羽衣甘蓝类受体激酶FERONIA基因克隆、表达及与相互作用蛋白分析

FERONIA(FER)受体激酶在有性生殖中的花粉与柱头信号识别中发挥重要作用,为分析类受体激酶FERONIA(FER)在羽衣甘蓝(Brassica oleracea var.acephala)授粉中的作用,以羽衣甘蓝自交不亲和系(S_(13-b)S_(13-b))为研究材料,采用RT-PCR从柱头中克隆得到BoFER基因,获得的BoFER cDNA序列,全长2682 bp编码893个氨基酸,其含有高度保守激酶结构域和Ser/Thr激酶结合位点。利用qRT-PCR技术对BoFER在授粉过程中的表达水平进行分析,结果发现BoFER在不亲和授粉过程其表达量逐渐升高,而在亲和授粉过程中其表达量逐渐降低。利用酵母双杂交分析BoFER与已知的自交不亲和相关蛋白的相互作用,结果显示BoFER与S位点受体激酶BoSRK13-b的激酶域存在相互作用。

巴西橡胶树FERONIA类受体激酶基因(HbFER)的克隆及表达分析

植物受体类激酶在植物的生长发育和抗逆等生理过程中起着重要作用。在橡胶树转录组测序的基础上,通过RACE技术和RT-PCR方法,扩增得到橡胶树的一个类受体激酶基因的全长,命名为HbFER。该片段包含1个2 679 bp的开放阅读框,可编码892个氨基酸。生物信息学分析结果表明:该氨基酸序列与拟南芥、番茄、甘蓝和杨树的FER蛋白高度同源,其同源性分别为74.5%、74.7%、73.4%、82.9%。HbFER蛋白除了含有Malectin_like和PTKc结构域外,还含有1个信号肽(含22个氨基酸)和2个跨膜结构域。半定量分析结果表明,HbFER基因在不同组织中的表达水平存在差异,叶片和花中的表达丰度最高,胶乳中的表达丰度最低,且该基因在叶片中的表达受植物激素水杨酸的诱导。

不同植物FERONIA基因密码子使用偏好性分析

为了了解植物FERONIA(FER)基因在长期进化过程中所形成的密码子使用模式及它们之间的亲缘关系,采用Codon W、CUSP在线程序和SPSS等分析软件对不同植物的FER基因密码子进行碱基组成分析、对应性分析、PR2-Plot分析、中性绘图分析和ENC-Plot分析并进行聚类分析。结果表明,单子叶植物FER基因密码子的平均G+C含量高于双子叶植物,且单子叶植物偏好使用以G/C结尾的密码子,而双子叶植物偏好使用以A/T结尾的密码子。对应性分析结果显示,在单子叶植物、双子叶植物中,向量轴1(Axis 1)可解释的变异分别占总变异的42.25%、28.56%。在单子叶植物中,Axis 1与总G+C含量、GC3含量、G3含量、C3含量呈极显著正相关(P<0.01),而在双子叶植物中则呈极显著负相关(P<0.01)。不同的碱基组成是单子叶植物和双子叶植物形成密码子使用偏好性的重要原因。PR2-Plot分析结果、中性绘图结果和ENC-Plot分析结果表明,自然选择是形成植物FER基因密码子使用偏好性的主要驱动力。基于编码序列(Coding sequence,CDS)构建的系统进化树比基于同义密码子相对使用度(RSCU)得出的聚类分析结果更接近传统的分类结果。本研究结果可为进一步开展植物分子进化和基因工程研究奠定基础。

Cytotoxicity evaluation and hepatoprotective potential of bioassay guided fractions from Feronia limmonia Linn leaf

Objective:To evaluate the cytotoxicity and hepatoprotective potentials of extracts,fractions or isolated compound from the leaves of Feronia limonia(F.limonia).Methods:Qualitative phytochemical analysis of extracts,fractions or compound was performed by means of thin layer chromatography and spectroscopic assays.The%purity of compound was measured by analytical HPLC.Extracts,fractions or compound have been individually evaluated for their cytotoxicity effects(10,20,100,250,500,750 and 1 000 μg/mL).Based on the inhibitory concentration(IC_(50)) obtained from the cell viability assay,graded concentrations of extracts,fractions or isolated compound were assessed(10,20,50,100,200 μg/mL) for its hepatoprotective potential against CCl_4-induced hepatotoxicity by monitoring activity levels of serum glutamatic pyruvatic transaminase(SGPT) and serum glutamic oxaloacetic transaminase(SGOT).Results:Results indicated that the methanol extract of F.limonia was non-toxic and hepatoprotective in nature as compared with the petroleum ether extract.The acetone fraction of methanolic extract also showed similar properties but the subsequent two fractions were cytotoxic.However,the pure compound isolated from the penultimate fraction of methanolic extract was non-toxic and hepatoprotective in nature.Biochemical investigations(SCOT,SCPT) further corroborated these cytological observations.Conclusions:It can be concluded from this study that F.limonia methanol extract,some fractions and pure isolated compound herein exhibit hepatoprotective activity.However,cytotoxicity recorded in the penultimate fraction and investigation of structural details of pure compound warrants further study.

Protective effect of aqueous extract of Feronia elephantum correa leaves on thioacetamide induced liver necrosis in diabetic rats

Objective:To evalueate hepatoprotective effects Feronia elephantum(F.elephantum)correa against thioacctamide(TA)induced liver necrosis in diabetic rats.Methods:Male wistar rats were made diabetic with alloxan(160 mg/kg)on day 0 of the study.They were intoxicated with hepatotoxicant(thioacetamide,300 mg/kg,ip)on day 9 of study to produce liver necrosis.Effects of 7 day daily once administration(day 2 to day 9)of EF(400 and 800 mg/kg,po)were evaluated on necorosis of liver in terms of mortality,liver volume,liver weight,serum aspartate aminotransferase(AST)and serum alanine transaminase(ALT),and histopathology of liver sections(for signs of necorosis and inflammation)on day-9 of the study.Separate groups of rats with treated only with alloxan(DA control),thioacetamide(TA control)and both(TA+DA control)were maintained.Results:FE significantly lowered the mortality rate and showed improvement in liver function parameters in TA-induced diabetic rats without change in liver weight,volume and serum glucose levels.Conclusions:FE showed promising activity against TA-induced liver necorsis in diabetic rats and so might be useful for prevention of liver complications in DM.

TLC Determination of Marmesin, a Biologically Active Marker from Feronia Limonia L.

Feronia limonia Linn. (Rutaceae) have gained traditional therapeutic importance owing to their high essential oil and coumarins content. Marmesin, a furanocoumarin was identified by TLC and isolated by column chromatography and further purified by Preparative TLC. Presently, there is no appropriate TLC based method available for standardization of F. limonia. A simple, sensitive and accurate high performance thin layer chromatographic (HPTLC) method has been developed for the estimation of marmesin in the methanolic extract of stem bark of Feronia limonia. HPTLC was performed on precoated silica gel 60F254 aluminium plates (20 cm × 20 cm) with Chloroform: Methanol (9.5:0.5), as mobile phase. Quantitative evaluation of the plate was performed in the absorption-reflection mode at 338 nm. The calibration curve was linear in the concentration range of 20 – 100 ng spot–1. The method was validated for precision, repeatability and accuracy. The technique has been applied, for the first time, for the estimation of marmesin. The proposed method was found to be robust, precise, and accurate, it therefore holds potential for detection, monitoring and quantification of marmesin in Feronia limonia and its related formulation.

Reversible antispermatogenic and antisteroidogenic activities of Feronia limonia fruit pulp in adult male rats

Objective:To explore the antispermatogenic and testicular antisleroidogenic activities of Feronia limonia fruit pulp southern India.Methods:Fourty Wistar male albino rats(Ratios norvegicus)were equally divided into four groups.Experimental groups were administered with the ethanolic extract of Feronia lirnonia(F.limoni)fruit pulp at doses of 250 and 500 nig/kg body weight once daily for 55 days.All treated rats had corresponding recovery groups.At the end of each treatment periods,various spermatological indices,tissue biochemicals and testicular enzymes levels were analysed.Blood profiles were also estimated.Results:Compared with the control,the F.lirnonia.fruit pulp at both dose leveb did not decrease body weight,which were associated with decline in epididymal sperm count,motility,viability and increased percent of abnormal sperm.Further,F.limonia.fruit pulp at 500 mg/kg body weight markedly reduced the epididymal and testicular protein content by 24.58%and 29.86%,respectively,as well as the glucose-6-phosphate dehydrogenase and△~5-3β-hydroxy steroid dehydrogenase)levels by 42.82%and 38.08%,respectively,while a significant elevation was observed in testicular cholesterol and ascorbic acid content.A gradual recovery of all parameters was observed after 55 days of treatment withdrawal.No significant alterations in haematological indices were observed.Conclusions:The present findings indicate that F.lirnonia fruit pulp may have reversible antispermatogenic and antisteroidogenic properties,and could partially support the traditional use as mate contraceptive.

In vitro antioxidant and cytotoxic activities of essential oil of Feronia elephantum Correa

Objective:To analyse the chemical composition and evaluation of antioxidant,cytotoxic and DN A fragmentation activities of essential oil of Feronia elephantum Correa.Methods:Chemical composition analysis of hydrodistilled essential oil was determined by gas chromatography-mass spectrometry and in vitro antioxidant activity of oil was determined by DPPH free radical,hydroxyly radical scavenging,metal chelating and prevention of deoxyribose degradation.Cytotoxicity and ONA fragmentation activities against breast cancer cells(MCF-7)were also analyzed.Resnlts:Gas chromatography-mass spectrometry analysis revealed the presence of 24compounds with caryophyllene oxide(62.29%)as major compound.A considerable antioxidant,cyotoxic and DN A fragmentation activities of oils was observed.Conclusiolis:The result of this study clearly indicates oil could be useful for food preservation and preparation.

Evaluation of Antibacterial and Antidiarrhoeal Activities of <i>Feronia limonia</i>Leaf Extract

The present study was carried out to investigate possible antibacterial and antidiarrhoeal activities of ethanol extract of Feronia limonia leaves. Phytochemical analysis of the crude extract was performed to detect presence of different kinds of phytoconstituents. The antibacterial activity was investigated against four Gram positive and four Gram negative bacteria by using disc diffusion method. The plant extract showed moderate antibacterial activity against Gram positive bacteria namely Staphylococcus saprophyticus and Staphylococcus pyogenes and all tested Gram negative bacteria namely Escherichia coli, Shigella boydii, Shigella dysentery and Shigella flexneri in dose dependant manner. The results of castor oil-induced diarrhoeal study showed that Feronia limonia extract significantly reduced the severity & frequency of diarrhoea in mice at a higher dose of 500 mg/kg compared with the standard drug loperamide (25 mg/kg). The present study clearly supports the medicinal value of this plant. The overall results indicate the possibility of presence of some active principles in the plant extract possessing antibacterial and antidiarrhoeal actions.

Antihepatotoxic Effect of Feronia Limonia Fruit against Carbon Tetrachloride Induced Hepatic Damage in Albino Rats

Feronia limonia Swingle, though mentioned as a high utility medicinal plant in Ayurveda for the treat-ment of liver disorders, but has no scientific investigations. Therefore, the present study was undertaken to investigate the hepatoprotective activity of extracts of Feronia limonia fruit on albino rat liver, damage induced by single dose of carbon-tetrachloride (1.5 ml/kg, p.o.). The oral administration of extracts (500 mg/kg, p.o.) for 7 days has shown significant hepatoprotective action. There is reduction in the elevated levels of serum enzymes such SGOT, SGPT, ALP and Serum bilirubin and elevation in level of GSH, GPX,GLY and total protein in Feronia limonia protected rats.

Synergistic Hepatoprotective Effect of Feronia fimonia L,, Citrullus colocynthis L,, and Tribulus terrestris L, against Paracetamol Induced Hepatotoxicity in Swiss Albino Rats

Paracetamol toxicity induced a significant rise in AST （aspartate transaminase）, ALT （alanine transaminase）, ALP （alkhaline phisphatase）, total bilirubin, total cholesterol, Triglycerides. Administration of 150 mg/kg b.w of synergistic ethanolic extracts of Feronia limonia L., Citrullus colocynthis L., and Tribulus terrestris L. effectively reduced these pathological damages caused by paracetamol intoxication. In addition to serum parameters treatment of 150 rng/kg b.w of synergistic ethanolic extract of Feronia limonia L., Citrullus colocynthis L. and Tribulus terrestris L. also promote the body weight in albino rats. Histopathological changes of the liver samples were compared with the normal control. From our results, we may infer that the mode of action of synergistic 90% ethanolic extracts of Feronia limonia L., Citrullus colocynthis L., and Tribulus terrestris L. （150 mg/kg b.w） in affording the in vivo hepatoprotective activity against paracetamol may be due to the cell membrane stabilization, hepatic cell regeneration and activation of antioxidative enzymes such as glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase.

水稻中类受体蛋白激酶FERONIA-like Receptor 1的表达纯化及抗体制备

类受体蛋白激酶参与调控植物的器官发育和抗逆性,是一种重要的蛋白激酶。通过对水稻中类受体蛋白激酶家族成员FERONIA-like receptor 1 (FLR1)进行蛋白质结构预测分析,发现FLR1蛋白具有跨膜结构,其N端在细胞膜外(FLR1-BW, 1~450 aa), C端在细胞膜内(FLR1-BN, 474~892 aa)。为了得到FLR1抗体用于其功能分析,将FLR1的N端和C端分别构建到原核表达载体pET-32a、pGEX-4T-1、pET-22b、pTriEx-4上,比较FLR1-BW和FLR1-BN在不同载体和不同条件下的表达效果。结果显示:FLR1-BW在pET-32a及pGEX-4T-1中能够有效表达,最优条件为16℃/16 h、0.5 mmol/L IPTG;而FLR1-BN蛋白表达后会对大肠杆菌产生细胞毒性,但在293细胞中能够表达。进一步利用纯化的FLR1-BW蛋白制备FLR1多克隆抗体,结果显示该抗体特异识别FLR1蛋白。本研究为类受体蛋白激酶的抗体制备提供了良好的思路并为研究FLR1功能奠定了基础。

FERONIA-mediated TIR1/AFB2 oxidation stimulates auxin signaling in Arabidopsis

The phytohormone auxin plays a pivotal role in governing plant growth and development.Although the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX(TIR1/AFB)receptors function in both the nucleus and cytoplasm,the mechanism governing the distribution of TIR1/AFBs between these cellular compartments remains unknown.In this study,we demonstrate that auxin-mediated oxidation of TIR1/AFB2 is essential for their targeting to the nucleus.We showed that small active molecules,reactive oxygen species(ROS)and nitric oxide(NO),are indispensable for the nucleo-cytoplasmic distribution of TIR1/AFB2 in trichoblasts and root hairs.Further studies revealed that this process is regulated by the FERONIA receptor kinase–NADPH oxidase signaling pathway.Interestingly,ROS and NO initiate oxidative modifications in TIR1C140/516 and AFB2C135/511,facilitating their subsequent nuclear import.The oxidized forms of TIR1C140/516 and AFB2C135/511 play a crucial role in enhancing the function of TIR1 and AFB2 in transcriptional auxin responses.Collectively,our study reveals a novel mechanism by which auxin stimulates the transport of TIR1/AFB2 from the cytoplasm to the nucleus,orchestrated by the FERONIA–ROS signaling pathway.

Structural and biochemical basis of Arabidopsis FERONIA receptor kinase-mediated early signaling initiation

Accumulating evidence indicates that early and essential events for receptor-like kinase(RLK)function involve both autophosphorylation and substrate phosphorylation.However,the structural and biochemical basis for these events is largely unclear.Here,we used RLK FERONIA(FER)as a model and crystallized its core kinase domain(FER-KD)and two FER-KD mutants(K565R,S525A)in complexes with ATP/ADP and Mg^(2+) in the unphosphorylated state.Unphosphorylated FER-KD was found to adopt an unexpected active conformation in its crystal structure.Moreover,unphosphorylated FER-KD mutants with reduced(S525A)or no catalytic activity(K565R)also adopt similar active conformations.Biochemical studies revealed that FER-KD is a dual-specificity kinase,and its autophosphorylation is accomplished via an intermolecular mechanism.Further investigations confirmed that initiating substrate phosphorylation requires autophosphorylation of the activation segment on T696,S701,and Y704.This study reveals the structural and biochemical basis for the activation and regulatory mechanism of FER,providing a paradigm for the early steps in RLK signaling initiation.

FERONIA Is a Key Modulator of Brassinosteroid and Ethylene Responsiveness in Arabidopsis Hypocotyls

Ethylene signaling is a complex pathway that has been intensively analyzed partly due to its importance to the manifestation of horticultural phenomena, including fruit ripening and tissue senescence. In order to further our un- derstanding of how this pathway is regulated, a screen for Arabidopsis mutants with increased ethylene response was conducted. From this, a mutant was identified as having a dark-grown hypocotyl that is indistinguishable from Col-0 wt in the presence of the ethylene perception inhibitor AgNO3, yet has extreme responsiveness to even low levels of ethylene. Map-based cloning of the mutation revealed a T-DNA insertion in the coding sequence of the receptor-like kinase FERONIA, which is required for normal pollen tube reception and cell elongation in a currently unknown capacity. In contrast to a previous report, analysis of our feronia knockout mutant shows it also has altered responsiveness to brassinosteroids, with etiolated fer-2 seedlings being partially brassinosteroid insensitive with regard to promotion of hypocotyl elonga- tion. Our results indicate that FERONIA-dependent brassinosteroid response serves to antagonize the effect of ethylene on hypocotyl growth of etiolated seedlings, with loss of proper brassinosteroid signaling disrupting this balance and leading to a greater impact of ethylene on hypocotyl shortening.

Nematode-Encoded RALF Peptide Mimics Facilitate Parasitism of Plants through the FERONIA Receptor Kinase

The molecular mechanism by which plants defend against plant root-knot nematodes(RKNs)is largely unknown.The plant receptor kinase FERONIA and its peptide ligands,rapid alkalinization factors(RALFs),regulate plant immune responses and cell expansion,which are two important factors for successful RKN parasitism.In this study,we found that mutation of FERONIA in Arabidopsis thaliana resulted in plants showing low susceptibility to the RKN Meloidogyne incognita.To identify the underlying mechanisms associated with this phenomenon,we identified 18 novel RALF-likes from multiple species of RKNs and showed that two RALF-likes(i.e.,MiRALF1 and MiRALF3)from M.incognita were expressed in the esophageal gland with high expression during the parasitic stages of nematode development.These nematode RALF-likes also possess the typical activities of plant RALFs and can directly bind to the extracellular domain of FERONIA to modulate specific steps of nematode parasitism-related immune responses and cell expansion.Genetically,both MiRALF1/3 and FERONIA are required for RKN parasitism in Arabidopsis and rice.Collectively,our study suggests that nematode-encoded RALFs facilitate parasitism via plant-encoded FERONIA and provides a novel paradigm for studying host-pathogen interactions.

花生响应干旱胁迫的FERONIA类受体蛋白激酶基因克隆与表达分析

FERONIA(FER)类受体蛋白激酶是CrRLK1L(Catharanthus roseus RLK1-like)激酶亚家族成员,在植物受精、细胞伸长、顶端生长以及非生物胁迫响应等方面起重要作用。本研究克隆了两个花生(Arachis hypogaea)FER类受体蛋白激酶基因AhFER1和AhFER2,它们的完整编码序列(CDS)和相应基因组DNA序列完全一致,说明AhFER1和AhFER2基因编码区均无内含子,分别包括2 655和2 640 bp的完整开放阅读框,编码的蛋白分别含885和880个氨基酸,分子量分别为99.58和98.96 kDa,等电点分别为6.5和6.22。生物信息学分析发现,AhFER1及AhFER2的氨基酸序列与其他植物FER蛋白均具有较高同源性,都包含malectin-like和蛋白激酶催化域两个保守结构域。AhFER1和AhFER2基因在花生幼苗的叶、茎和根中的表达水平有差异:AhFER1基因在叶中表达量最高,其次是根,茎中表达量最低;而AhFER2基因则在茎中表达量最高,其次是叶,表达量最低是根。干旱胁迫对花生茎和根中AhFER1基因的表达以及花生叶、茎和根中AhFER2基因的表达均无影响,但干旱胁迫显著增强花生叶中AhFER1基因的表达,说明AhFER1基因可能在花生叶响应干旱胁迫时起重要作用。

FERONIA is involved in phototropin 1-mediated blue light phototropic growth in Arabidopsis

Plant shoot phototropism is triggered by the formation of a light-driven auxin gradient leading to bending growth.The blue light receptor phototropin 1(phot1)senses light direction,but how this leads to auxin gradient formation and growth regulation remains poorly understood.Previous studies have suggested phot1’s role for regulated apoplastic acidification,but its relation to phototropin and hypocotyl phototropism is unclear.Herein,we show that blue light can cause phot1 to interact with and phosphorylate FERONIA(FER),a known cell growth regulator,and trigger downstream phototropic bending growth in Arabidopsis hypocotyls.fer mutants showed defects in phototropic growth,similar to phot1/2 mutant.FER also interacts with and phosphorylates phytochrome kinase substrates,the phot1 downstream substrates.The phot1-FER pathway acts upstream of apoplastic acidification and the auxin gradient formation in hypocotyl under lateral blue light,both of which are critical for phototropic bending growth in hypocotyls.Our study highlights a pivotal role of FER in the phot1-mediated phototropic cell growth regulation in plants.

How a single receptor‑like kinase exerts diverse roles:lessons from FERONIA

FERONIA(FER)is a member of the Catharanthus roseus receptor-like kinase 1-like(CrRLK1L)protein subfamily,which participates in reproduction,abiotic stress,biotic stress,cell growth,hormone response,and other molecular mecha-nisms of plants.However,the mechanism by which a single RLK is capable of mediating multiple signals and activat-ing multiple cellular responses remains unclear.Here,we summarize research progress revealing the spatial–temporal expression of FER,along with its co-receptors and ligands determined the function of FER signaling pathway in multiple organs.The specificity of the FER signaling pathway is proposed to operate under a four-layered mechanism:(1)Spatial–temporal expression of FER,co-receptors,and ligands specify diverse functions,(2)Specific ligands or ligand combinations trigger variable FER signaling pathways,(3)Diverse co-receptors confer diverse FER perception and response modes,and(4)Unique downstream components that modify FER signaling and responses.Moreover,the regulation mechanism of the signaling pathway-appears to depend on the interaction among the ligands,RLK receptors,co-receptors,and downstream components,which may be a general mechanism of RLKs to maintain signal specificity.This review will provide a insight into understanding the specificity determination of RLKs signaling in both model and horticultural crops.

The journey to glory: receptor-like kinases in pollen tube growth

Double fertilization in angiosperm is a prerequisite for seed and fruit development and has been under close scrutiny in the past decades. Communications between male and female gametophytes are especially important in achieving successful reproduction, in which receptor-like kinases are dominant players. Here, we review progress on the functionality of receptor-like kinases(RLKs) in pollen tube growth and point out challenges facing the study of RLKs in pollen biology.