甘薯sporamin蛋白的功能及其在抗虫转基因植物中的应用

Sporamin蛋白是甘薯块根中一种主要的可溶性蛋白,具有块根特异表达、伤害诱导表达等生物学特性,并具有胰蛋白酶抑制剂活性。sporamin具有的胰蛋白酶抑制剂活性使其广泛应用于近年植物抗虫转基因的研究。该文就其序列结构特性、表达调控、生物学功能,以及在抗虫转基因领域的研究进展进行了综述。     

Identification and characterisation of Kunitz-type plasma kallikrein inhibitors unique to Oxyuranus sp. snake venoms

As part of a wider study on Australian snake venom components, we have identified and characterised Kunitz-type protease inhibitors from the venoms of Oxyuranus scutellatus and Oxyuranus microlepidotus (Australian taipans) with plasma kallikrein inhibitory activity. Each inhibitor had a mass of 7 kDa and was purified from the venom as part of a protein complex. Mass spectrometry and N-terminal sequencing was employed to obtain amino acid sequence information for each inhibitor and a recombinant form of the O. scutellatus inhibitor, termed TSPI, was subsequently expressed and purified. TSPI was investigated for inhibition against a panel of 12 enzymes involved in haemostasis and estimates of the K_i value determined for each enzyme. TSPI was found to be a broad spectrum inhibitor with most potent inhibitory activity observed against plasma kallikrein that corresponded to a K_i of 0.057 ± 0.019 nM. TSPI also inhibited fibrinolysis in whole blood and prolonged the intrinsic clotting time. These inhibitors are also unique in that they appear to be found only in Oxyuranus sp. venoms.     

Physico-chemical and antifungal properties of protease inhibitors from Acacia plumosa

This study was aimed at investigating the purification, biological activity, and some structural properties of three serine protease inhibitors isoforms, denoted ApTIA, ApTIB, and ApTIC from Acacia plumosa Lowe seeds. They were purified from the saline extract of the seeds, using Superdex-75 gel filtration and Mono-S ion exchange chromatography. They were further investigated by mass spectrometry, spectroscopic measurements, surface plasmon resonance, and inhibition assays with proteases and phytopathogenic fungi. The molecular mass of each isoform was estimated at ca. 20 kDa. Each contained two polypeptide chains linked by a disulfide bridge, with different isoelectric points that are acidic in nature. The N-terminal sequences of both chains indicated that they were Kunitz-type inhibitors. Circular dichroism (CD) analyses suggested the predominance of both disordered and beta-strands on ApTI isoforms secondary structure, as expected for β-II proteins. In addition, it was observed that the proteins were very stable, even at either extreme pH values or at high temperature, with denaturation midpoints close to 75 °C. The isoinhibitors could delay, up to 10 times, the blood coagulation time in vitro and inhibited action of trypsin (Ki 1.8 nM), α-chymotrypsin (Ki 10.3 nM) and kallikrein (Ki 0.58 μM). The binding of ApTIA, ApTIB, and ApTIC to trypsin and α-chymotrypsin, was investigated by surface plasmon resonance (SPR), this giving dissociation constants of 0.39, 0.56 and 0.56 nM with trypsin and 7.5, 6.9 and 3.5 nM with α-chymotrypsin, respectively. The growth profiles of Aspergillus niger, Thielaviopsis paradoxa and Colletotrichum sp. P10 were also inhibited by each isoforms. These three potent inhibitors from A. plumosa may therefore be of great interest as specific inhibitors to regulate proteolytic processes.     

Molecular cloning of Kunitz-type proteinase inhibitor group B genes from potato

Eighteen clones representing copies of four Kunitz-type proteinase inhibitor group B genes (PKPI-B) obtained by polymerase chain reaction cloning of potato (Solanum tuberosum L. cv. Istrinskii) genomic DNA were sequenced and analyzed. Three new genes were found and named PKPI-B1, PKPI-B2, and PKPI-B10: these were represented by five, two, and seven clones, respectively. The remaining four clones corresponded to the formerly characterized PKPI-B9 gene. These data show that at least four PKPI-B encoding genes are harbored in the genome of potato cv. Istrinskii. Their analysis suggests that variability of PKPI-B encoding genes in potato is limited and could be explained by cross-hybridization events in the ancestor forms rather than by random mutagenesis.     

Inhibition of Tryptase TL2 from Human T4+ Lymphocytes and Inhibition of HIV-1 Replication in H9 Cells by Recombinant Aprotinin and Bikunin Homologues

The serine esterase TL₂ from human T4+ lymphocytes is a binding component to HIV-1 glycoprotein gp120 and seems to play a role in the HIV-1 infection mechanism. Recombinant variants of the Kunitz-type serine proteinase inhibitor aprotinin were investigated for their ability to inhibit tryptase TL₂ and the binding of gp120 to this enzyme. Furthermore, the viral replication of HIV-1 was investigated in H9 cell cultures under the influence of recombinant aprotinin and bikunin variants. In contrast to native aprotinin, the recombinant variant [Arg¹⁵, Phe¹⁷, Glu⁵²]aprotinin with a reactive-site sequence homologous to the V3 loop of HIV-1 gp120 showed a specific inhibition of tryptase TL₂ (>80%). However, the [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin variant with hydrophobic subsites was the most potent inhibitor of the binding of gp120 to tryptase TL₂ (68%). Our results show that the enzyme activity of purified tryptase TL₂ is inhibited not only by variants with basic amino acids, but also those with hydrophobic residues in the reactive-site region. Therefore, tryptase TL₂ is not a typical trypsin-like or chymotrypsin-like protease. Investigations on inhibition of HIV-1 replication in H9 cell cultures showed that tryptase TL₂ is involved in the mechanism of virus internalization into human lymphocytes. The [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin showed a significant retardation of syncytium formation over a period of 5 days in a 1 M concentration. Similar investigations were performed with recombinant variants of bikunin, the light chain of human inter--trypsin inhibitor. Only the single-headed variant [Arg⁹⁴]⁸²bikunin inhibited slightly the syncytium formation over a period of 2 days in a 2.2 M concentration. Wild-type bikunin and all full-length variants showed no effect, possibly due to steric hindrance by the second domain of the double-headed inhibitor.     

胰蛋白酶抑制剂基因siRNA表达体系的构建

以大豆基因组DNA为模板,利用聚合酶链式反应(PCR)技术克隆了大豆胰蛋白酶抑制剂基因KSTI3的全长DNA片段,并将其构建到pMD18-T vector上。核苷酸序列测定结果表明:该基因片段全长654bp,与已发表的KSTI3基因序列同源性达99%。将反义 正义基因片段插入到pBI121 35S启动子下,构建重组质粒pBIKSTI3。通过冻融法将该重组质粒转入农杆菌EHA105中,获得了siRNA表达体系。利用农杆菌介导法将带有pBIKSTI3的菌株转化大豆,从2棵再生植株中得到2 100bp的特异性扩增条带,而未转化的植株中无该片段的产生。