决明胰蛋白酶抑制剂基因的克隆及其原核表达载体构建

[目的]克隆决明胰蛋白酶抑制剂全长cDNA序列,并构建原核表达载体。[方法]从决明种子中提取胰蛋白酶抑制剂总RNA,通过RT-PCR得到胰蛋白酶抑制剂cDNA,纯化后与PMD19-T载体连接,转化至大肠杆菌DH5α,获得了决明胰蛋白酶抑制剂基因的全长序列,并将该序列克隆到原核表达载体pET-28a(+)中,构建重组质粒pET-28a(+)/COTI。[结果]决明胰蛋白酶抑制剂基因核苷酸长度为630bp,编码一条长度为209个氨基酸的多肽。决明胰蛋白酶抑制剂与不同植物来源的Kunitz蛋白酶抑制剂有高度的同源性,表明其属于Kunitz蛋白酶抑制剂家族成员。所获重组质粒pET-28a(+)/COTI经过双酶切鉴定,其含有目的片段,且重组质粒构建正确。[结论]克隆了决明胰蛋白酶抑制剂的全长cDNA序列,并成功构建了含有该基因的原核表达载体,这为该基因的进一步表达及功能鉴定奠定了基础。     

甜荞胰蛋白酶抑制剂cDNA片段的克隆及序列特征

采用盐析、凝胶过滤和离子交换层析等方法从甜荞中纯化出荞麦胰蛋白酶抑制剂(buckwheat trypsm inhibitor,BTI),经活性鉴定该抑制剂属丝氨酸类蛋白酶抑制剂家族。为了获得BTI的基因序列,并弄清其在体内的表达调控机制,应用RTPCR和3’-RACE等方法,直接体外扩增该抑制剂基因,首次获得总长为361bp的DNA片段(GenBank登录号为AY335158),并命名为BTI-W1。该片段包括一个183bp的开放阅读框,编码61个氨基酸。由该基因推导的氨基酸序列与已报道的荞麦胰蛋白酶抑制剂BTI-2的氨基酸序列的同源性达100％。BTI-WI基因的获得,对于深入开展荞麦胰蛋白酶抑制剂结构与功能关系的研究具有重要意义,也为荞麦植物资源的开发利用建立了前期研究基础。     

水稻EPSP合酶基因的克隆、结构分析和定位

5-烯醇丙酮莽草酸-3-磷酸(EPSP)合酶是芳香族氨基酸合成途径中的一个关键酶, 该基因在植物抗除草剂基因工程中具有重要的应用价值. 根据水稻EPSP合酶基因的EST序列设计探针, 在水稻TAC基因组文库中筛选到16个阳性克隆. 对阳性克隆E11进行亚克隆, 由此获得了由3661核苷酸组成的水稻EPSP合酶基因全序列. 序列分析和同源性比较揭示, 该基因由8个外显子和7个内含子组成. 以窄叶青8号/京系17组合构建的DH群体和分子图谱将水稻EPSP合酶基因定位于水稻第6条染色体的上端.     

小麦尿卟啉原Ⅲ合成酶基因克隆及序列分析

根据水稻已公布的尿卟啉原Ⅲ合成酶(UROS)基因和小麦EST的保守序列,设计特异性引物对小麦尿卟啉原Ⅲ合成酶基因的部分片段进行克隆,得到了364 bp的cDNA(命名为UROS1)。以UROS1作为种子进行电子克隆,得到一段长为1210 bp的cDNA序列,并设计特异性引物克隆到1个1077 bp cDNA序列。对该片段分析结果表明,克隆得到的小麦UROS基因包含了信号肽区和全长的成熟肽区。小麦UROS基因与水稻UROS基因的同源性为86%左右,其推导氨基酸序列与水稻和拟南芥蛋白序列同源性分别约91%和79%。动物、植物以及微生物间核酸序列的保守性较低,氨基酸序列保守性也不高,但都存在UROS保守结构域(Hem D)。进化分析显示,该酶在不同物种间的进化速度差异较大。     

天花粉胰蛋白酶抑制剂基因的克隆及DNA序列分析

从天花粉块茎中分离纯化的天花粉胰蛋白酶抑制剂ＴＴＩ是目前已知的最小的蛋白酶抑制剂,它属于南瓜族蛋白酶抑制剂家族;由２７个氨基酸残基组成,含三对二硫键。本文用ＰＣＲ方法扩增一特异探针;结合传统的筛库方法,从天花粉ｃＤＮＡ基因库中筛选到含ＴＴＩ基因的克隆,经序列测定,得到了ＴＴＩ的ｃＤＮＡ全序列。其读框编码区编码的是一个由６５个氨基酸组成的Ｐｒｅ－Ｐｒｏ－ＴＴＩ,Ｐｒｅ与Ｐｒｏ分别含有２４个和１４个氨基酸。由ｃＤＮＡ序列推论的氨基酸序列和已测定的氨基酸序列完全相同。     

小麦一个新BBI型蛋白酶抑制剂基因TaUES的克隆及初步分析

根据小麦基因表达芯片结果,以山融3号小麦叶片为试验材料,克隆获得在盐胁迫处理24 h时表达显著提高的基因TaUES(up-regulated expression under saline-stress in wheat,ID:KC408382)。序列分析显示,TaUES编码一个富含半胱氨酸的97个氨基酸的多肽,该多肽含有1个BowB结构域和10个保守的半胱氨酸残基;与小麦或其他物种BBI型蛋白酶抑制剂氨基酸序列具有较高的同源性。推测TaUES是小麦中一种新的BBI型蛋白酶抑制剂基因。基因表达分析表明,TaUES基因参与盐和干旱胁迫应答。异源过表达转基因功能初步分析表明,过表达TaUES转基因烟草后代株系耐盐性提高。     

稻瘟菌侵染诱导水稻凝集素基因的表达

利用mRNA差异显示技术(DDRT-PCR),从非亲和性稻瘟菌生理小种131侵染的水稻品种爱知旭(Oryza sati-va L. cv.Aichi-asahi)叶片中分离了8个诱导差异表达的cDNA片段.对这8个差示片段进行了回收、重扩增和克隆,以其中一个长度为321碱基并与甘露糖结合水稻凝集素和水稻盐诱导蛋白基因高度同源的差示片段为探针,筛选水稻非亲和性cDNA文库,获得12个阳性克隆.序列测定和数据库查询表明该基因的cDNA与水稻凝集素基因的cDNA及盐诱导蛋白基因的cDNA核苷酸同源性高达96%,推定的氨基酸序列与甘露糖结合水稻凝集素的氨基酸序列一致,与水稻盐诱导蛋白仅相差2个氨基酸.Southern杂交显示该基因在水稻基因组中有两个同源拷贝数,Northern杂交表明非亲和性稻瘟菌侵染可强烈诱导该基因表达.因此推测该基因参与了水稻对稻瘟菌侵染的防御反应.     

凡纳滨对虾虾头内源性蛋白酶同源性分析

以凡纳滨对虾 (Litopenaeus vannamei shrimp) 虾头为原料,采用Q- Sepharose F F和Sephadex G-150对虾头内源碱性蛋白酶进行了纯化,通过SDS-PAGE测定分子量为79.95 kD|采用DEAE-Sepharose F.F和Sephadex G-100对内源酸性蛋白酶进行了纯化,通过SDS-PAGE测定分子量为27.45 kD. 利用HPLC-ESI-MS/MS对虾头内源碱性和酸性蛋白酶同源性进行了初步分析,将检测到内源性蛋白酶的部分氨基酸序列分别与不同物种的胰蛋白酶和胃蛋白酶氨基酸序列于Vector NTI suite 8.0软件上进行序列比对. 结果表明,内源性碱性蛋白酶与猪胰蛋白酶具有很高的同源性,均含有氨基酸序列LSSPATLNSRVATVSLPR|内源性酸性蛋白酶与非洲蟾蜍胃亚蛋白酶具有很高的同源性,均含有氨基酸序列EFGLSETEPGTNF.     

光滑鳖甲丝氨酸蛋白酶抑制剂基因ApSerpin-FA72的克隆、表达及功能验证

【目的】本研究旨在对光滑鳖甲Anatolica polita borealis丝氨酸蛋白酶抑制剂基因进行克隆及表达分析,以验证光滑鳖甲丝氨酸蛋白酶抑制剂的功能。【方法】利用PCR和cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术克隆获得光滑鳖甲丝氨酸蛋白酶抑制剂基因。采用生物信息学方法对该基因及其编码蛋白的基本性质进行预测和分析,同时构建其编码产物的系统进化树;构建光滑鳖甲丝氨酸蛋白酶抑制剂蛋白重组表达载体,表达、纯化蛋白进行功能验证。【结果】获得光滑鳖甲丝氨酸蛋白酶抑制剂基因Ap Serpin-FA72(Gen Bank登录号:MF188125),其基因编码序列长为1 176 bp,编码由391个氨基酸残基组成的多肽,蛋白理论分子量为43.7 k D,理论等电点为5.14,包含一个由21个氨基酸组成的信号肽。As Serpin-FA72属亲水蛋白,分泌到胞外发挥作用,可能具有胁迫应答的功能,与赤拟谷盗Triboloum castaneum Serpin的同源性最高。纯化得到的融合蛋白Trx A-Ap Serpin-FA72大小约为63.7 k D。功能验证表明,重组蛋白Trx A-Ap SerpinFA72对胰蛋白酶及胰凝乳蛋白酶活性均有抑制作用。【结论】光滑鳖甲丝氨酸蛋白酶抑制剂基因的表达产物对胰蛋白酶及胰凝乳蛋白酶活性具有抑制作用,表明其可能对消化类丝氨酸蛋白酶活性起抑制作用,对其功能活性的验证有助于深入研究Ap Serpin-FA72与丝氨酸蛋白酶之间的关系。     

三角帆蚌KuSPI基因的克隆及表达分析

Kuntiz型丝氨酸蛋白酶抑制剂(Kuntiz-type serine proteinase inhibitor,KuSPI)又称胰腺胰蛋白酶抑制剂,能够和相应的蛋白酶互作来调控生物体内的级联反应。本研究通过RACE克隆得到三角帆蚌KuSPI cDNA全序列共624 bp,包含38 bp和148 bp的5'和3'端UTR,ORF(open reading frame)438 bp,编码氨基酸145个,分子量为16 397.5 u,KuSPI氨基酸序列的同源性分析后发现其包含1个典型Kuntiz结构域,属于典型的KuSPI基因。qRT-PCR检测表明:KuSPI基因在闭壳肌、外套膜、肠、斧足、肝、血液、鳃、性腺和肾脏中均有表达,在外套膜中最高。利用嗜水气单胞菌进行诱导后发现,KuSPI基因在外套膜中表达量变化最大,呈现明显的先升高再降低趋势。外套膜是三角帆蚌先天免疫的首道防线,我们推测Ku SPI基因在其免疫反应中起到重要作用。     

Molecular Cloning and Sequence Analysis of a Gene Encoding Rice Proteinase Inhibitor

With the primers designed basing on the terminal amino acid sequences of rice proteinase inhibitors and the preferred codons of rice genes, a new gene coding for a rice proteinase inhibitor has been amplified and cloned from Oryza sativa var. japonica (cv. Zhonghua 8) using PCR technique. The gene contains 408 basepairs and encodes 133 amino acid residues. The deduced amino acid sequence with duplicated Bowman-Birk type structure and active sites specific to trypsin has relatively high homology with that of proteinase inhibitors from wheats, beans etc. As for rice, the new gene shares 74.8% homology with a rice bran trypsin inhibitor reported previously. The evolutionary characteristics of the proteinase inhibitor family has also been discussed.     

Purification and some properties of two proteinase inhibitors (DE-1 and DE-3) from Erythrina latissima (broad-leaved erythrina) seed

Two proteinase inhibitors, DE-1 and DE-3, were purified from Erythrina latissima seeds. Whereas DE-1 inhibits bovine chymotrypsin and not bovine trypsin, DE-3 inhibits trypsin but not chymotrypsin. The molecular weights and the amino acid compositions of the two inhibitors resemble the corresponding properties of the Kunitz-type proteinase inhibitors. The N-terminal primary structure of DE-3 showed homology with soybean trypsin inhibitor (Kunitz) and also with the proteinase inhibitors (A-II and B-II) from Albizzia julibrissin seed.     

Two kunitz-type proteinase inhibitors from potato tubers

Two proteinase inhibitors have been isolated from tubers of potato (Solanum tuberosum). Based on N-terminal amino acid sequence homologies, they are members of the Kunitz family of proteinase inhibitors. Potato Kunitz inhibitor-1 (molecular weight 19,500, isoelectric point 6.9) is a potent inhibitor of the animal pancreatic proteinase trypsin, and its amino terminus has significant homology to a recently characterized cathepsin D Kunitz inhibitor from potato tubers (Mares et al. [1989] FEBS Lett 251:94-98). Potato Kunitz inhibitor-2 (molecular weight 20,500, isoelectric point 8.6) is an inhibitor of the microbial proteinase subtilisin Carlsberg; its amino terminus is almost identical to an abundant 22 kilodalton protein from potato tubers (Suh et al. [1990] Plant Physiol 94:40-45) and has significant homology to other Kunitz-type subtilisin inhibitors from small grains. Both Kunitz inhibitors are abundant proteins of the cortex of potato tubers.     

The complete amino acid sequence of barley trypsin inhibitor

The amino acid sequence of barley trypsin inhibitor has been determined. The protein is a single polypeptide consisting of 121 amino acid residues and has Mr = 13,305. No free sulfhydryl groups were detected by Ellman's reagent, which indicates the presence of five disulfide bridges in the molecule. The primary site of interaction with trypsin was tentatively assigned to the arginyl-leucyl residues at positions 33 and 34. On comparison of the sequence of this inhibitor with those of other proteinase inhibitors, we found that the barley trypsin inhibitor could not be classified into any of the established families of proteinase inhibitors (Laskowski, M., Jr., and Kato, I. (1980) Annu. Rev. Biochem. 49, 593-626) and that this inhibitor should represent a new inhibitor family. On the other hand, this trypsin inhibitor showed a considerable similarity to wheat alpha-amylase inhibitor (Kashlan, N., and Richardson, M. (1981) Phytochemistry (Oxf.) 20, 1781-1784) throughout the whole sequence, suggesting a common ancestry for both proteins. This is the first case of a possible evolutionary relationship between two inhibitors directed to totally different enzymes, a proteinase and a glycosidase.     

The amino acid sequence of the double-headed protein proteinase inhibitor from dog submandibular glands, I. Structural homology to the pancreatic secretory trypsin inhibitors (author's transl)]

The primary structure of the broad specificity proteinase inhibitor from dog submandibular glands was elucidated. The inhibitor consists of a single polypeptide chain of 117 amino acids which is folded into two domains (heads) connected by a peptide of three amino acid residues. Both domains I and II show a clear structural homology to each other as well as to the single-headed pancreatic secretory trypsin inhibitors (Kazal type). The trypsin reactive site (-Cys-Pro-Arg-Leu-His-Glx-Pro-Ile-Cys-) is located in domain I and the chymotrypsin reactive center (-Cys-Thr-Met-Asp-Tyr-Asx-Arg-Pro-Leu-Tyr-Cys-) in domain II, cf. the Figure. The inhibitor is thus double-headed with two independent reactive sites. Whereas head I is responsible for the inhibition of trypsin and plasmin, head II is responsible for the inhibition of chymotrypsin, subtilisin, elastase and probably also Aspergillus oryzae protease and pronase. Remarkably, the structural homology exists also to the single-headed acrosin-trypsin inhibitors from seminal plasma[12] and the Japanese quail inhibitor composed of three domains[13].     

A possible evolutionary relationship between plant trypsin inhibitor, alpha-amylase inhibitor, and mammalian pancreatic secretory trypsin inhibitor (Kazal)

The amino acid sequence of the carboxyl-terminal half of barley trypsin inhibitor was found to be significantly similar to the whole sequence of bovine pancreatic secretory trypsin inhibitor (Kazal). Kazal type inhibitors and related proteins are known for the extraordinary mode of divergence among animals, and the present observation extends this to a plant for the first time. The present observation together with our previous finding of sequence homology between barley trypsin inhibitor and wheat alpha-amylase inhibitor (Odani, S., Koide, T., & Ono, T. (1982) FEBS Lett. 141, 279-282) suggest an unusual evolutionary relationship between cereal enzyme inhibitors and animal proteinase inhibitors of the Kazal type.     

Proteinase inhibitors from the tropical sea anemone Radianthus macrodactylus: Isolation and characteristic

Two new serine proteinase inhibitors (RmIn I and RmIn II) from the tropical sea anemone Radianthus macrodactylus have been isolated and characterized. The purification procedure includes polychrome-1 hydrophobic chromatography, Superdex Peptide 10/30 FPLC, and Nucleosil C(18) reverse-phase HPLC. The molecular masses of RmIn I, RmIn II, and the complexes RmIn II/trypsin and RmIn I,II/alpha-chymotrypsin have been determined. The K(i) values of RmIn I and RmIn II for trypsin and alpha-chymotrypsin have been determined. The polypeptides RmIn I and RmIn II are shown to be nontoxic and to exhibit antihistamine activity. The N-terminal amino acid sequences of RmIn I (GICSEPIVVGPCKAG-) and RmIn II (GSTCLEPKVVGPCKA-) have been determined. A high homology of the amino acid sequences is demonstrated for the proteinase inhibitors produced by such evolutionarily distant species as coelenterates, reptiles, and mammals.     

The complete amino acid sequence of the major Kunitz trypsin inhibitor from the seeds of Prosopsis juliflora.

The major inhibitor of trypsin in seeds of Prosopsis juliflora was purified by precipitation with ammonium sulphate, ion-exchange column chromatography on DEAE- and CM-Sepharose and preparative reverse phase HPLC on a Vydac C-18 column. The protein inhibited trypsin in the stoichiometric ratio of 1:1, but had only weak activity against chymotrypsin and did not inhibit human salivary or porcine pancreatic alpha-amylases. SDS-PAGE indicated that the inhibitor has a Mr of ca 20,000, and IEF-PAGE showed that the pI is 8.8. The complete amino acid sequence was determined by automatic degradation, and by DABITC/PITC microsequence analysis of peptides obtained from enzyme digestions of the reduced and S-carboxymethylated protein with trypsin, chymotrypsin, elastase, the Glu-specific protease from S. aureus and the Lys-specific protease from Lysobacter enzymogenes. The inhibitor consisted of two polypeptide chains, of 137 residues (alpha chain) and 38 residues (beta chain) linked together by a single disulphide bond. The amino acid sequence of the protein exhibited homology with a number of Kunitz proteinase inhibitors from other legume seeds, the bifunctional subtilisin/alpha-amylase inhibitors from cereals and the taste-modifying protein miraculin.     

Wound-induced proteinase inhibitors from tomato leaves. II. The cDNA-deduced primary structure of pre-inhibitor II

A cDNA containing the complete amino acid-coding region of wound-induced tomato Inhibitor II was constructed in the plasmid pUC9. The open reading frame codes for 148 amino acids including a 25-amino acid signal sequence preceding the N-terminal lysine of the mature Inhibitor II. The Inhibitor II sequence exhibits two domains, one domain having a trypsin inhibitory site and the other a chymotrypsin inhibitory site, apparently evolved from a smaller gene by a process of gene duplication and elongation. The amino acid sequence of tomato leaf Inhibitor II exhibits homology with two small proteinase inhibitors isolated from potato tuber and an inhibitor from eggplant. The small potato tuber inhibitors are homologous with 33 amino acids of the N-terminal domain and 19 amino acids from the C-terminal domain. Two identical nucleotide sequences of Inhibitor II cDNA in the 3' noncoding region were present that were also found in an Inhibitor I cDNA. These include an atypical polyadenylation signal, AATAAG, and a 10-base palindromic sequence, CATTATAATG, for which no function is yet known.     

Amino acid sequence of rat epidermal thiol proteinase inhibitor

The complete amino acid sequence of rat epidermal thiol proteinase inhibitor was determined. The unique 103-residue sequence was derived by analysis of two peptides generated by limited proteolysis of the native inhibitor with Staphylococcus aureus V8 protease and of three cyanogen bromide fragments. The protein has a high degree of sequence homology to either rat liver or human leucocyte inhibitor but is not identical and may represent a new type of low molecular weight thiol proteinase inhibitors.