Regeneration of Populus nigra transgenic plants expressing a Kunitz proteinase inhibitor (KTi3) gene

Transgenic poplar (Populus nigra, cv. Jean Pourtet) plants were recovered as a result of Agrobacterium tumefaciens-mediated transformation performed with EHA105 pBI-KUN strain. Plasmid pBI-KUN contains a 650 bp insert derived from the soybean (Glycine max L.) KTi₃, gene, coding for a Kunitz trypsin proteinase inhibitor. A total of 58 independent transgenic lines were obtained from 200 co-cultivated leaf explants. Southern blot hybridization analysis demonstrated the presence of KTi₃ gene in the poplar genome. Northern blot analysis of different kanamycin-resistant plantlets confirmed the accumulation of KTi₃ mRNA and revealed different levels of expression. The trypsin inhibitory activity was determined in poplar transgenic tissues by means of specific assay. Moreover, the trypsin-like digestive proteinases of the polyphagous moth Lymantria dispar (Lepidoptera, Lymantriidae) and Clostera anastomosis (Lepidoptera, Notodontidae) were detected and inhibited in vitro by Kunitz proteinase inhibitor from selected transgenic plants. Two insect bioassays were performed on P. nigra transgenic plant lines, using larvae of the above mentioned insects. In both cases larval mortality and growth as well as pupal weight were not significantly affected when the insects were fed on transgenic leaves and control leaves, respectively.     

Adaptation of Spodoptera exigua (Lepidoptera: Noctuidae) to barley trypsin inhibitor BTI-CMe expressed in transgenic tobacco

Nicotiana tabacum plants were transformed with the cDNA of barley trypsin inhibitor BTI-CMe under the control of the 35S CaMV promoter. Although the transgene was expressed and the protein was active in the homozygous lines selected, growth of Spodoptera exigua (Lepidoptera: Noctuidae) larvae reared on transgenic plants was not affected. The protease activity in larval midgut extracts after 2 days feeding on transformed tobacco leaves from the highest expressing plant showed a reduction of 25% in the trypsin-like activity compared to that from insects fed on non-transformed controls. The susceptibility of digestive serine-proteases to inhibition by BTI-CMe was confirmed by activity staining gels. This decrease was compensated with a significant induction of leucine aminopeptidase-like and carboxipeptidase A-like activities, whilechymotrypsin-, elastase-, and carboxipeptidase B-like proteases were not affected.     

Effects of soybean Kunitz trypsin inhibitor on the cotton boll weevil (Anthonomus grandis)

The cotton boll weevil, Anthonomus grandis, is an economically important pest of cotton in tropical and subtropical areas of several countries in the Americas, causing severe losses due to their damage in cotton floral buds. Enzymatic assays using gut extracts from larval and adult boll weevil have demonstrated the presence of digestive serine proteinase-like activities. Furthermore, in vitro assays showed that soybean Kunitz trypsin inhibitor (SKTI) was able to inhibit these enzymes. Previously, in vivo effects of black-eyed pea trypsin chymotrypsin inhibitor (BTCI) have been demonstrated towards the boll weevil pest. Here, when neonate larvae were reared on an artificial diet containing SKTI at three different concentrations, a reduction of larval weight of up to 64% was observed for highest SKTI concentration 500 microM. The presence of SKTI caused an increase in mortality and severe deformities of larvae, pupae and adult insects. This work therefore represents the first observation of a Kunitz trypsin inhibitor active in vivo and in vitro against A. grandis. Bioassays suggested that SKTI could be used as a tool in engineering crop plants, which might exhibit increased resistance against cotton boll weevil.     

Accumulation of a chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests

A member of the potato proteinase inhibitor II (PPI II) gene family that encodes for a chymotrypsin iso-inhibitor has been introduced into tobacco (Nicotiana tabacum) usingAgrobacterium tumefaciens-mediated T-DNA transfer. Analysis of the primary transgenic plants (designated R₀) confirmed that the introduced gene is being expressed and the inhibitor accumulates as an intact and fully functional protein. For insect feeding trials, progeny from the self-fertilization of R₀ plants (designated R₁) were used. Leaf tissue, either from transgenic or from control (non-transgenic) plants, was fed to larvae ofChrysodeixis eriosoma (Lepidoptera: Noctuidae, green looper),Spodoptera litura (F.) (Lepidoptera: Noctuidae) andThysanoplusia orichalcea (F.) (Lepidoptera: Noctuidae) and insect weight gain (increase in fresh weight) measured. Consistently,C. eriosoma larvae fed leaf tissue from transgenic plants expressing thePPI II gene grew slower than insects fed leaf tissue from non-transgenic plants or transgenic plants with no detectablePPI II protein accumulation. However, larvae of bothS. litura andT. orichalcea consistently demonstrated similar or faster growth when fed leaf tissue from transgenic plants compared with those fed non-transgenic plants. In agreement with the feeding trials, the chymotrypsin iso-inhibitor extracted from transgenic tobacco effectively retarded chymotrypsin-like activity measured inC. eriosoma digestive tract extracts, but not in extracts fromS. litura. We conclude, therefore, that for certain insects the use of chymotrypsin inhibitors should now be evaluated as an effective strategy to provide field resistance against insect pests in transgenic plants, but further, that a single proteinase inhibitor gene may not be universally effective against a range of insect pests. The significance of these observations is discussed with respect to the inclusion of chymotrypsin inhibitors in the composite of insect pest resistance factors that have been proposed for introduction into crop plants.     

Reversible denaturation of the soybean Kunitz trypsin inhibitor

The soybean Kunitz trypsin inhibitor (SKTI) is a beta-sheet protein with unusual stability to chemical and thermal denaturation. Different spectroscopic criteria were used to follow the thermal denaturation and renaturation of SKTI. Upon heating to 70 degrees C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 degrees C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 degrees C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 degrees C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered beta-sheet proteins.     

Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor CMe

Proteinase inhibitors have been proposed to function as plant defence agents against herbivorous pests. We have introduced the barley trypsin inhibitor CMe (BTI-CMe) into wheat (Triticum aestivum L.) by biolistic bombardment of cultured immature embryos. Of the 30 independent transgenic wheat lines selected, 16 expressed BTI-CMe. BTI-CMe was properly transcribed and translated as indicated by northern and western blot, with a level of expression in transgenic wheat seeds up to 1.1% of total extracted protein. No expression was detected in untransformed wheat seeds. Functional integrity of BTI-CMe was confirmed by trypsin inhibitor activity assay. The significant reduction of the survival rate of the Angoumois grain moth (Sitotroga cerealella, Lepidoptera: Gelechiidae), reared on transgenic wheat seeds expressing the trypsin inhibitor BTI-CMe, compared to the untransformed control confirmed the potential of BTI-CMe for the increase of insect resistance. However, only early-instar larvae were inhibited in transgenic seeds and expression of BTI-CMe protein in transgenic leaves did not have a significant protective effect against leaf-feeding insects.     

幼虫密度对斜纹夜蛾抗病能力的影响

为了阐明斜纹夜蛾Spodoptera litura Fabricius幼虫密度对其抗病能力的影响,在室内条件下(温度23℃±1℃,相对湿度75%)对不同幼虫密度(1、2、5、10、15头/皿(直径为12cm))饲养的斜纹夜蛾幼虫抵抗斜纹夜蛾核型多角体病毒侵染的能力及其免疫指标进行了研究。结果表明:幼虫密度对斜纹夜蛾幼虫接种核型多角体病毒后的存活率、存活时间及血淋巴酚氧化酶活性影响显著。随着幼虫密度的增加,接种核型多角体病毒后幼虫的存活率降低,存活时间缩短。当幼虫密度达到15头/皿时,幼虫存活率显著低于其它幼虫密度。不同幼虫密度幼虫的存活时间以1头/皿的最高,15头/皿的最低,且二者之间差异显著。幼虫血淋巴中酚氧化酶活性随幼虫密度的增加而明显降低,当幼虫密度达到5头/皿时,幼虫酚氧化酶活性显著低于1头/皿的。另外,幼虫溶菌酶活性和血细胞总数受幼虫密度影响不显著。不同密度幼虫抗病性的变化与其血淋巴中酚氧化酶活性的变化趋势较为一致。所以斜纹夜蛾幼虫抗病能力的降低可能与幼虫酚氧化酶活性的下降有关。因此,幼虫密度是影响斜纹夜蛾幼虫抗病性变化的重要因子。     

Sweet potato (Ipomoea batatas) trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera litura

A sweet potato (Ipomoea batatas cv. Tainong 57) trypsin inhibitor gene was introduced into tobacco plants (Nicotiana tabaccum cv. W38) by Agrobacterium tumefaciens– mediated transformation. From 30 independent transformants, three lines with high level of expression were further analyzed. The trypsin inhibitor gene, under control of the 35S CaMV promoter, led to the production of the trypsin inhibitor proteins up to 0.2% of the total protein. In insecticidal bioassays of transgenic tobacco plants, larval, growth of Spodoptera litura (F.), the tobacco cutworm, was severely retarded as compared to their growth on control plants. This observation implied that expression of sweet potato trypsin inhibitor can provide an efficient method for crop protection. Received: 29 July 1996 / Revision received: 15 November 1996 / Accepted: 8 December 1996     

The expression of a mammalian proteinase inhibitor,bovine spleen trypsin inhibitor in tobacco and its effects on Helicoverpa armigera larvae

The cDNA for bovine spleen trypsin inhibitor (SI), a homologue of bovine pancreatic trypsin inhibitor (BPTI), including the natural mammalian presequence was expressed in tobacco using Agrobacterium tumefaciens-mediated transformation. Stable expression required the N-terminal targeting signal presequence although subcellular localization was not proven. SI was found to exist as two forms, one coinciding with authentic BPTI on western blots and the second marginally larger due to retention of the C-terminal peptide. Both were retained on a trypsin-agarose affinity gel and had inhibitory activity. Newly emergent leaves contained predominantly the large form whereas senescent leaves had little except the fully processed form present. Intermediate-aged leaves showed a gradual change indicating that a slow processing of the inhibitor peptide was occurring. The stability of SI was shown by the presence of protein at high levels in completely senescent leaves. Modifications to the cDNA (3 and 5 changes and minor codon changes) resulted in a 20-fold variation in expression. Expression of modified SI in transgenic tobacco leaves at 0.5% total soluble protein reduced both survival and growth of Helicoverpa armigera larvae feeding on leaves from the late first instar. In larvae surviving for 8 days, midgut trypsin activity was reduced in SI-tobacco fed larvae, while chymotrypsin activity was increased. Activities of leucine aminopeptidase and elastase-like chymotrypsin remained unaltered. The use of SI as an insect resistance factor is discussed.     

Absorption and fluorescence spectra ofS-quinolylethylated Kunitz soybean trypsin inhibitor

Disulfide bonds in soybean trypsin inhibitor (Kunitz) were simultaneously reduced and alkylated using tri-n-butylphosphine and 2-vinylquinoline at pH 7.6 in 0.11 M Tris-4.4 M urea, 41% ethanol. The resulting S--2-quinolylethylated protein (2-QE-STI) has a new absorption peak at 315–318 nm. Its quinoline fluorescence can be excited above 310 nm independently of intrinsic protein fluorescence. Free 2-quinolylethylcysteine (2-QEC) shows unexpectedly weak fluorescence. Quinoline absorption in 2-QEC and 2-QE-STI changes with pH. The apparentpK values determined spectrophotometrically are near 5 for 2-QEC and 3 for 2-QE-STI. Fluorescence decreased with increasing pH and in the presence of chloride ions. Both structural and charge effects thus appear to influence the absorption and fluorescence of the quinoline group. Corrected fluorescence emission (excited at 316 nm) of neutral 2-QE-STI diluted in 0.1 N H₂SO₄ was directly proportional to concentration in the range 0.4–8 m 2-QEC. The 2-QEC content of the protein derivative determined by UV absorption at pH 1.5 was in agreement with the expected value of four residues per mole. Fluorescence measurements ofS-2-quinolylethylated proteins may be especially useful as a sensitive, specific assay for cyst(e)ine residues.Reference to a company or product name does not imply approval or recommendation of the product by the U.S. Department of Agriculture to the exclusion of others that may be suitable.Abbreviations used are Mops: 3-(N-morpholino)propanesulfonic acid; STI: soybean trypsin inhibitor (Kunitz); 2-PE-STI:S--2-pyridylethylated STI; 2-QEC:S--(2-quinolylethyl)-l-cysteine; 2-QE-STI:S--2-quinolylethylated STI; TosPheCH₂-trypsin: bovine trypsin treated withp-toluenesulfonyl phenylalanine chloromethyl ketone.     

寄主植物对斜纹夜蛾酯酶和乙酰胆碱酯酶活性的影响

利用生物测定与生物化学的方法,就取食不同寄主植物的2个斜纹夜蛾[Spodoptera litura(Fab.)]品系对丙溴磷和灭多威的敏感性及其体内的酯酶与乙酰胆碱酯酶的活性变化进行了研究。结果表明,取食不同寄主植物的斜纹夜蛾对丙溴磷和灭多威的敏感性不同。在敏感品系中,取食不同食料的斜纹夜蛾后代对灭多威的敏感性顺序为:烟草<棉花<大豆<人工饲料;对丙溴磷而言,敏感性顺序则为:棉花<烟草<大豆<人工饲料。而在田间抗性品系中,对丙溴磷的敏感性顺序为:棉花<烟草<人工饲料<大豆;对灭多威的敏感性顺序为:棉花<烟草<大豆<人工饲料。此外,在田间抗性和敏感品系中,取食不同食料的斜纹夜蛾体内的酯酶活性之间均存在显著差异,对其乙酰胆碱酯酶的活性也有程度不同的诱导效应,但并不会引起乙酰胆碱酯酶的质变。     

铅胁迫对斜纹夜蛾生长发育与生殖的影响

在植食性昆虫斜纹夜蛾幼虫标准人工饲料中添加不同浓度的重金属铅(Pb),研究Pb胁迫对其生长发育与生殖的影响.结果表明:斜纹夜蛾不同发育阶段(幼虫、蛹和成虫)的存活率和体质量随着饲料中Pb浓度的增加而下降,引起存活率显著下降的最低Pb胁迫浓度是100 mg·kg-1,引起体质量显著减少的最低Pb胁迫浓度是50 mg·kg-1.在取食Pb浓度为25～ 200 mg·kg-1的人工饲料后,斜纹夜蛾成虫的产卵天数显著减少;产卵力和生育力随着饲料中Pb浓度的增加而显著下降;1000粒卵的平均质量显著低于对照;卵孵化率显著下降.重金属Pb胁迫对斜纹夜蛾生长发育和生殖具有显著的抑制作用.     

大豆胰蛋白酶抑制剂和防御信号物质对斜纹夜蛾幼虫保护酶的影响

以广食性害虫斜纹夜蛾为研究对象,连续6代自二龄或三龄开始用含有大豆胰蛋白酶抑制剂(soybean trypsin inhibitor,SBTI)的人工饲料喂养幼虫,饲养至五龄时测定SBTI对幼虫保护酶的影响。结果表明,SBTI抑制斜纹夜蛾幼虫SOD、CAT活性,随着饲养代数的增加,SBTI的抑制效果降低;SBTI可显著升高斜纹夜蛾幼虫POD活性。预先接触外源植物防御信号物质茉莉酸甲酯和水杨酸甲酯48h均可显著影响斜纹夜蛾幼虫体内保护酶SOD、POD、CAT活性,且可显著减缓SBTI对斜纹夜蛾幼虫SOD、POD、CAT活性的作用效果。表明,斜纹夜蛾取食SBTI后能够调节自身的保护酶系统,逐步适应蛋白酶抑制剂的毒害,而预先接触植物防御信号物质可增强其对植物防御蛋白的适应能力。     

Avidin expressed in transgenic tobacco leaves confers resistance to two noctuid pests,Helicoverpa armigera and Spodoptera litura

Fertile transgenic tobacco plants with leaves expressing avidin in the vacuole have been produced and shown to halt growth and cause mortality in larvae of two noctuid lepidopterans, Helicoverpa armigera and Spodoptera litura. Late first instar H. armigera larvae and neonate (<12-h-old) S. litura larvae placed on leaves excised from T₀ tobacco expressing avidin at 3.1–4.6M (moles/kg of fresh leaf tissue) had very poor growth over their first 8 days on the leaves, significant numbers had died by days 11 or 12 and all were dead by day 22 (H. armigera) or day 25 (S. litura). Similar results were obtained when late first instar H. armigera larvae were placed on leaves from T₁ plants expressing avidin at six different average concentrations, ranging from 3.7 to 17.3M. Two larvae on the lowest expressing leaves survived to pupation, but there was total mortality among the other groups and no relationship between avidin concentration and the effects on the larvae. Synergistic effects between avidin-expressing tobacco plants and a purified Bt toxin, Cry1Ba, were demonstrated. Late instar H. armigera larvae fed with leaves from T₂ plants expressing avidin at average concentrations of either <5.3 or >12.9M, and painted with Cry1Ba protein at a rate equivalent to an expression level of 0.5% of total leaf protein, died significantly faster than larvae given either of the two treatments alone. Larvae fed with avidin-expressing leaves painted with the protease inhibitor, aprotinin, at a rate equivalent to 1% of total leaf protein had mortality similar to those given avidin-leaves alone. There was no evidence of antagonism between these two proteins.     

Laboratory evaluation of flight activity of the common cutworm, Spodoptera litura (Lepidoptera: Noctuidae)

Abstract  The flight activity of Spodoptera litura in tethered conditions is evaluated using a computer-mediated flight-mill in the laboratory. The 3–4-day-old moths fly longer and farther than those of other ages. Male and female moths exhibit similar flight activity. Mating status does not influence the flight duration and distance of 2-day-old females. However, these two flight parameters with a 6-day-old mated female is significantly lower than that of unmated ones. The optimum temperature for flight ranged from 16–24°C, whereas the optimum RH ranged from 60%–100%. During 72-h period, the total flight duration and distance of 1-day-old male and female moths were 19.6 h (± 5.8) and 83.3 km (± 28.4), and 24.0 h (± 7.0) and 105.4 km (± 37.4), respectively. These results indicate that S. litura has a great potential to undertake long-distance migratory flights.     

Sub-lethal effects of SpltMNPV infection on developmental stages of Spodoptera litura (Lepidoptera: Noctuidae)

Application of different doses of SpltMNPV on final instars of Spodoptera litura showed dose-related mortality. A significant increase in the time for pupation and progeny mortality as well as reduced fecundity was observed in surviving larvae. Impact of sublethal doses on subsequent generation needs attention in designing baculovirus-based pest management.     

Entity evidence for differentiation between Tia and Tib types of soybean Kunitz trypsin inhibitor: detection of a novel transitional variant type between Tia and Tib in wild soybean (Glycine soja Sieb. & Zucc.)

Soybean Kunit trypsin inhibitor (SKTI) has several polymorphic types. Of these SKTI, there are large differences of nine amino acid substitutions between Tia and Tib. So far no transitional type between them has been found. A novel transitional intermediate variant between Tia and Tib was detected in 11 lines from 720 Japanese wild soybeans (Glycine soja Sieb. & Zucc.). This variant showed identical electrophoretic mobility to Tib in the Davis system polyacrylamide gel electrophoresis (PAGE), but higher electric points than other SKTI proteins (Tia, Tib, Tic) in isoelectric focusing PAGE. The genetic analysis of SKTI in F₂ seeds from a cross between the novel variant type and Tib showed that this variant type is inherited as codominant alleles in a multiple allelic system at an SKTI locus. This variant also showed inhibitory activity to trypsin. We propose the genetic symbol Ti b ⁱ⁵ for this novel variant. The sequence analysis of Tib ⁱ⁵ revealed that six nucleotides were different between Tib ⁱ⁵ and Tia, and the nucleotides of these mutated positions were identical to Tib. This causes substitution of five amino acids at the residue position 62 (Tyr→Phe), 74 (Ser→Arg), 114 (Met→Val), 120 (Leu→Ile) and 137 (Pro→Thr). These substitutive amino acids are completely in accord with the amino acids of Tib, showing that Tib ⁱ⁵ is an intermediate between Tia and Tib types. Tib ⁱ⁵ type is widely distributed throughout seven separate areas from northeast to southwest Japan with a 1.5% frequency of total materials examined. This indicated that Tib ⁱ⁵ type did not originate from a recent mutation event, but had spread in wild soybean from ancient times.     

黄皮种子提取物对斜纹夜蛾幼虫的杀虫活性及有效成分鉴定

【目的】测定黄皮Clausena lansium种子提取物对斜纹夜蛾Spodoptera litura幼虫的拒食活性,对有效成分进行分离、鉴定,并进一步测定其拒食活性、生长发育抑制活性及细胞毒性,为筛选斜纹夜蛾生态防治植物源杀虫剂提供依据。【方法】采用叶蝶法测定黄皮种子甲醇提取物及其石油醚、乙酸乙酯、正丁醇和水4种溶剂萃取物对斜纹夜蛾3龄幼虫的非选择性拒食活性和生长发育抑制活性;用硅胶柱层析进行石油醚萃取物和乙酸乙酯萃取物拒食活性成分分离;用核磁共振谱(NMR)进行拒食有效成分结构鉴定;用MTT法测定拒食活性成分对SL细胞毒性。【结果】黄皮种子甲醇提取物对斜纹夜蛾3龄幼虫具有较强的拒食活性,活性成分主要分布于石油醚相和乙酸乙酯相中;石油醚相和乙酸乙酯相经硅胶柱层析分离得10个馏分,以黄色晶体馏分2的拒食活性最强;经分离纯化及核磁共振氢谱和碳谱鉴定,黄皮新肉桂酰胺B(lansiumamide B)为主要的拒食活性成分,对斜纹夜蛾3龄幼虫48 h拒食中浓度为214.95μg/m L,同时具有较强的细胞毒性,对SL细胞48 h抑制中浓度为10.09μg/m L。【结论】黄皮种子提取物对斜纹夜蛾幼虫具有较强的拒食活性及细胞毒性,黄皮新肉桂酰胺B是主要活性成分之一,作为植物源杀虫剂开发具有较好的发展前景。