水稻cDNA文库的构建及巯基蛋白酶抑制剂cDNA的分离

取开花后2周左右的水稻未成熟种子提取总RNA,分离mRNA,进而反转录合成cDNA并定向插入λgt22A克隆载体,经体外包装建成水稻未成熟种子的表达型 cDNA文库,经测定库容量达 1.5 × 10⁶ Pfu.进一步人工合成水稻巯基蛋白酶抑制剂(Oryzacystatin)编码基因5＇-端保守的21Nt的寡核苷酸经末端标记作为探针,从 2.1×10⁴Pfu中筛选出 9个阳性克隆,经鉴定其中 8个含有完整的水稻巯基蛋白酶抑制剂编码序列,对 1号克隆的序列分析表明,其除含有 309 bp的水稻巯基蛋白酶抑制剂编码序列外,在 5＇-端及 3＇-端还分别含有 84 bp和 497 bp的非编码序列,其中 3＇-端带有AATAAAA两个加尾信号以及31 Nt的Poly(A)尾,同水稻巯基蛋白酶抑制剂基因组序列比较,编码区碱基序列完全一致,但5＇-端和3＇-端非编码区有明显差异,其中加尾信号以后的cDNA碱基序列中含有大量的不连续插入.     

水稻巯基蛋白酶抑制剂的分子修饰与其对稻瘟病菌的抑制作用

水稻巯基蛋白酶抑制剂（ＣＰＩ）经用二硫苏糖醇,对氯汞苯甲酸和碘乙酸修饰后,对木瓜蛋白酶的抑制活性并无改变;用Ｎ－乙基顺丁烯二酰亚胺与ＣＰＩ反应,可以测出ＣＰＩ分子内有１９个巯基被修饰,被修饰后,抑制活性仍无改变,表明水稻ＣＰＩ的抑制活性不需要巯基参与;应用Ｎ－溴代丁二酰亚胺与ＣＰＩ反应,可测出ＣＰＩ分子内有２个Ｔｒｐ被修饰,修饰后,抑制活性全部丧失,表明Ｔｒｐ是保持抑制活性所必需的基团。水稻巯基蛋白酶抑制剂和丝氨酸蛋白酶抑制剂对稻瘟病菌丝体的生长均有抑制作用,但后者的抑制作用比前者更强,若将两种抑制剂混合使用,则对稻瘟病菌丝体的抑制作用非常强烈;当抑制剂加入量达７２μｇ时,即可产生明显的抑制作用。     

水稻巯基蛋白酶抑制剂cDNA在大肠杆菌中的表达

根据从水稻未成熟种子cDNA文库中筛选出的水稻巯基蛋白酶抑制剂(Oryzacys-tatin)cDNA序列,利用聚合酶链反应(PCR)技术扩增出Oryzacystatin编码区,插入到温度敏感型大肠杆菌表达载体的PtPL启动子下游．该质粒带有温度敏感型阻遏子的编码基因cIts857。转化大肠杆菌DH5a后。通过升温诱导,Oryzacystatin在大肠杆菌中获得高教表达,SDS—PAGE表明分子量约为12kDa。与预期结果一致,表达量占细菌可溶性蛋白总量的10％以上,对巯基蛋白酶的抑制活性检测表明,可溶性蛋白组分对木瓜蛋白酶有明显的抑制活力。     

水稻疏基蛋白酶抑制剂的分子修饰与其对稻瘟病菌的抑制作用

水稻巯基蛋白酶抑制剂经用二硫苏糖醇,对氯汞苯甲酸和碘乙酸修饰后,对木瓜蛋白酶的抑制活性并无改变;用Ｎ－乙基顺丁烯二酰亚胺与ＣＰＩ反应,可以测出ＣＰＩ分子内有１９个巯基被修饰,被修饰后,抑制活性仍无改变,表明水稻ＣＰＩ的抑制活性不需要巯基参与;应用Ｎ－溴代丁二酰亚胺与ＣＰＩ反应,可测出ＣＰＩ分子内有２个Ｔｒｐ被修饰,修饰后,抑制活性全部丧失,表明Ｔｒｐ是保持抑制活性所必需的基因,水稻巯基蛋白酶抑制剂     

双价抗虫基因叶绿体共转化植株抗虫性及其后代表型分析

利用基因枪法将含有水稻巯基蛋白酶抑制剂(Oryzacystatin,OC)基因烟草叶绿体表达载体和含有苏云金芽孢杆菌晶体毒蛋白基因(Bt cry IAc)烟草叶绿体表达载体,共转化烟草叶绿体,获得壮观霉素抗性植株。转基因植株抗棉铃虫试验表明,转双价抗虫基因植株比转单一抗虫基因植株具有更强的杀虫活性。转基因植株后代Southern检测及其遗传学分析试验证明,双价抗虫基因可以稳定地遗传给后代,且表现为叶绿体特有的母系遗传规律。 Abstract:The Bt gene and OC gene were cotransformed to tobacco chloroplast with particle bombardment method and spectinomycin resistance tobacco seedlings were obtained.Bioassays showed that the transgenic tobacco containing both genes had enhanced toxicity to the larvae of cotton bollworm (helicoverpa zea) by comparison with the plants containing only Bt or OC gene.Southern-blotting analysis and genetic analysis of progenies showed that the Bt and OC gene expressed and was inherited maternally to the progenies.     

一种水稻蛋白酶抑制剂基因的克隆及其结构分析

参照水稻蛋白酶抑制剂部分氨基酸序列 ,利用水稻偏爱密码子设计引物 ,经 PCR扩增 ,从我国水稻 (Oryza sativa)品种“中花 8号”中克隆到一个长 40 8bp的基因。序列测定和分析表明 ,克隆到的是一个未见报道的新的水稻蛋白酶抑制剂基因 ,该基因编码了一个由 1 33个氨基酸组成 ,具有重复双功能结构域和以抑制胰蛋白酶为主的活性中心的包曼 -伯克 (Bowman- Birk)型蛋白酶抑制剂 ,该基因推导的氨基酸序列与大麦、小麦、豆类等的某些蛋白酶抑制剂的氨基酸序列具有较高的同源性 ,与该家族的水稻的一种胰蛋白酶抑制剂氨基酸全序列同源性高达 75%。     

小麦旗叶老化期间的内肽酶

小麦叶片中存在着内肽酶,其最适pH为4．8,最适反应温度为45℃。小麦旗叶全展以后,净光合速率和总可溶性蛋白质含量下降,而内肽酶比活上升。用蛋白质合成抑制剂处理的结果表明,有内肽酶的从头合成;用激活制和抑制剂处理的结果表明,小麦叶片中至少有3种类型的内肽酶（即巯基蛋白酶、丝氨酸蛋白酶和金属蛋白酶）,而在蛋白质降解中起主要作用的是巯基蛋白酶。另外,氨基酸对内肽酶有保护作用,激素对内肽酶具有调节作用,活性氧对内肽酶的活力上升也有促进作用。     

水稻RubisCO的纯化及其与烟草RubisCO性质的比较

利用蔗糖密度梯度离心和DEAE-Sepharose fast flow柱层析等步骤从水稻叶片中纯化了RubisCO。此法不仅快速,而且酶的收得率高,酶的比活达1.15 μmol CO_2 min~(-1) mg~(-1)。 水稻RubisCO的热稳定性比烟草酶差,在活化时对Mg~(2 )较敏感。水稻和烟草RubisCO钝化态时总巯基数和表面巯基数相同,然而当酶活化后,水稻酶表面巯基数增加,而烟草酶则减少。当这些表面巯基被修饰后,水稻酶活性损失60％而烟草酶活力仅损失15％。水稻和烟草RubisCO的远紫外CD光谱有明显的区别,这显示了两者在二级结构、酶比活和性质上的重大差别。     

缺硫对镉胁迫下水稻幼苗非蛋白巯基物质含量和谷胱甘肽硫转移酶活性的影响

通过设置缺硫(S)处理,研究了镉(Cd)胁迫下水稻生长情况、幼苗Cd和非蛋白巯基含量以及谷胱甘肽硫转移酶(GST)活性的动态变化.结果表明:Cd胁迫明显抑制了水稻生长,显著诱导了巯基物质[非蛋白巯基(NPT)、谷胱甘肽(GSH)、植物螯合肽(PC)]的合成,GST活性表现出先升后降的趋势.缺S处理下,尽管水稻根部对Cd的吸收和向地上部的转运都有所增加,但Cd胁迫程度并未明显增强,巯基物质含量明显降低,根部GST活性提高.表明巯基物质和GST在水稻抗Cd胁迫过程中互为补充,在一定程度上减轻了Cd的毒性效应.     

水稻巯基蛋白酶抑制剂的纯化及其性质研究

水稻的糠皮和胚经生理盐水浸取、离心后的上清液加热至８０℃处理１０ｍｉｎ,离心获得的上清液调ｐＨ至８．０,得到沉淀。沉淀溶解于０．０１ｍｏｌ／ＬＨＣｌ,经透析冷冻干燥得水稻巯基蛋白酶抑制剂（ＣＰＩ）粗品;粗品再经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱线性离子梯度洗脱和ＳｅｐｈａｄｅｘＧ－１００柱分子筛层析,即可获得在ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和ＨＰＬＣ上均为单一蛋白带的ＣＰＩ样品。经上述步骤,ＣＰＩ可被纯化５８倍。经ＳｅｐｈａｄｅｘＧ－１００和ＳＤＳ－ＰＡＧＥ测定其分子量均为１２０００,Ｎ末端氨基酸为Ｐｒｏ,等电点５．６．水稻ＣＰＩ经１００℃处理１０ｍｉｎ后,其抑制活性无任何变化,在ｐＨ２．０～９．０之间,活性也不发生改变,但ｐＨ在９．０以上,其活性逐渐下降,水稻ＣＰＩ对木瓜蛋白酶是一种高亲和性的抑制剂,它对木瓜蛋白酶和无花果蛋白酶有强抑制作用,对菠萝蛋白酶仅有弱抑制作用,但对胰蛋白酶则全无抑制作用;其抑制类型属竞争性抑制剂类型,Ｋ＿ｉ值约３．５×１０￣（－８）ｍｏｌ／Ｌ对木瓜蛋白酶的抑制摩尔比约为１：１。     

Co‐expression of the proteinase inhibitors oryzacystatin I and oryzacystatin II in transgenic potato alters Colorado potato beetle larval development

Colorado potato beetle (CPB; Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II (OCI and OCII) have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato (Solanum tuberosum L.) cultivars Desiree, Draga?evka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition of cysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long‐term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae.     

Cloning, in silico characterization and interaction of cysteine protease and cystatin for establishing their role in early blight disease in tomato

Cysteine protease (CP) and Cysteine protease inhibitor (CPI) or cystatin constitute a critical point in programmed cell death (PCD), a basic biological phenomenon which takes place in the plants, when they are exposed to varying biotic and abiotic stresses. In the present study we isolated and cloned cDNAs encoding cysteine protease and cystatin from early blight infected tomato plants. Using computational biology tools the sequence-structure-function relationships for the tomato cystatin and cysteine protease were elucidated. Interaction between the cystatin and cysteine protease of host and pathogen is higher as compared to interaction shown by cystatin and cysteine protease within the host. The interaction energy of (a)tomato cystatin—tomato cysteine protease, (b)tomato cystatin—fungal cysteine protease and (c)tomato cysteine protease—fungal cystatin are ?319.33 Kcal/mol, ?504.71 Kcal/mol and ?373.731 Kcal/mol respectively. Comparative protein sequence analysis with different plant cystatins and cysteine protease were also done with the sequences of cystatin and cysteine protease isolated from tomato. Structures for all the cystatin and cysteine protease were modeled along with their interactions with fungal cystatin and cysteine protease in order to explore the structural variability and its manifestation at the functional level. This helped to relate the already known functions of these proteins with their sequences as well as the predicted structures. This also served to better understand the CP-CPI interaction operational in developing this protein family and its implication in plant defense during fungal pathogenesis in tomato plants.     

Cystatin B homolog from rock bream Oplegnathus fasciatus: genomic characterization, transcriptional profiling and protease-inhibitory activity of recombinant protein

Cysteine proteases are present in all living organisms and, in animals, function in a vast array of physiological and pathological processes. Cysteine protease inhibitors act upon the cysteine proteases to regulate their activity. The cystatin superfamily of cysteine protease inhibitors has members represented in all living organisms studied to date. Here, we report the identification of a new member of the family 1 cystatin in Oplegnathus fasciatus rock bream (denoted as RbCyt B) and the characterization at the molecular level. The complete genomic sequence of RbCyt B consists of three exons and a promoter region. The open reading frame (ORF) encodes for a 100 amino acids length polypeptide with a single cystatin-like domain and a cysteine protease inhibitor signature motif. The conserved N-terminal glycine, glutamine-valine-glycine motif, QxVxG, and a variant of the proline-tryptophan, PW, motif were identified. RbCyt B showed closest phylogenetic distance to Dicentrarchus labrax cystatin B, and shared up to 73% amino acid identity and 90% amino acid similarity with known cystatin B genes. RbCyt B mRNA expression was detected in nine different tissues and was highly expressed in liver, spleen, gill, brain, intestine, kidney, head kidney, and blood, as compared with muscle. In vivo immune stimulation with Edwardsiella tarda bacteria caused significant up-regulation of RbCyt B mRNA in head kidney and spleen at 24h post-infection (P<0.05). Recombinant RbCyt B was expressed in Escherichia coli, and the purified protein demonstrated 82% papain inhibitory activity at 500 × 10(-3) μg μL(-1) in a concentration-dependent manner. These results suggest that RbCyt B is a member of family 1 cystatin with high homology to cystatin B, and is a biologically active protein possessing papain inhibitory activity and potentially involved in immune responses against invading Gram-negative bacteria in rock bream.     

Ectopic phytocystatin expression leads to enhanced drought stress tolerance in soybean (Glycine max) and Arabidopsis thaliana through effects on strigolactone pathways and can also result in improved seed traits

Ectopic cystatin expression has long been used in plant pest management, but the cysteine protease, targets of these inhibitors, might also have important functions in the control of plant lifespan and stress tolerance that remain poorly characterized. We therefore characterized the effects of expression of the rice cystatin, oryzacystatin‐I (OCI), on the growth, development and stress tolerance of crop (soybean) and model (Arabidopsis thaliana) plants. Ectopic OCI expression in soybean enhanced shoot branching and leaf chlorophyll accumulation at later stages of vegetative development and enhanced seed protein contents and decreased the abundance of mRNAs encoding strigolactone synthesis enzymes. The OCI‐expressing A. thaliana showed a slow‐growth phenotype, with increased leaf numbers and enhanced shoot branching at flowering. The OCI‐dependent inhibition of cysteine proteases enhanced drought tolerance in soybean and A. thaliana, photosynthetic CO₂ assimilation being much less sensitive to drought‐induced inhibition in the OCI‐expressing soybean lines. Ectopic OCI expression or treatment with the cysteine protease inhibitor E64 increased lateral root densities in A. thaliana. E64 treatment also increased lateral root densities in the max2‐1 mutants that are defective in strigolactone signalling, but not in the max3‐9 mutants that are defective in strigolactone synthesis. Taken together, these data provide evidence that OCI‐inhibited cysteine proteases participate in the control of growth and stress tolerance through effects on strigolactones. We conclude that cysteine proteases are important targets for manipulation of plant growth, development and stress tolerance, and also seed quality traits.     

Inhibition of cysteine proteinases by a protein inhibitor from potato

The inhibitory specificity of a protein from potato tubers that inhibits cysteine proteinases (potato cysteine proteinase inhibitor, PCPI) has been compared with that of chicken egg-white cystatin. Most proteinases that are inhibited by cystatin were also inhibited by PCPI, but the potato inhibitor inhibited stem bromelain and fruit bromelain, which are not inhibited by cystatin, and for which no protein inhibitor of comparable potency has previously been described. In contrast, papaya proteinase IV was unaffected by PCPI as it is by the cystatins, and the exopeptidase, dipeptidyl peptidase I, is inhibited by cystatins, but was unaffected by PCPI. The differences in inhibitory specificity between these proteins may well reflect differences between superfamilies of cysteine proteinase inhibitors.     

Expression of Chicken Cystatin for Improving Insect Resistance in Rice

To become mature and infectious, many viruses and insects require proteolytic cleavage, which can be specifically inhibited by proteinase inhibitors. Oryzacystatin (OC), the first-described cystatin originating from rice seed, consists of two molecular species, OC-I and OC-II, both of which have antiviral activity. These intrinsic rice cystatins show a narrow inhibition spectrum and ordinarily are present in rice seeds at insufficient levels for inhibiting the cysteine proteinases of rice insect pests. In addition, our comparison of inhibitory activity (Ki value) showed that chicken cystatin (Ki 5 × 10-12 M) was more powerful than other cystatins, such as OC-I (Ki 3.02 × 10-8 M) and OC-II (l(i 0.83 × 10-8 M). Chicken cystatin also possesses a wide inhibitory spectrum against various cysteine proteinases. Here, we introduced the insecticidal chicken cystatin 8ene into rice plants to improve their insect resistance. Four highly expressive, independent transgenic lines were identified. Molecular analyses revealed that the transferred 8ene was expressed stably in the independent transgenic lines. Therefore, introducing the insecticidal cysteine proteinase inhibitor 8ene into rice plants can be part of a general development strategy for pest control.     

Oryzacystatin I expressed in transgenic potato induces digestive compensation in an insect natural predator via its herbivorous prey feeding on the plant

We observed recently that the rice cysteine proteinase inhibitor, oryzacystatin I (OCI) expressed in transgenic potato does not affect growth and development of the two-spotted stinkbug predator (Perillus bioculatus) via its herbivorous prey feeding on the plant. Here we monitored the inhibitory activity of recombinant OCI along this potato --> herbivore --> predator continuum, to determine if the absence of effect was associated with a digestive compensatory response of the predator following inhibition of its proteinases by the recombinant cystatin. After confirming that OCI is present in the plant, and ingested in an active form by potato beetle larvae, quantitative and electrophoretic assays allowed us to determine that the recombinant cystatin (representing about 0.8% of total soluble proteins in leaves) was entirely bound to a approximately 30-kDa target proteinase in the prey's midgut, forming a sodium dodecyl sulphate (SDS)-stable complex detected on immunoblots with an anti-OCI polyclonal antibody. Despite the apparent absence of free, residual OCI in the beetle's midgut, digestive protease activity in the predator, known to include OCI-sensitive activity, was altered negatively when the prey was fed the modified plant. This inhibitory process at the third trophic level was accompanied by a compensatory response in the predator, by which serine-type proteinases were synthesized de novo. Overall, our data suggest that the affinity between OCI and the predator's OCI-sensitive proteinases is: (i) as strong as (or stronger than) the affinity between OCI and the potato beetle 30-kDa-sensitive proteinase; and (ii) stronger than the affinity between these enzymes and the plant endogenous homologue of OCI, potato multicystatin, induced in the plant by potato beetle feeding. Our results also show that predatory organisms can adapt their digestive metabolism to the presence of plant antidigestive proteins ingested by their herbivorous preys. In a broader context, this study stresses the need to monitor the inhibitory effects of PI-expressing plants not only on the herbivorous insects targeted, but also on the organisms likely to consume these pests in the environment.     

cDNA cloning, identification and characterization of a novel cystatin from the tentacle of Cyanea capillata

Cystatin is of interest from biochemical and evolutionary prospective, and also has been applied in biotechnology. In this paper, a novel cystatin was found by EST sequence analysis of the cDNA library of Cyanea capillata tentacle. The sequence of a full-length cDNA clone contained an open reading frame encoding a putative 18-residue signal peptide and a mature protein of 113 amino acids, which showed only 26% identities to Family 2 cystatins and had its own characteristic enzyme-binding motifs, Ser(97)-Trp(98), which had not been found in any other known cystatins. Thus, the novel cystatin cloned from jellyfish was designated as cystatin J, which may belong to a new family of cystatin, called Family 4. The mature cystatin J was produced in Escherichia coli as a thioredoxin (Trx) fusion protein using the pET expression system and purified by affinity and cation exchange chromatography. The recombinant cystatin J of approximately M(r) = 12,800 displayed an obvious inhibition of papain (K(i) value below 0.5 nM), in competition with substrate. Thus, the recombinant cystatin J was a functional cystatin in spite of relatively lower sequence similarity with other cystatins. Activity of the novel cystatin was stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at >50 degrees C.     

Identification of the duplicated segments in rice chromosomes 1 and 5 by linkage analysis of cDNA markers of known functions

We mapped two loci for ADP-ribosylation factor homologues (ARF1, ARF2) and two loci for cysteine proteinase inhibitors (oryzacystatin-I and -II: OCI, OCII) by linkage analysis of restriction fragment length polymorphism loci in rice (Oryza sativa L.) genomic DNAs using their cDNAs as probes.Oc-1 andArf-2 were found to be closely located to each other on chromosome 1, whileOc-2 andArf-1,both found on chromosome 5, were also located close to each other. The map distances are about 2 cM in both pairs. In each chromosome, theArf locus was located about 27 cM from that of the aldolase gene (Ald-2 in chromosome 1 andAld-1 in chromosome 5). These three genes are in the same order,Ald-Arf-Oc, but in opposite orientations relative to the distal ends of the linkage group. The presence of two sets of three linked genes on chromosomes 1 and 5 strongly suggests a structural similarity of the blocks of the two chromosomes, which probably reflects duplication of the segment. A recent investigation by other workers has shown that these rice blocks correspond to two regions in maize chromosomes 8 and 6, that have previously been shown to share many duplicated nucleotide sequences. It is therefore very likely that the duplication of the region occurred before the divergence of rice and maize during the evolution of the subfamilies of the grasses (Gramineae). In view of a recently discovered possible structural similarity between the small GTP-binding protein superfamily, which includesArf andras proteins, and the cystatin family, the close linkage ofOc andArf loci found in the present study suggests a possible cluster of genes related to the small GTP-binding proteins.