Co-delivery of PROTAC and siRNA via novel liposomes for the treatment of malignant tumors.

Targeted elimination of damaged or overexpressed proteins within the tumor serves a pivotal role in regulating cellular function and restraining tumor cell growth. Researchers have been striving to identify safer and more effective methods for protein removal. Here, we propose the synergistic employment of a small molecule degrading agent (PROTAC) and siRNA to attain enhanced protein clearance efficiency and tumor therapeutic effects. Co-delivery liposomes were prepared to facilitate the efficient encapsulation of PROTAC and siRNA. Specifically, the cationic liposome significantly improved the solubility of the insoluble PROTAC (DT2216). The cationic polymer (F-PEI) achieved efficient encapsulation of the nucleic acid drug, thereby promoting endocytosis and enhancing the therapeutic impact of the drug. Both in vivo and in vitro experiments demonstrated remarkable degradation of target proteins and inhibition of tumor cells by the co-delivery system. In conclusion, the co-delivery liposomes furnished a nano-delivery system proficient in effectively encapsulating both hydrophilic and hydrophobic drugs, thereby presenting a novel strategy for targeted combination therapy in treating tumors.

[Mechanism of Huachansu Injection against colorectal cancer based on network pharmacology and cellular experimental].

This study aimed to elucidate the mechanism of Huachansu Injection(HCSI) against colorectal cancer(CRC) using network pharmacology, molecular docking technology, and cellular experimental. This research group initially used LC-MS/MS to detect the content of 16 bufadienolides in HCSI. Ten bufadienolide components were selected based on a content threshold of greater than 10 ng·mL~(-1). Their potential targets were further predicted using the SwissTargetPrediction database. CRC-related targets were obtained through GeneCards, OMIM, TTD, and PharmGKB databases. The intersection targets of HCSI in the treatment of CRC were obtained through Venny. The "active component-target-disease" network and target protein-protein interaction(PPI) network were constructed via Cytoscape software. Core targets were screened based on the degree values. Gene Ontology(GO) function and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were performed on these key targets. Molecular docking was conducted using AutoDock software on major bufadienolide active components and key targets. Different concentrations of HCSI, psi-bufarenogin(BUF), and bufotalin(BFT) were tested for their effects on cell viability, migration, and apoptosis rates in CRC HCT116 cells. Western blot was conducted to detect the expression of proteins related to the PI3K/Akt/mTOR signaling pathway in HCT116 cells. Eight main active components of HCSI, including arenobufagin, BUF, and BFT, as well as 20 key targets of HCSI in combating CRC, such as EGFR, IL6, and mTOR, were identified. Based on KEGG pathway enrichment and molecular docking results, the PI3K/Akt/mTOR signaling pathway was selected for further verification. Cellular experimental demonstrated that HCSI, BUF, and BFT significantly inhibited the proliferation and migration abilities of HCT116 cells, induced apoptosis in these cells, and downregulated the expression of PI3K/Akt/mTOR pathway-related proteins. This result suggests that HCSI, BUF, and BFT may exert their anti-CRC effects by regulating the PI3K/Akt/mTOR signaling pathway through targets such as mTOR and PIK3CA. This study provides theoretical evidence for exploring the active ingredients and mechanism of HCSI against CRC.

Mechanism of Biqi capsules in the treatment of gout based on network pharmacology and experimental verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Gout is a crystal-related arthropathy caused by monosodium urate (MSU) deposition, resulting from purine metabolism disorders and hyperuricemia (HUA). Gout belongs to the traditional medicine category of Bi syndrome. Biqi capsules (BQ) is a traditional Chinese medicine formula used to treat Bi syndrome. The BQ prescription is derived from the ancient prescription of Hua Tuo, a famous physician in the Han Dynasty. AIM OF THE STUDY: To study the effect and mechanism of BQ in treating acute gouty arthritis (AGA) and HUA. MATERIALS AND METHODS: Analyzing BQ's signaling pathways for gout treatment via network pharmacology. The HUA model was induced orally with adenine and potassium oxonate. The rat AGA model was established by MSU injection. In vitro, MH7A and RAW 246.7 cells were treated with LPS and MSU. Serum uric acid, creatinine, and urea nitrogen levels were evaluated. Kidney and ankle joint pathology was observed via HE staining. Inflammatory signaling pathway proteins, epithelial-mesenchymal transition (EMT) pathway proteins, and uric acid metabolism-related proteins were detected by Western blot. RESULTS: 1780 potential targets for gout treatment were identified, and 1039 target proteins corresponding to BQ's active ingredients were obtained. Pathway enrichment analysis revealed BQ improved gout mainly through inflammatory pathways. Experimental results showed BQ could reduce serum uric acid level and increase uric acid clearance rate by regulating the expression of adenosine deaminase (ADA), and organic anion transporter 1 (OAT1) and glucose transporter 9 (GLUT9) in HUA mice. BQ could improve renal function and injury by inhibiting the NLRP3 pathway in HUA mice' kidneys. Additionally, BQ could alleviate ankle joint swelling and synovial injury, inhibit the TLR4/NLRP3 pathway, and reduce levels of inflammatory factors including interleukin 6 (IL-6), interleukin 1ß (IL-1ß), and tumor necrosis factor-alpha (TNF-α) in AGA rats. The main component of BQ, brucine, could inhibit the activation of NLRP3/NF-κB pathway induced by MSU and reduce the expression level of inflammatory factors (IL-6, IL-1ß, and TNF-α) in macrophages. Brucine could inhibit the activation of the EMT pathway and reduce the expression level of inflammatory factors (IL-6, TNF-α) in human fibroblast-like synoviocytes (MH7A cells) induced by MSU. CONCLUSIONS: BQ effectively reduced serum uric acid levels, improved kidney and joint damage, and ameliorated the inflammatory response caused by MSU. Its main component, brucine, effectively improved the inflammatory response and reduced the invasive ability of synoviocytes induced by MSU.

A hydrogen generator composed of poly (lactic-co-glycolic acid) nanofibre membrane loaded iron nanoparticles for infectious diabetic wound repair.

Diabetic wound, which is chronic skin disease, poses a significant challenge in clinical practice because of persistent inflammation and impaired angiogenesis. Recently, hydrogen has emerged as a novel therapeutic agent due to its superior antioxidant and anti-inflammatory properties. In this study, we engineered a poly (lactic-co-glycolic acid) (PLGA) electrospun nanofibre membrane loaded with citric acid (CA) and iron (Fe) nanoparticles, referred to as Fe@PLGA + CA. Our in vitro assays demonstrated that the Fe@PLGA + CA membrane continuously generated and released hydrogen molecules via a chemical reaction between Fe and CA in an acidic microenvironment created by CA. We also discovered that hydrogen can ameliorate fibroblast migration disorders by reducing the levels of matrix metalloproteinase 9 (MMP9). Furthermore, we confirmed that hydrogen can scavenge or biochemically neutralise accumulated reactive oxygen species (ROS), inhibit pro-inflammatory responses, and induce anti-inflammatory reactions. This, in turn, promotes vessel formation, wound-healing and accelerates skin regeneration. These findings open new possibilities for using elemental iron in skin dressings and bring us one step closer to implementing hydrogen-releasing biomedical materials in clinical practice.

Bioactive Nanofiber-Hydrogel Composite Regulates Regenerative Microenvironment for Skeletal Muscle Regeneration after Volumetric Muscle Loss.

Volumetric muscle loss (VML) is a severe form of muscle trauma that exceeds the regenerative capacity of skeletal muscle tissue, leading to substantial functional impairment. The abnormal immune response and excessive reactive oxygen species (ROS) accumulation hinder muscle regeneration following VML. Here, an interfacial cross-linked hydrogel-poly(ε-caprolactone) nanofiber composite, that incorporates both biophysical and biochemical cues to modulate the immune and ROS microenvironment for enhanced VML repair, is engineered. The interfacial cross-linking is achieved through a Michael addition between catechol and thiol groups. The resultant composite exhibits enhanced mechanical strength without sacrificing porosity. Moreover, it mitigates oxidative stress and promotes macrophage polarization toward a pro-regenerative phenotype, both in vitro and in a mouse VML model. 4 weeks post-implantation, mice implanted with the composite show improved grip strength and walking performance, along with increased muscle fiber diameter, enhanced angiogenesis, and more nerve innervation compared to control mice. Collectively, these results suggest that the interfacial cross-linked nanofiber-hydrogel composite could serve as a cell-free and drug-free strategy for augmenting muscle regeneration by modulating the oxidative stress and immune microenvironment at the VML site.

CHIP suppresses the proliferation and migration of A549 cells by mediating the ubiquitination of eIF2α and upregulation of tumor suppressor RBM5.

The protein kinase RNA-like endoplasmic reticulum kinase (PERK)-eukaryotic translation initiation factor 2 subunit α (eIF2α) pathway plays an essential role in endoplasmic reticulum (ER) stress. When the PERK-eIF2α pathway is activated, PERK phosphorylates eIF2α (p-eIF2α) at Ser51 and quenches global protein synthesis. In this study, we verified eIF2α as a bona fide substrate of the E3 ubiquitin ligase carboxyl terminus of the HSC70-interaction protein (CHIP) both in vitro and in cells. CHIP mediated the ubiquitination and degradation of nonphosphorylated eIF2α in a chaperone-independent manner and promoted the upregulation of the cyclic AMP-dependent transcription factor under endoplasmic reticulum stress conditions. Cyclic AMP-dependent transcription factor induced the transcriptional enhancement of the tumor suppressor genes PTEN and RBM5. Although transcription was enhanced, the PTEN protein was subsequently degraded by CHIP, but the expression of the RBM5 protein was upregulated, thereby suppressing the proliferation and migration of A549 cells. Overall, our study established a new mechanism that deepened the understanding of the PERK-eIF2α pathway through the ubiquitination and degradation of eIF2α. The crosstalk between the phosphorylation and ubiquitination of eIF2α shed light on a new perspective for tumor progression.

Genome evolution during bread wheat formation unveiled by the distribution dynamics of SSR sequences on chromosomes using FISH.

BACKGROUND: During the bread wheat speciation by polyploidization, a series of genome rearrangement and sequence recombination occurred. Simple sequence repeat (SSR) sequences, predominately located in heterochromatic regions of chromosomes, are the effective marker for tracing the genomic DNA sequence variations. However, to date the distribution dynamics of SSRs on chromosomes of bread wheat and its donors, including diploid and tetraploid Triticum urartu, Aegilops speltoides, Aegilops tauschii, Triticum turgidum ssp. dicocoides, reflecting the genome evolution events during bread wheat formation had not been comprehensively investigated. RESULTS: The genome evolution was studied by comprehensively comparing the distribution patterns of (AAC)n, (AAG)n, (AGC)n and (AG)n in bread wheat Triticum aestivum var. Chinese Spring and its progenitors T. urartu, A. speltoides, Ae. tauschii, wild tetroploid emmer wheat T. dicocoides, and cultivated emmer wheat T. dicoccum. Results indicated that there are specific distribution patterns in different chromosomes from different species for each SSRs. They provided efficient visible markers for identification of some individual chromosomes and SSR sequence evolution tracing from the diploid progenitors to hexaploid wheat. During wheat speciation, the SSR sequence expansion occurred predominately in the centromeric and pericentromeric regions of B genome chromosomes accompanied by little expansion and elimination on other chromosomes. This result indicated that the B genome might be more sensitive to the "genome shock" and more changeable during wheat polyplodization. CONCLUSIONS: During the bread wheat evolution, SSRs including (AAC)n, (AAG)n, (AGC)n and (AG)n in B genome displayed the greatest changes (sequence expansion) especially in centromeric and pericentromeric regions during the polyploidization from Ae. speltoides S genome, the most likely donor of B genome. This work would enable a better understanding of the wheat genome formation and evolution and reinforce the viewpoint that B genome was originated from S genome.

Xiaoaiping injection enhances paclitaxel efficacy in ovarian cancer via pregnane X receptor and its downstream molecules.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoaiping injection, a traditional Chinese medical injection extracted from root of Marsdenia tenacissima (Roxb.) Moon, has been exclusively used on curing malignant tumor in China and as adjuvant therapeutic agent for chemotherapeutics, including paclitaxel. AIM OF THE STUDY: The goal of this study was to investigate the synergistic inhibitory efficacy of Xiaoaiping injection and paclitaxel on ovarian cancer. The mechanism may be associated with nuclear receptor pregnane X receptor (PXR) regulating its downstream molecules. MATERIALS AND METHODS: In vitro, MTT assay, flow cytometry and Hoechst dyeing were used to evaluate the SK-OV-3 cell proliferation, apoptosis and cell cycle respectively. The mRNA and protein expression of PXR and its downstream CYP450 enzymes, transporters and Bcl-2 families were measured by qRT-PCR and Western blot. Rhodamine 123 efflux experiment was conducted to detect the P-gp efflux ability. PXR plasmid and PXR siRNA were transiently transfected into SK-OV-3 cells respectively to establish PXR-overexpressed or PXR-interfered cells. In vivo, xenograft tumor mice model was established by SK-OV-3 cells to estimate the antitumor effect of Xiaoaiping injection combined with paclitaxel. The expressions of PXR and its downstream molecules in tumor tissues were determined to further clarify the potential mechanism. RESULTS: Xiaoaiping injection significantly enhanced the anti-proliferation, pro-apoptosis effect of paclitaxel on SK-OV-3 cells. The synergetic effect was displayed by Xiaoaiping injection inhibiting paclitaxel-induced PXR and CAR expression, which subsequently inhibited CYP450 enzymes CYP2C8 and CYP3A4, transporter P-gp and anti-apoptotic proteins Bcl-2 and Bcl-xl in SK-OV-3 cells. In PXR-overexpressed cells, Xiaoaiping injection down-regulated the expression of PXR and its downstream molecules. The result of xenograft tumor model showed that Xiaoaiping injection combined with paclitaxel enhanced anti-tumor effect on ovarian cancer in vivo. CONCLUSIONS: Xiaoaiping injection enhances anti-tumor effect of paclitaxel by inhibiting cell proliferation, inducing apoptosis process. The mechanism may be associated with Xiaoaiping injection inhibiting PXR and its downstream metabolic enzymes CYP2C8, CYP3A4, transporter P-gp and anti-apoptosis protein Bcl-2.

Xiao-Ai-Ping Injection Enhances Effect of Paclitaxel to Suppress Breast Cancer Proliferation and Metastasis via Activating Transcription Factor 3.

Chemotherapy is an effective treatment for invasive breast cancer. Paradoxically, many recently published findings showed that the first-line chemotherapeutic agent paclitaxel (PTX) showed pro-metastatic effects in the progress of treating breast cancer. Xiao-Ai-Ping (XAP) injection, composed of a traditional herbal medicine, Marsdenia tenacissimae extract, is known to exert antitumor effects on various cancers. However, there are few experimental studies on breast cancer. The underlying mechanism of the antitumor effect of XAP combined with chemotherapy agents has not been fully understood. In the present study, we sought to find the antitumor effects of XAP combined with PTX in vitro and in vivo. The data demonstrated that the combination of XAP with PTX resulted in remarkable enhancement of the pro-apoptotic, migration-inhibiting, and anti-invasive effects of PTX in vitro. Significantly, further study showed the overexpression of ATF3 in PTX-treated cell, while XAP counteracted the change of ATF3 induced by PTX. Moreover, it showed that combination treatment could promote the inhibition of tumor growth in MDA-MB-231 cell xenograft mouse model. Compared with PTX treatment, the downregulation of ATF3 indicated that ATF3 played a pivotal role in the combination of XAP with PTX to exert a synergistic effect. Overall, it is expected that PTX combined with XAP may serve as an effective agent for antitumor treatment, and dampening ATF3 maybe a potential strategy to improve the efficacy of PTX.

Hydroxycamptothecin Inhibits Peritendinous Adhesion via the Endoplasmic Reticulum Stress-Dependent Apoptosis.

Traumatic peritendinous fibrosis is a worldwide clinical problem resulting in severe limb disability. Hydroxycamptothecin (HCPT) is an anti-neoplastic drug widely exploited in clinical practice. It has shown potential of anti-fibrosis in recent years. We previously demonstrated that HCPT inhibited the characterization of fibrosis in vitro. However, it is still unclear whether it ameliorates peritendinous adhesion in an in vivo animal tendon injury model. The underlying mechanism is also worth investigating. The present study aims to determine whether HCPT inhibits tendon adhesion and to explore the underlying mechanisms. In a rat tendon injury model, we observed that topical application of HCPT significantly attenuated peritendinous adhesion as revealed by the results of macroscopic observation, biomechanical, histological, immunohistochemical evaluation, western blot, and quantitative PCR (q-PCR) analyses. Furthermore, western blot and q-PCR analyses revealed that this phenomenon is correlated with HCPT activation of endoplasmic reticulum (ER) stress. In addition, in vitro studies show that HCPT significantly inhibits fibroblast proliferation and induces apoptosis by reducing the expression of extracellular matrix (ECM) proteins COL3A1 and α-smooth muscle actin (α-SMA). Finally, we employed small interfering RNA (siRNA) to target inositol requiring kinase 1 (IRE1) and activated transcription factor 6 (ATF-6) to verify that the effect of inhibitory fibrosis of HCPT disappears after knockdown of ATF-6 and IRE1, thereby suggesting that an anti-fibrotic effect of HCPT is mediated by the ER-dependent apoptotic pathway. In conclusion, our results indicate that HCPT inhibits peritendinous fibrosis through the ER-dependent apoptotic pathway and might serve as a potential solution to prevent traumatic peritendinous adhesion.

Effects of diosmetin on nine cytochrome P450 isoforms, UGTs and three drug transporters in vitro.

Diosmetin (3', 5, 7-trihydroxy-4'-methoxyflavone), a natural flavonoid from traditional Chinese herbs, has been used in various medicinal products because of its anticancer, antimicrobial, antioxidant, estrogenic and anti-inflammatory activity. However, flavonoids could affect the metabolic enzymes and cause drug-drug interactions (DDI), reducing the efficacy of co-administered drugs and potentially resulting in serious adverse reactions. To evaluate its potential to interact with co-administered drugs, the IC50 value of phase I cytochrome P450 enzymes (CYPs), phase II UDP-glucuronyltransferases (UGTs) and hepatic uptake transporters (organic cation transporters (OCTs), organic anion transporter polypeptides (OATPs) and Na+-taurocholate cotransporting polypeptides (NTCPs)) were examined in vitro by LC-MS/MS. Diosmetin showed strong inhibition of CYP1A2 in a concentration-dependent manner. The intensity of the inhibitory effect was followed by CYP2C8, CYP2C9, CYP2C19 and CYP2E1. For CYP2A6, CYP2B6, CYP2D6 and CYP3A4, diosmetin was found to have no significant inhibitory effects, and the induction effect on CYPs was not significant. For UGTs, diosmetin had a minimal inhibitory effect. In addition, the inhibitory effects of diosmetin on OATP and OCT1 were weak, and it had little effect on NTCP. This finding indicated that drug-drug interactions induced by diosmetin may occur through co-administration of drugs metabolized by CYP1A2.

Identification and characterization of high-molecular-weight glutenin subunits from Agropyron intermedium.

High-molecular-weight glutenin subunit (HMW-GS) is a primary determinant of processing quality of wheat. Considerable progress has been made in understanding the structure, function and genetic regulation of HMW-GS in wheat and some of its related species, but less is known about their orthologs in Agropyron intermedium, a useful related species for wheat improvement. Here seven HMW-GSs in Ag. intermedium were identified using SDS-PAGE and Western blotting experiments. Subsequently, the seven genes (Glu-1Aix1 ∼ 4 and Glu-1Aiy1 ∼ 3) encoding the seven HMW-GSs were isolated using PCR technique with degenerate primers, and confirmed by bacterial expression and Western blotting. Sequence analysis indicated that the seven Ag. intermedium HMW-GSs shared high similarity in primary structure to those of wheat, but four of the seven subunits were unusually small compared to the representatives of HMW-GS from wheat and two of them possessed extra cysteine residues. The alignment and clustering analysis of deduced amino acid sequences revealed that 1Aix1 and 1Aiy1 subunits had special molecular structure, belonging to the hybrid type compounding between typical x- and y-type subunit. The xy-type subunit 1Aix1 is composed of the N-terminal of x-type and C-terminal of y-type, whereas yx-type subunit 1Aiy1 comprises the N-terminal of y-type and C-terminal of x-type. This result strongly supported the hypothesis of unequal crossover mechanism that might generate the novel coding sequence for the hybrid type of HMW-GSs. In addition to the aforementioned, the other novel characteristics of the seven subunits were also discussed. Finally, phylogenetic analysis based on HMW-GS genes was carried out and provided new insights into the evolutionary biology of Ag. intermedium.

Microdissection and chromosome painting of the alien chromosome in an addition line of wheat--Thinopyrum intermedium.

In this study, chromosome painting was developed and used to identify alien chromosomes in TAi-27, a wheat--Thinopyrum intermedium addition line, and the chromosomes of the three different genomes of Th. Intermedium. The smallest alien chromosome of TAi-27 was microdissected and its DNA amplified by DOP-PCR was used as a probe to hybridize with metaphase chromosomes of TAi-27 and Th. intermedium. Results showed that hybridization signals were observed in all regions of a pair of the smallest alien chromosomes and the pericentromeric area of another pair of alien chromosomes in TAi-27, indicating that the probe from microdissected chromosome is species specific. In Th. intermedium, 14 chromosomes had wide and strong hybridization signals distributed mainly on the pericentromere area and 9 chromosomes with narrow and weak signals on the pericentromere area. The remaining chromosomes displayed a very weak or no signal. Sequential FISH/GISH on Th. intermedium chromosomes using the DNAs of microdissected chromosome, Pseudoroegneria spicata (St genome) and pDbH12 (a J(s) genome specific probe) as the probes indicated that the microdissected chromosome belonged to the St genome, three genomes (J(s) , J and St) in Th. intermedium could be distinguished, in which there is no hybridization signal on J genome that is similar to the genome of Th. bessarabicum. Our results showed that the smallest alien chromosomes may represent a truncated chromosome and the repetitive sequence distribution might be similar in different chromosomes within the St genome. However, the repetitive sequence distributions are different within the J(s) genome, within a single chromosome, and among different genomes in Th. intermedium. Our results suggested that chromosome painting could be feasible in some plants and useful in detecting chromosome variation and repetitive sequence distribution in different genomes of polyploidy plants, which is helpful for understanding the evolution of different genomes in polyploid plants.

Draft genome of the wheat A-genome progenitor Triticum urartu.

Bread wheat (Triticum aestivum, AABBDD) is one of the most widely cultivated and consumed food crops in the world. However, the complex polyploid nature of its genome makes genetic and functional analyses extremely challenging. The A genome, as a basic genome of bread wheat and other polyploid wheats, for example, T. turgidum (AABB), T. timopheevii (AAGG) and T. zhukovskyi (AAGGA(m)A(m)), is central to wheat evolution, domestication and genetic improvement. The progenitor species of the A genome is the diploid wild einkorn wheat T. urartu, which resembles cultivated wheat more extensively than do Aegilops speltoides (the ancestor of the B genome) and Ae. tauschii (the donor of the D genome), especially in the morphology and development of spike and seed. Here we present the generation, assembly and analysis of a whole-genome shotgun draft sequence of the T. urartu genome. We identified protein-coding gene models, performed genome structure analyses and assessed its utility for analysing agronomically important genes and for developing molecular markers. Our T. urartu genome assembly provides a diploid reference for analysis of polyploid wheat genomes and is a valuable resource for the genetic improvement of wheat.

Molecular analysis of three new receptor-like kinase genes from hexaploid wheat and evidence for their participation in the wheat hypersensitive response to stripe rust fungus infection.

Considerable progress has been made in understanding the function of receptor-like kinase (RLK) genes in model plants. However, much less is known about these genes in crop species. Here we report the characterization of three new wheat RLK genes (TaRLK-R1, 2 and 3). The primary structure of the putative proteins TaRLK-R1, 2 and 3 contained a signal peptide, a cysteine-rich extracellular domain, a transmembrane domain, and a predicted intracellular kinase domain. The fusions between TaRLK-R1, 2 or 3 and the green fluorescence protein (GFP) were targeted to the plasma membrane; such targeting required the signal peptide, extracellular domain and transmembrane domain. Transcription of TaRLK-R1, 2 and 3 was found mainly in the green organs, and was regulated by light. Transcript levels of TaRLK-R1, 2 and 3 increased during the hypersensitive reaction (HR) to stripe rust fungus. In addition, the TaRLK-R3 transcript level was also upregulated by abiotic stresses. Further experiments revealed that the recombinant kinase domain of TaRLK-R3 exhibited auto-phosphorylation activity in vitro. Knocking down the transcript levels of TaRLK-R1, 2 or 3 individually or all together by virus-induced gene silencing compromised the wheat HR to stripe rust fungus. The demonstration of TaRLK-R1, 2 and 3 as positive contributors in the wheat HR to stripe rust fungus suggests a new direction for further functional studies of this important family of RLK genes, and may facilitate the breeding of wheat varieties resistant to stripe rust disease.

[Genome constitution of Agropyron elongatum 4x by biochemical and SSR markers].

We analyzed the electrophoritic patterns of 16 kinds of isozyme and three kinds of storage protein in Agropyron elongatum 2x and Ag. elongatum 4x by isoelectric focusing (IEF) and SDS-PAGE, respectively. The results showed three types of electrophoritic patterns. In the first type, the zymogram phenotypes are identical between Ag. elongatum 2x and Ag. elongatum 4x. Two kinds of isozyme belong to this type, which accounts for 10.5% of all the biochemical markers analyzed. In the second type, Ag. elongatum 4x showed a phenotype which is made up of combinations of the all of Ag. elongatum 2x bands and the specific bands. Ten kinds of isozyme and three kinds of storage protein fall into this class, which is the most part and accounts for 63.2% of all the markers analyzed. In the third type, Ag. elongatum 2x and Ag. elongatum 4x showed some identical bands and their own specific bands. Five kinds of isozyme were classified to this group,which accounts for 26.3% of all the markers analyzed. The results above suggested that Ag. elongatum 4x is a allotetraploid composed of one genome originated from Ag. elongatum 2x and another unknown genome which is apparently distinct from the St, J and N genomes of relative species. Otherwise, SSR primers were used to analyze the relation of Ag. elongatum 2x and Ag. elongatum 4x. The amplification results of most primers showed that Ag. elongatum 2x and Ag. elongatum 4x have some identical bands and Ag. elongatum 4x have some specific bands. This result validated the conclusion from biochemical marker analyses that Ag. elongatum 4x is a allotetraploid.

[Evolution of partial promoter region of HMW glutenin genes from E and E1 genome of Agropyron elongatum].

The partial promoter regions of HMW glutenin subunit genes were cloned form the genomes E (in diploid Agropyron elongatum) and E1 (in tetraploid Agropyron elongatum) by PCR approach. There was only one nucleotide acid difference in the promoter sequences of x-type subunits between the two genomes; moreover, the promoter sequences of the two y-type subunits were completely identical. Although these promoter regions were very similar to each other, differences still existed in sequence size and the kind of nucleotide acid between the x-type and y-type subunits. It was speculated that the E1 genome in tetraploid Agropyron elongatum was probably originated from E genome in diploid species. The comparisons of these subunits with some of those from A, B, D and G genome of Triticeae demonstrated that the sequences of their partial promoter regions were conserved and shared a high homology more than 90%. The phylogenetic analysis based on the sequences in this region indicated that the y-type HMW glutenin subunits of Agropyron elongatum species were different from other subunits, whereas the x-type subunits of them were most closely related to that from the B genome.