically important non-edible oilseed crop.Its seed oils are rich in hydroxy fatty acid,which are highly valuable with a wide range of industrial applications.Sucrose transportation is critical in regulating the growth,development and oilseed yield in castor bean.The transporters or carriers(SUTs or SUCs)play a central role in orchestrating sucrose allocation and aiding in plant adaptation to diverse stresses.In this study,based on castor bean genome,three RcSUCs(RcSUC2,RcSUC3 and RcSUC4)were identified and characterized.The expressional profiles of RcSUCs in different tissues such as leaf,stem,root,phloem and seed tissues exhibited a distinct divergence of gene expression,suggesting that the functions of RcSUC2,RcSUC3 and RcSUC4 are differentiated into long or short-distance transportation among tissues.Additionally,under abiotic stresses including hot temperature,low temperature,drought and salt stresses,the sugar allocation among leaf,stem and roots was tested.The expressional changes of Rc SUCs in leaf,stem and root tissues were associated with sugar transportation and allocation.Taken together,the differential expression of Rc SUCs among tissues responsing to abiotic stress suggested functional differences in sucrose transport and redistribution in different tissues.This study is helpful to understand the physiological and molecular mechanisms of sucrose transportation and allocation among tissues in heterotrophic oilseeds,and could provide clues for genetic improvement and optimization of cultivation practices.

Lipases in the castor bean seed of Chinese varieties:Activity comparison,purification and characterization

The lipase activities in the three main Chinese castor bean varieties [ZiBi 05 （ZB 5）,TongBi 06 （TB 6）,JinBi 02 （JB 2）] were investigated.Two lipases were found in extracts from the endosperms of all the three varieties.One lipase is located in the lipid bodies,showing higher activity at pH 4.5 and exhibiting the highest activity at day 1 of germination.The other lipase is located in the particulates,showing higher activity at pH 9.5 and particularly active at day 4 of germination.The lipid bodies from ZB 5 show the highest lipase activity at day 1 and pH 4.5 among the three varieties during the 8 d of germination.The acid lipase located in the lipid bodies of ZB 5 of germination 1 d was purified to homogeneity by DEAE-sepharose CL-6B and butyl-sepharose CL-4B chromatography after isolation,delipidation and solubilization,with a purification factor of 16.1 and overall activity recovery of 17.7%.The characterizations （such as optimum pH,pH stability,optimum temperature,thermal stability,effect of metal ions,organic solvents stability,substrate specificity） of the purified acid lipase were conducted in detail.A combination of the characterized properties may make this enzyme a useful biocatalyst for industrial application.

Effects of compression ratio on variation of stresses and residual oil of cake in pressing process of castor beans and its curve fitting

The relationships among compression ratio and stress, compression ratio and residual oil of cake in pressing process of castor beans were studied using the test equipment under different states of oilseeds and ways of pressing manners. The results show that variation of stress increases nonlinearly and residual oil rate decreases with the increase of compression ratio. Lower residual oil of cake was obtained by pressing gently and frequently. Curve fitting on both relationships had been built and parameters for the model were obtained by least square procedure and deepening research on pressing process of the castor beans for castor oil. By assuming that the value of oil production is equivalent to the value of energy consumption, the critical compression ratio of intact seeds is 6.2 while that of crushed seeds is 3.6.

<i>Wrinkled</i>1 (WRI1) Homologs, AP2-Type Transcription Factors Involving Master Regulation of Seed Storage Oil Synthesis in Castor Bean (<i>Ricinus communis</i>L.)

Among APETALA2 (AP2)-type plant specific transcription factor family, WRINKLED1 (WRI1), has appeared to be a master gene transcriptionally regulating a set of carbon metabolism- and fatty acid synthesis (FAS)-related genes responsible for seed specific triacylglycerols (TAGs) storage in oil plants. B3 type transcription factors, such as ABI3 and FUS3, are known to be involved in seed development, such as seed storage protein synthesis and maturation. Based on the recent whole genome sequence data of castor bean (Ricinus communis L.), putative WRI1 homologs (RcWRI1, RcWRI2) specifically expressed in castor bean seed have been identified by comparing organ specific expression profiles among seed development-related transcription factors, seed storage specific genes (Ricin, RcOleosin) and a set of FAS genes including genes for sucrose synthase (RcSUS2), biotin carboxyl carrier protein (a subunit of acetyl-CoA carboxylase, RcBCCP2) and ketoacyl-acyl carrier protein synthase (RcKAS1). Immunoreactive signals with WRI1, FUS3 and ABI5-related polypeptides were also detected in seed specifically, consistent with the expression profiles of seed development-related genes. The WRI1 binding consensus sites, [CnTnG](n)(7)[CG], designated as the AW-box, were found at the promoter region of RcBCCP2 and RcKAS1. Thus, RcWRI1 possibly play a pivotal role in seed specific TAGs storage during seed development by directly activating FAS -related genes.

蓖麻根腐病抗性鉴定及其SSR标记的初步建立

蓖麻根腐病是茄腐镰孢菌(Fusarium solani)引起的根部病害,严重影响蓖麻产量。抗源缺乏制约了抗病品种的选育。为寻找抗病种质、建立抗性分子标记,该研究对252份蓖麻材料的抗性进行了表型和分子标记鉴定。结果表明:(1)浓度为1×10^(6)个·mL^(-1)的孢子悬浮液灌根是一种有效的接种方法;以接种后枯萎天数为基础的5级评价法,可作为鉴定标准。(2)鉴定出130份抗病材料,其中高抗为105份。(3)野生材料中抗病材料比率(66%)远高于栽培材料(35%),建议将野生材料,尤其是中国华南野生材料的研究利用作为今后抗病育种的重要方向。(4)初步建立了8个与抗性关联的SSR标记。该研究结果提供了有效的根腐病抗性鉴定方法和评价标准,筛选出了一批育种迫切需要的抗病基因资源,初步建立了可用于辅助选择的SSR标记,为蓖麻抗根腐病育种奠定了重要基础。

绿豆、蓖麻对轻度重金属污染农田修复效果的影响

为探索不同植物对轻度重金属污染农田修复情况,筛选出适用该地区的吸附植物,设置对照组CK、绿豆组、蓖麻组3个处理,3次重复,每个试验小区40 m^(2),分析种植绿豆和蓖麻对土壤中5种重金属含量影响;绿豆和蓖麻籽实、秸秆、地上部对5中重金属积累情况;不同处理对5种重金属的地上部富集系数影响。种植绿豆、蓖麻均能降低土壤1重金属含量,与CK相比,绿豆组Cr、Pb、Cd和Hg分别降低11.84%、26.27%、56.97%、33.33%,蓖麻组分别降低5.35%、20.85%、33.94%、33.33%,绿豆组和蓖麻组相比,土壤Cr降低了6.32%;土壤Pb降低了6.85%;土壤Cd降低了34.86%;土壤As降低了3.29%。绿豆籽实5种重金属含量均小于绿豆秸秆,蓖麻籽实5种重金属含量均小于蓖麻秸秆,重金属在植物体内器官转移程度呈逐渐减小状态。蓖麻籽实Cr和Pb的含量仅为绿豆的0.19、0.37倍、Cd含量是绿豆的1.50倍、Hg含量与绿豆秸秆中Hg含量相当。蓖麻秸秆中Cr、Pb、Cd、As的含量分别仅为绿豆的0.44、0.17、0.30、0.69倍,蓖麻秸秆中Hg含量和绿豆秸秆中Hg含量相当。由此可见,绿豆对Cr、Pb、Cd和As的地上部富集能力高于蓖麻,二者对Hg的富集能力相当。绿豆和蓖麻对重金属均有较好吸附作用,绿豆吸附效果优于蓖麻。

世界特种油料种质资源保存概况

为加强特种油料种质资源的收集保存,更好地为育种研究利用提供服务,本文阐述了特种油料种质资源在我国及世界上重点国家的保存情况。美国、印度、欧盟、中国等13个国家共保存向日葵、红花、亚麻(含纤用)、蓖麻及苏子等特种油料种质资源约9万份,其中亚麻30000多份,向日葵21800多份,红花15000多份,蓖麻约5000多份,苏子近900份。欧盟、美国、俄罗斯和加拿大是亚麻资源的主要保存国家(地区);向日葵资源主要集中在美国、欧盟和中国;红花种质主要分布在印度、美国、中国和俄罗斯;蓖麻种质以中国、美国和印度居多。比较这些国家所拥有的特种油料种质资源数量,美国位居第一,保存数量最多,超过22000份;印度其次;欧盟、中国和俄罗斯居中。中国保存特油作物种质资源(蓖麻、向日葵、红花、苏子)8400余份,其中21%为国外种质,国内种质主要来源于湖北省、华北、东北、西北和西南地区。本文为我国特种油料种质资源的引进、收集保存提出了建议。

蓖麻蒴果剥壳力学特性的试验

为了实现蒴果壳与蓖麻籽成功剥离,载荷的大小以及载荷来自的方位相当重要.在蓖麻蒴果壳的物理特性测定分析基础上,运用接触力学对蓖麻蒴果脐部与顶部和腰部受到压载荷时,进行力学特性的试验分析与研究.结果显示蒴果在脐部与顶部受到压载荷作用时,果壳破裂的位置在蒴果顶部中缝处,中果皮内面产生裂纹而使蓖麻籽在果壳中脱出;蒴果在腰部受到压载荷作用时,果壳破裂的位置出现在蒴果中果皮中缝的两端,蒴果四周角靠近直线处也有裂纹而使蓖麻籽在果壳中脱出,蓖麻蒴果脐部与顶部受力大于腰部受力.

乙醇浸提法提取蓖麻饼粕总黄酮的工艺研究

本文采用乙醇浸提法提取蓖麻饼粕中的黄酮类化合物,在单因素实验基础上,通过正交实验对蓖麻饼粕总黄酮的提取条件进行优化,得到乙醇浸提法提取蓖麻饼粕总黄酮的最优工艺为:乙醇浓度70%,料液比1∶25(g/m L),提取温度75℃,提取时间3h,此时总黄酮的提取率为2.50%,换算成含量为25.03mg/g。该工艺条件下总黄酮的提取率较高,连续两次浸提提取率可达总提取率的96.01%,且提取方法简单。本研究对蓖麻饼粕总黄酮工业化生产具有一定的应用价值。

棉籽和蓖麻籽的冷榨试验与数值模拟

探讨棉籽和蓖麻籽植物油料的冷榨应力-应变关系、应力-压缩比关系以及加载速率对应力-应变关系和应力-压缩比关系的影响,探讨临界压榨压力选取问题。进行了棉籽和蓖麻籽冷榨试验,采用川北方程建立双曲线型应力-应变模型和压缩比的理论计算模型。研究表明,加载速率对油料破碎应力、应变以及应力-应变关系有影响,速率越大,破碎应力和应变越大;棉籽和蓖麻籽的模拟结果与实测值比较吻合。根据压缩比变化率及其斜率曲线近似确定出棉籽和蓖麻籽工程临界压榨压力分别为70 MPa和100 MPa。

蓖麻杂交种的SSR鉴定及遗传变异分析

采用SSR标记对蓖麻CSR24×CSR181杂交所得的F1种子进行分析,为蓖麻早期杂种鉴定和遗传变异分析奠定技术与理论基础。结果表明:(1)各位点鉴定所得结果基本一致,除RCM207和RC129位点鉴定的杂种率未超过10%外,其它位点的杂种率都十分接近,在13.46%～17.27%之间。(2)少数个体在相关位点发生了变异,在引物RC242的扩增图谱中有4个单株出现了双亲特异条带的缺失,产生了双亲都没有的新条带;在引物Rco23、Rco26、Rco29、RC129、RCM613和RCM999的扩增结果中出现了父本特异条带的缺失,同时产生了一条新条带。(3)多样性及UPGMA聚类分析表明杂交后代遗传变异显著,子代个体与亲本间的遗传相似系数在0.45～1.0之间,个体间差异明显。

蓖麻SnRK2基因家族的鉴定和特征分析

SnRK2是一类植物特有的Ser/Thr类蛋白激酶。为研究其在蓖麻抗逆过程中的作用,基于蓖麻的基因组序列信息,利用生物信息学方法鉴定了6个蓖麻SnRK2蛋白激酶成员。分析发现这些蛋白具有亲水性属性,基因结构分析表明,植物SnRK2基因在进化过程中具有很高的保守性;系统与进化分析发现植物SnRK2蛋白在单、双子叶植物中分化显著,可能是独立演化的结果。高通量RNA-seq测序结果显示,大部分SnRK2基因在检测的各个组织中均有表达,且在不同组织间或种子发育的不同阶段没有明显的组织特异性;外源ABA处理种子后,Group 2的基因表达显著上调。进一步利用半定量RT-PCR技术检测外源(100μmol/L)ABA、(250mmol/L)Na Cl和(4℃)冷胁迫处理的幼苗,发现Group 2中的基因29908.m006067被这3种胁迫强烈激活,基因29772.m000313对ABA和冷胁迫有较强的响应。

蓖麻提取物对鼠抗生育作用的实验研究

利用蓖麻提取物对昆明种小鼠进行了短期与长期的抗生育实验，研究发现蓖麻提取物（蓖麻油和蓖麻蛋白）对小鼠有明显的抗生育作用。蓖麻蚩白及其与蓖麻油的混合物在抗早孕方面的效果均可达到100％，蓖麻油抗着床的效果也可达到100％。蓖麻油长期抗鼠生育效果明显，在210d（正常小鼠的妊娠期是21～23d）内有效降低小鼠生育代数与产仔数，生育抑制率达80％以上。蓖麻提取物对离体小鼠子宫的影响也非常显著，通过增强小鼠子宫内部收缩有效减少着床机率。在中止妊娠的实验中发现，服用了蓖麻蛋白及其与蓖麻油的混合物的小鼠子宫内没有着床位点。

胺鲜酯和镉对蓖麻幼苗光合生理特性的影响

以蓖麻(Ricinus communis L.)为研究对象,通过水培试验研究胺鲜酯(DA-6)和镉(Cd)对蓖麻生长和光合生理特性的交互作用,探讨DA-6浸种对植物Cd毒害是否具有缓解作用。研究结果表明,50μmol.L-1CdCl2对蓖麻幼苗生长和光合作用具有抑制作用。Cd处理显著降低蓖麻叶片的光合速率(Pn)、气孔导度(Gs)和蒸腾速率(E),而胞间CO2浓度(Ci)则明显增大;在Cd胁迫下,蓖麻叶片的叶绿素a、b、a+b、类胡萝卜素含量以及叶绿素/类胡萝卜素比值均显著降低,而叶绿素a/b比值则显著增大;Cd胁迫导致F0增大,Fm、Fv/Fm、Fv/F0和ΦPS II显著降低。无论是否添加Cd,DA-6对蓖麻幼苗的生长均没有显著影响;同时,DA-6处理对蓖麻幼苗的镉积累也没有显著影响。在正常情况下,DA-6处理能提高蓖麻叶片的光合色素含量,但叶绿素a/b比值、叶绿素/类胡萝卜素比值以及叶绿素荧光参数和气体交换参数均没有显著影响。在Cd胁迫下,10和1000mg.L-1 DA-6处理不仅导致光合色素含量(Chl a、Chl b、Chl a+b和Car)和气体交换参数(Pn、E和Gs)降低,同时也导致Fm、Fv/Fm、Fv/F0和ΦPS II降低。可见DA-6不仅不能缓解Cd对蓖麻幼苗的毒害,而且通过降低光合色素含量和PS II的光化学活性,加重了Cd对光合作用的抑制作用。