二化螟滞育生物钟蛋白TIME-EA4基因的克隆及时空和温度诱导表达分析

【目的】生物测时蛋白TIME-EA4是昆虫特有的一种滞育生物钟蛋白。本文旨在克隆二化螟Chilo suppressalis TIME-EA4基因,研究其在二化螟不同发育时期和不同组织中的时空表达模式及在不同田间种群和不同温度诱导下的滞育和非滞育种群中的表达水平。【方法】通过RACE技术克隆二化螟TIME-EA4基因全长c DNA序列,利用荧光定量PCR的方法检测TIME-EA4基因在二化螟不同发育时期、6龄幼虫不同组织中的表达模式,及在不同季节采集的田间种群和不同温度处理1h后的滞育和非滞育种群6龄幼虫中的表达量变化。【结果】从二化螟中克隆获得TIME-EA4的c DNA序列(Gen Bank登录号:KU356855),全长821 bp,开放阅读框516 bp,编码172个氨基酸;TIME-EA4基因在二化螟不同发育时期和不同组织中均有表达,且在早期蛹、雌雄成虫以及6龄幼虫的头部、脂肪体和中肠中表达水平较高,同时在滞育幼虫组织中的表达高于非滞育幼虫。此外,TIME-EA4基因在滞育种群中的表达量是非滞育种群的3倍左右,同时在非滞育种群中,该基因的表达明显受10℃及以下低温的诱导,且在10℃时表达量最高,而在滞育种群中该基因的表达受温度影响不明显。【结论】结果说明TIME-EA4基因与二化螟的抗低温胁迫以及低温滞育相关。本研究为TIME-EA4在农业害虫滞育的分子作用机制研究奠定了基础。     

Twelve nuclear microsatellite loci for rice stem borer (Chilo suppressalis W.)

We developed 12 polymorphic microsatellite markers for the rice stem borer Chilo suppressalis (Walker). These loci were screened for 96 individuals from eight populations across China. The total number of alleles ranged from 3 to 33 and the expected heterozygosity at these loci ranged from 0.131 to 0.671. These molecular markers will be useful for fully investigating the population structure and host preference of C. suppressalis at fine spatial scales.     

Cloning of heat shock protein genes (hsp70, hsc70 and hsp90) and their expression in response to larval diapause and thermal stress in the wheat blossom midge,Sitodiplosis mosellana

Sitodiplosis mosellana Géhin, one of the most important pests of wheat, undergoes obligatory diapause as a larva to survive unfavorable temperature extremes during hot summers and cold winters. To explore the potential roles of heat shock proteins (hsp) in this process, we cloned full-length cDNAs of hsp70, hsc70 and hsp90 from S. mosellana larvae, and examined their expression in response to diapause and short-term temperature stresses. Three hsps included all signature sequences of corresponding protein family and EEVD motifs. They showed high homology to their counterparts in other species, and the phylogenetic analysis of hsp90 was consistent with the known classification of insects. Expression of hsp70 and hsp90 were highly induced by diapause, particularly pronounced during summer and winter. Interestingly, hsp70 was more strongly expressed in summer than in winter whereas hsp90 displayed the opposite pattern. Abundance of hsc70 mRNA was comparable prior to and during diapauses and was highly up-regulated when insects began to enter the stage of post-diapause quiescence. Heat-stressed over-summering larvae (⩾30 °C) or cold-stressed over-wintering larvae (⩽0 °C) could further elevate expression of these three genes, but temperature extremes i.e. as high as 45 °C or as low as −15 °C failed to trigger such expression patterns. Notably, hsp70 was most sensitive to heat stress and hsp90 was most sensitive to cold stress. These results suggested that hsp70 and hsp90 play key roles in diapause maintenance and thermal stress; the former may be more prominent contributor to heat tolerance and the latter for cold tolerance. In contrast, hsc70 most likely is involved in developmental transition from diapause to post-diapause quiescence, and thus may serve as a molecular marker to predict diapause termination.     

Seasonal changes in glycerol content and enzyme activities in overwintering larvae of the Shonai ecotype of the rice stem borer,Chilo suppressalis Walker

Enzymes associated with glycogen metabolism and glycerol synthesis in larvae of the Shonai ecotype of the rice stem borer, Chilo suppressalis, were investigated over the winter in 2000-2001. Glycerol content was scarcely detected in September and October, rapidly increased in November and December, peaked in January, and then decreased. Glycogen was converted to glycerol over the winter until February, and glycerol was reconverted to glycogen in March. The trehalose content remained constant over the winter. The activities of enzymes associated with glycerol synthesis changed with the season. Glycerol accumulation was accomplished by activation of glycogen phosphorylase, inhibition of fructose-1,6-bisphosphatase and pyruvate kinase, and activation of enzymes associated with glycerol synthesis, mainly glyceraldehyde-3-phosphatase and polyol dehydrogenase with glyceraldehyde activity. These changes led to a diversion of triose phosphates into the pathway of glycerol synthesis. However, activities of the two initial enzymes of the hexose monophosphate shunt were not activated and remained relatively constant, but high during the period of active glycerol synthesis. Both decreasing temperature in the field and the transition from the diapause to the post-diapause state may be responsible for the changes in activities of enzymes associated with glycerol synthesis.     

Expression of the maize proteinase inhibitor (mpi) gene in rice plants enhances resistance against the striped stem borer (Chilo suppressalis): effects on larval growth and insect gut proteinases

The maize proteinase inhibitor (mpi) gene was introduced into two elite japonica rice varieties. Both constitutive expression of the mpi gene driven by the maize ubiquitin 1 promoter and wound-inducible expression of the mpi gene driven by its own promoter resulted in the accumulation of MPI protein in the transgenic plants. No effect on plant phenotype was observed in mpi-expressing lines. The stability of transgene expression through successive generations of mpi rice lines (up to the T(4) generation) and the production of functional MPI protein were confirmed. Expression of the mpi gene in rice enhanced resistance to the striped stem borer (Chilo suppressalis), one of the most important pests of rice. In addition, transgenic mpi plants were evaluated in terms of their effects on the growth of C. suppressalis larvae and the insect digestive proteolytic system. An important dose-dependent reduction of larval weight of C. suppressalis larvae fed on mpi rice, compared with larvae fed on untransformed rice plants, was observed. Analysis of the digestive proteolytic activity from the gut of C. suppressalis demonstrated that larvae adapted to mpi transgene expression by increasing the complement of digestive proteolytic activity: the serine and cysteine endoproteinases as well as the exopeptidases leucine aminopeptidase and carboxypeptidases A and B. However, the induction of such proteolytic activity did not prevent the deleterious effects of MPI on larval growth. The introduction of the mpi gene into rice plants can thus be considered as a promising strategy to protect rice plants against striped stem borer.     

二化螟性信息素应用技术:笼罩诱捕器和筒形诱捕器

除目前在我国普遍使用的水盆诱捕器 ,本文还介绍了两种诱捕器———笼罩诱捕器和筒形诱捕器。 3种诱捕器的诱蛾效果比较研究表明 :( 1 )笼罩诱捕器中使用铁纱所制的诱笼的诱蛾量较多 ,同时其锥体的锥角以 60°左右为宜 ;( 2 )筒形诱捕器的诱蛾量略高出笼罩诱捕器 ;( 3) 3种诱捕器中 ,水盆诱捕器的诱蛾效果较好 ,筒形诱捕器次之 ,而笼罩诱捕器较差。     

扬州地区水稻二化螟寄生蜂种类及主要寄生蜂发生动态

本文报道了江苏扬州地区水稻二化螟Chilo suppressalis(Walker)寄生蜂主要有10种,即中华钝唇姬蜂Eriborus sinicus(Holmgren)、二化螟盘绒茧蜂Cotesia chilonis(Munakata)、稻螟小腹茧蜂Microgaster russata Haliday、螟甲腹茧蜂Chelonus munakatae Munakata、中华茧蜂Amyosoma chinensis(Szepligeti)、螟黄足盘绒茧蜂Cotesia flavipes(Cameron)、夹色姬蜂Auberteterus alternecoloratus(Cushman)、桑蟥聚瘤姬蜂Iseropus(Gregopimpla)kuwanae(Viereck)、螟蛉瘤姬蜂Itoplectis naranyae(Ashmead)以及二化螟盘绒茧蜂的重寄生蜂绒茧灿金小蜂Trichomalopsis apanteloctena(Crawford)。田间系统调查表明:扬州地区7-9月水稻二化螟寄生蜂的主要种类为二化螟盘绒茧蜂、中华钝唇姬蜂、螟甲腹茧蜂、稻螟小腹茧蜂、螟蛉瘤姬蜂、桑蟥聚瘤姬蜂,但这些寄生蜂对一代水稻二化螟幼虫的寄生率较低,对二代水稻二化螟幼虫的寄生率高。水稻二化螟越冬期的系统调查表明:扬州地区二化螟越冬幼虫的寄生蜂主要有3种,即二化螟盘绒茧蜂、中华钝唇姬蜂和稻螟小腹茧蜂,其中二化螟盘绒茧蜂和中华钝唇姬蜂是寄生水稻二化螟越冬幼虫的优势种。水稻二化螟越冬幼虫的寄生率调查表明,扬州地区水稻二化螟越冬幼虫的寄生率在不同年份间有一定的波动,但一般都在15%以上,有的甚至高达89.88%,因此寄生蜂对二化螟越冬幼虫具有很好的控制作用,能有效压低来年水稻二化螟的田间越冬基数。此外,3种寄生蜂出茧时间在1月和2月之间达到最长,分别达到19.79 d、17.26 d和27.00 d,该结果表明,在扬州地区这3种寄生蜂可能都存在滞育现象。     

Cloning and nucleotide sequencing of three heat shock protein genes (hsp90, hsc70, and hsp19.5) from the diamondback moth, Plutella xylostella (L.) and their expression in relation to developmental stage and temperature

Heat shock protein genes, hsp90, hsc70, and hsp19.5, were cloned and sequenced from the diamondback moth, Plutella xylostella (L.) by RT-PCR and RACE method. The cDNA sequence analysis of hsp90 and hsp19.5 revealed open reading frames (ORFs) of 2,151 and 522 bp in length, which encode proteins with calculated molecular weights of 82.4 and 19.5 kDa, respectively. Analysis of cDNA from hsc70 revealed an ORF of 1,878 bp coding a protein with a calculated molecular weight of 69.3 kDa. Furthermore, the analysis of genomic DNA from hsc70 confirmed the presence of introns while no introns were apparent in hsp90 and hsp19.5. Southern blot analysis suggested the presence of multiple copies of each gene family in the DBM genome. Detectable expression of hsp19.5 was observed at the pupal stage while expression of hsp90 and hsc70 was detected at both pupal and adult stages. At adult stage, females showed a higher expression of hsp90 and hsc70 than males. An increased expression was observed in all three genes after exposure to a high temperature in both sexes. These results suggest that in addition to a heat shock response, these HSP genes might be involved in other functions during the course of development in DBM.     

Effects of diapause and cold-acclimation on the avoidance of freezing injury in fat body tissue of the rice stem borer, Chilo suppressalis Walker

Overwintering freeze-tolerant larvae of Chilo suppressalis can survive at -25 degrees C, but non-diapausing larvae cannot. We reported earlier that to prevent intracellular freezing, which causes death in overwintering larvae of the Saigoku ecotype distributed in southwestern Japan, water leaves and glycerol enters fat body cells through water channels during freezing. However, it is still unclear how diapause and low-temperature exposure are related to the acquisition of freeze tolerance. We compared the extent of tissue damage, accumulation of glycerol, and transport of glycerol and water in fat body tissues between cold-acclimated and non-acclimated non-diapausing and diapausing larvae. The tissue from cold-acclimated diapausing larvae could survive only when frozen in Grace's insect medium with 0.25 M glycerol at -20 degrees C. The protection provided by glycerol was offset by mercuric chloride, which is a water-channel inhibitor. Fat body tissue isolated from non-acclimated diapausing larvae was injured by freezing even though glycerol was added to the medium, but the level of freezing injury was significantly lower than in non-diapausing larvae. Radiotracer assays in cold-acclimated diapausing larvae showed that during freezing, water left the cells into the medium and glycerol entered the cells from the medium at the same time. Therefore, in Saigoku ecotype larvae of the rice stem borer, both diapause and cold-acclimation are essential to accumulate glycerol and activate aquaporin for the avoidance of freezing injury.     

环链棒束孢hsp90基因的克隆及冷热胁迫下的表达特征分析

通过本地Blast筛选转录组数据库方法,首次克隆了环链棒束孢热休克蛋白90基因全长cDNA序列,命名为Ichsp90（GenBank登录号KT944289）。克隆结果表明,该序列含有2 284个碱基,包括一个含2 097个碱基的开放阅读框,编码699个氨基酸,推测蛋白的分子量为79.23kDa,等电点（pI）为4.86,且含有5个Hsp90家族特征基序和胞质特征序列MEEVD,推导的氨基酸序列与其他丝状真菌相似性在92%-96%之间。用qRT-PCR方法分析了冷热胁迫下,该基因在环链棒束孢中的相对表达情况,结果表明：在4℃冷胁迫下15min检测到Ichsp90表达量下降到最低点,为对照的-1.8倍;随后表达量开始上升,至120min表达量是对照的1.07倍。在39℃高温胁迫下,60min Ichsp90表达量达到最高峰,为对照样品的5.02倍;随后表达量开始下降,至110min为对照样品的2.46倍。因此推测,Ichsp90基因在环链棒束孢抵抗外界温度胁迫中发挥重要的作用。     

Effects of delayed mating and male mating history on longevity and reproductive performance of the rice stem borer, Chilo suppressalis (Walker) (Lep., Pyralidae)

Abstract:  The mating potential, effects of delayed mating and male mating history on longevity and reproductive performance of female rice stem borer (RSB), Chilo suppressalis , were investigated under laboratory conditions. Given the opportunity, RSB males copulated an average of 2.7 times, while females generally mated only once. Females were more severely affected by mating delay than males in terms of female longevity and reproductive performance. With increasing age at mating, females' longevity increased, while oviposition period, fecundity and egg fertility decreased. Mean fecundity and egg fertility of females mated 1 and 7 days after emergence were 251.3% and 99.2%, and 96.2% and 75.5% respectively. Both were reduced significantly when female mating was delayed beyond 4 days after emergence. However, irrespective of the first mating age and different mating history of the males, the results indicated that both did not result in a significant difference in the quality of their contributions to female longevity and reproductive performance. The results obtained in this study are discussed in relation to the potential effect on pheromone control of RSB.     

二化螟Minus-C气味结合蛋白的分子克隆及功能鉴定

气味结合蛋白（odorant binding proteins, OBPs）在昆虫对寄主气味的感受中起重要作用, 但有关Minus-C OBP及其功能的报道很少。本研究通过基因组数据分析并利用RACE技术, 克隆和鉴定了二化螟Chilo suppressalis (Walker)的一个Minus-C OBP基因, 命名为CsupOBP1（GenBank登录号： KC492498）。CsupOBP1基因的开放阅读框长423 bp, 编码141个氨基酸, 其中N端的18个氨基酸为预测的信号肽序列, 成熟蛋白序列中具有4个保守的半胱氨酸位点。实时定量PCR分析显示, 该基因在幼虫头部及成虫雌雄足、 翅和雄性触角等化感组织中高表达, 其中在雄虫触角内的表达量显著高于雌虫触角。利用荧光竞争结合实验对CsupOBP1重组蛋白与38种化合物的结合特性的测定表明, 重组CsupOBP1与β 紫罗兰酮的结合能力最强（Ki=9.53 μmol/L）。触角电位测定表明, 二化螟成虫可对β-紫罗兰酮产生显著的触角电生理反应, 但雄虫反应明显强于雌虫, 与结合试验及雄虫触角中CsupOBP1的表达量显著高于雌虫触角的测定结果相一致。鉴于β-紫罗兰酮是水稻等植物中普遍存在的一种芳香气味组分, 推测CsupOBP1可能通过对该气味的结合和运输, 从而在二化螟对寄主植物的嗅觉定向中起作用。     

Role of membrane transport of water and glycerol in the freeze tolerance of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae)

Overwintering larvae of the rice stem borer, Chilo suppressalis accumulate glycerol and are freezing tolerant to about -25 degrees C. However, non-diapausing larvae cannot accumulate glycerol and are killed by freezing. We compared the extent of tissue damage, the effects of glycerol concentration, and the transport of glycerol and water in fat body tissues from these larvae at selected freezing temperatures. Tissues from overwintering larvae, but not non-diapausing larvae, survive when frozen at -20 degrees C with 0.25 M glycerol, but the protection afforded by glycerol is offset by the water-channel inhibitor mercuric chloride. Glycerol in higher concentration (0.75 M) affords some protection even to the fat body of non-diapausing larvae. Radiotracer assays of overwintering larvae show that water leaves the tissues during freezing while glycerol enters, and that mercuric chloride disrupts this process. Transport is also disrupted after lethal freezing at -35 degrees C. Therefore, membrane transport of water and glycerol is involved in the avoidance of freezing injury to fat body cells of the rice stem borer, apparently by mediating the replacement of water with glycerol in freezing-tolerant tissues.     

Over-expression of a small heat shock protein,sHSP17.7, confers both heat tolerance and UV-B resistance to rice plants

Exposure of rice (Oryza sativa L.) seedlings to a high temperature (42°C) for 24 h resulted in a significant increase in resistance to UV-B damage. UV-B resistance was enhanced in parallel with the period of heat treatment. sHSP17.7 was isolated from heated rice seedlings, and the influence of rice sHSP17.7 expression on the viability of E. coli under heat-shock conditions was assessed. After heating, the survival rate of sHSP17.7 cells was 2-fold higher than that of the control cells. The molecular chaperone activity of sHSP17.7 was investigated using catalase as a substrate. Recombinant sHSP17.7 had heat-stable chaperone properties that were capable of protecting stressed catalase from precipitation. sHSP17.7 was overexpressed in the rice cultivar Hoshinoyume, by Agrobacterium-mediated transformation, under the control of a CaMV 35S promoter. Transgenic rice plants with increased levels of sHSP17.7 protein exhibited significantly increased thermotolerance compared to untransformed control plants. The level of increased thermotolerance was correlated with the level of increased sHSP17.7 protein in the transgenic plants. The transgenic rice plant with the highest constitutive expression of sHSP17.7 had significantly greater resistance to UV-B stress than untransformed control plants. Increase in the degree of resistance of transgenic plants to UV-B was accompanied by an increase in production of sHSP17.7 protein.     

Effects of Bt transgenic rice line on stripe stem borer,Chilo suppressalis and its consequences on egg parasitoid,Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae) in the laboratory

Interactions among Chilo suppressalis (Walker), its larval parasitoid Trichogramma brassicae Bezdenko and insect-resistant transgenic rice lines were evaluated under laboratory conditions. The results showed that all neonates and second larvae of stem borer were dead regardless of being fed rotationally or permanently on Bt rice, but 18% and 28% of the third and fourth instar larvae could complete development and turned to adults, respectively, when fed rotationally. In the second year of project, third instar larvae of stem borer were used to evaluate Bt rice impact on T. brassicae. The result showed that emergence rate was lower, the abnormal female rate increased and adult female longevity of the wasp decreased significantly in comparison with the conventional variety. There was no significant difference in gender ratio and parasitism rate under Bt rice treatment in comparison with conventional variety.     

Cloning and expression of heat shock protein genes in two whitefly species in response to thermal stress

Two whitefly species, Trialeurodes vaporariorum and Bemisia tabaci biotype B were shown to have different temperature tolerance and seasonal dynamics. To determine whether this variation in thermal tolerance is related to different expression patterns of heat shock protein (hsp) genes during temperature stress, we obtained complete cDNA sequences for hsp90, hsp70 and hsp20, and analysed their expression profiles across temperature gradients by real‐time quantitative polymerase chain reaction (PCR). Six full‐length cDNAs were cloned and sequenced from these two species. The full‐length cDNAs of hsp90s contain 2166 and 2157 bp open‐reading frames (ORF) which encode proteins with calculated molecular weights of 83 013 and 82 857 Da in T. vaporariorum and B. tabaci, respectively. The 1947 and 1959 bp ORFs of whitefly hsp70s comprise 649 and 653 amino acids with the calculated masses of 70 885 and 71 008 Da in T. vaporariorum and B. tabaci, respectively. Both complete cDNAs of hsp20 of T. vaporariorum and B. tabaci contain 585 bp ORFs and deduced amino acid sequences had molecular weights of 21 559 and 21 539 Da, respectively. The hsp expression profile results showed that temperatures for onset (T_on) or maximal (T_max) induction of hsp expression in T. vaporariorum were generally 2–6°C lower than those in B. tabaci. These results suggest that the T_on (or T_max) of hsps can represent the differences in temperature tolerance of these two whitefly species, and may be used to determine their natural geographical distribution and natural population seasonal dynamics. Significant upregulation of most hsps were observed when temperature stress was lifted, except that hsp70 and hsp20 of B. tabaci did not respond to the cold stress, indicating that response to heat and cold stress may have a different genetic and physiological basis in two whitefly species. These results highlight the importance of understanding the complexity of the heat shock response across multiple isoforms while attempting to link them to whole‐organism traits such as thermal tolerance.     

谢氏宽漠王HSP70基因cDNA片段的克隆及热激条件下的表达

谢氏宽漠王Mantichorula semenowi Reitter是一种沙漠指示性甲虫。本研究由该种甲虫体内克隆到两种不同的HSP70基因片段,分别为MsHSP70和MsHSC70。同源性发现表明这两个基因片段与已报道的其他昆虫的热休克蛋白核苷酸序列高度同源。半定量RT-PCR分析显示：经42℃热激1 h 后立即诱导MsHSP70表达至最高峰;在恢复到室温的1～4 h 内MsHSP70表达量逐渐降低,但仍然高于未热激对照组。而MsHSC70在42℃热激1 h后表达受到抑制,但在恢复2 h和4 h时有少量的表达,分别仅为未热激对照组的0.25和0.28倍。结果提示MsHSP70和MsHSC70在保护细胞方面具有不同的作用。本实验结果为谢氏宽漠王在极端的沙漠环境胁迫下的抗逆适应性研究提供了理论基础。     

热应激和吡虫啉对大豆蚜hsp70和hsc70基因mRNA表达的影响

【目的】昆虫在高温或农药的胁迫下,通过高效表达热休克蛋白(HSP)等建立应激自我保护机制。本研究为从转录组水平上认识大豆蚜Aphis glycines在热应激和吡虫啉胁迫下hsp70和hsc70 mRNA表达分子机制,进而寻找自我保护应激反应中的薄弱环节,为大豆蚜的生物防治提供理论基础。【方法】采用同源克隆、RACE技术和实时荧光定量PCR等方法研究不同热激时间和热激后不同恢复时间及不同吡虫啉浓度对大豆蚜4龄若虫hsp70和hsc70的表达影响。【结果】37℃热激后,大豆蚜4龄若虫中hsp70表达量先上调,1 h时升至对照组的10.36倍（P<0.05）,然后逐渐下降。同样热激后恢复时间的长短对大豆蚜若蚜中hsp70的表达具有显著影响。热激处理后,大豆蚜若蚜中hsp70立即大量表达,表达量为对照组的8.78倍（P<0.05）,随后表达量下降至对照组水平,而hsc70的表达量并没有显著变化（P>0.05）。大豆蚜若蚜受吡虫啉的胁迫时,其hsp70和hsc70的表达量受吡虫啉的浓度及胁迫的时间的影响,呈现先升高后下降的趋势,具有明显的短期效应。【结论】吡虫啉诱导大豆蚜hsp70和hsc70表达量的上调;而热胁迫对hsp70和hsc70 mRNA具有不同的表达模式,高温可以诱导hsp70的表达,但对hsc70没有明显的诱导作用。