摘要
麦长管蚜翅型具有二型现象,有翅型和无翅型之分。翅型分化是一个受多因子影响的调控体系决定的,二型的比例随内因和外界环境的变化而变化。因此利用分子生物学手段从基因水平上开展蚜虫翅型分化的机理研究,对丰富麦蚜基础生物学具有重要意义,并为今后基于蚜虫翅型分化调控研发新的蚜虫防控措施奠定理论基础。
本文以麦长管蚜翅型分化相关基因Cdc42为对象,采用原位杂交技术和实时荧光定量RT-PCR技术确定该基因在麦长管蚜中的组织定位及相对表达量。并通过体外涂抹保幼激素及其类似物测定其对麦长管蚜的翅型分化的调控作用。取得如下研究结果:
采用原位杂交技术,对Cdc42基因在二型麦长管蚜中的分布进行组织定位,结果表明在滴加杂交液的组织切片被特异性的染成棕褐色,而阴性对照的组织切片没有被染色。由此表明原位杂交技术可以灵敏检测到Cdc42基因在该蚜虫不同组织中的表达状况。原位杂交结果显示,Cdc42基因在有翅蚜和无翅蚜的头部和腹部均有表达,头部的表达集中在两复眼之间,腹部的表达几乎全集中在伪胚胎;有翅蚜的胸部几乎全部都有表达,根据着色点数量推断有翅蚜的胸部表达量最高;而无翅蚜的胸部几乎没有表达。
再运用实时荧光定量RT-PCR技术确定该基因的相对表达量,结果表明Cdc42基因在麦长管蚜不同组织及不同龄期均有表达,若蚜期高于成蚜期,有翅蚜高于无翅蚜,该基因在有翅蚜与无翅蚜胸部的表达成显著差异;从伪胚胎开始就有较高的转录水平。
对2龄若蚜经体外涂抹保幼激素JHⅢ及其类似物ZR-515的滴度效益结果表明,随着激素浓度的增加,无翅和有翅麦长管蚜成虫整虫Cdc42相对表达量逐渐减低,均低于正常有翅蚜的表达量,无翅蚜中的表达量只有正常表达量的千分之一,各浓度之间差异极显著;并且无翅型麦长管蚜成虫的个体数量不断增加,有翅蚜数量逐渐减少,各浓度之间差异极显著。
本研究为探讨建立微型昆虫基因的准确组织定位和定量分析方法奠定了基础,也为深入开展保幼激素、Cdc42基因与翅型分化的关系提供了理论依据。
The English grain aphid, Sitobion avenae, is of wing dimorphism, namely, winged and wingless aphid. The aphid’s wing differentiation is manipulated by a complicate factors system, and the ratio of the wing dimorphism depends on the intrinsic and external environment factors. So in order to uncover the mechanism of aphid’s wing differentiation, through the related genes expression level by molecular biology approach, which will be very important to enrich the basical biology of aphid, and also benefit to further develop new method to control the aphid based on the knowledge of the manipulation of aphid’s wing differentiation.
In this experiment thesis, Cdc42 gene tissue location and accurate relative expression levels of S. avenae were determined by using in situ hybridization (ISH) and real-time fluorescence quantitative PCR method to determine the Cdc42 gene in the location and relative expression levels of the organization in Sitobion avenae. Then the change dynamic of Cdc42 gene expression, and population and the ratio of alatae and apterae aphid were detected with dose variation of juvenile hormone (JH) and its analogues by smearing 2 instar nymphae aphid. The aim is to understand preliminarily for the interaction of JH in vitro, the Cdc42 gene expression, and the function of manipulation of wheat aphid’s wing dimorphism. The major results are as follows:
First, ISH results showed that drip liquid hybridization specificity of tissue biopsies were specific stained in brown with nucleic acid probe of Cdc42 gene, while without the same staining characteristic on the negative control, which indicated that ISH could be sensitive to identify the tissue locaton of the gene in the aphid. ISH results showed that Cdc42 expressed in the head and abdomen parts of the aphid, and focused between the two complicated eyes by aphid’s head.In the abdomen the gene expressions were almost concentrated the pseudo-embryos. According to the brown staining. The Cdc42 gene specific stainging spread all over on the alate thorax part, and it was deduced by the number staining dot, that the highest expression level occurred on the thorax part of alate aphids, but hardly not expressed on the thorax part of apterae aphid.
Tissue and stage specific transcriptive levels were determined by real-time fluorescence quantitative PCR and showed that Cdc42 was transcripted in multiples tissues and at different stages. There was higher transcriptive level in the nymphae stages than that in adult stage, and higher in alate aphid than that of apterous one, and significantly higher on the thorax part of alate than that in apterae aphid. The gene’s expression start up at the aphid’s pseudo-embryo stage, and even with very high expressed level in the stage.
Application juvenile hormone (JH) and its analogues ZR-515 on aphid’s thorax in vitro by three concentrations, to study the impact of JH dose on the expression of Cdc42 gene and wing differentiation, the results showed that with the dose increasing of JHⅢand its analogues ZR-515, the expression level of Cdc42 gene in the whole part of alate and apterae aphid decreasing gradually, which were significant lower in the three treated concentration than that in control of alate aphid, by real-time fluorescence quantitative PCR detection, and the expression level of Cdc42 in the treated apterae aphid was only one thousandth of the level compared with aphid in control.There were also significant difference among the different treatments. The number of apterae adults increasing, but the number of alate aphids decreasing under the JH and ZR-515 treatments.
In this thesis, a micro-insect gene tissue localization and quantitative analysis, method has been established, and the results provide a primary data for the interaction of JH, Cdc42 gene and wing differentiation in aphid.
引文
1.曹敏.禾谷缢管蚜翅型分化相关基因的cDNA克隆及分析.硕士学位论文.北京:中国农业大学,2004.
2.陈小芬,杨玉荣.原位杂交检测hsp70 mRNA在黑腹果蝇胚胎中的表达[J].厦门大学学报(自然科学版), 2008, 59~64.
3.戴华国,吴晓毅.褐飞虱体内保幼激素滴度变化及其与翅型分化的关系[J].昆虫学报,2001,44(1): 27~32.
4.丁格[美]编著,巫国瑞译著.昆虫迁飞和滞育的进化.科学出版社.北京. 1984, 74~85.
5.杜建光,王群,程遐年.褐飞虱翅型分化的组织学研究[J].昆虫学报,1997,Vol.40, Suppl: 135~138.
6.冯永淼,闫素梅.利用实时荧光定量RT-PCR测定肉鸡组织中CaBp基因的相对表达.[J].黑龙江畜牧兽医,2007,8:27~30.
7.高书晶,庞保平,周晓榕,董奇彪.麦田昆虫群落结构及多样性的季节动态[J].昆虫知识,2006,43(3):295~299.
8.韩俊英,曾瑞萍.荧光定量PCR技术及其应用[J].国外医学,遗传学分册, 2000, 23 (3): 117~120.
9.蒋春燕,王泰健,王琴.实时荧光定量PCR技术[J].动物医学进展, 2005, 26 (12): 97~101.
10.蒋红玲.麦长管蚜G蛋白β、γ亚基的基因克隆及相互作用蛋白的筛选.博士学位论文.北京:中国农业科学院,2004.
11.李红梅.麦蚜迁飞的生理生化特性研究.硕士学位论文.北京:中国农业科学院,2002.
12.李红梅,程登发,田喆,孙京瑞.禾谷缢管蚜体内保幼激素的变化趋势[J].植物保护学报,2004,31(4):371~376.
13.刘学英.禾谷缢管蚜地理种群及翅型分化分子机制的初步研究.硕士学位论文.北京:中国农业科学院,2002.
14.刘光杰.褐飞虱成虫翅型分化研究[J].昆虫知识,2000,37(3):186~190.
15.刘树生,吴晓晶.温度对桃蚜和萝卜蚜翅型分化的影响[J].昆虫学报,1994,37(3):292~297.
16.吕利华.大豆蚜有翅蚜产生的原因[J].昆虫学报,1993,36(2):143~148.
17.陆良勇,黄伟达,刘尚廉.应用RNA原位核酸杂交检测乳腺癌石蜡切片中ER mRNA、PRmRNA表达[M].中国组织化学与细胞化学杂志,2002,3,11(1):92~97.
18.马巨法,唐健,胡国.稻褐飞虱翅二型现象[J].昆虫知识.1995,467~469.
19.马蕊.麦蚜唾液酶诱导小麦防御反应机制研究.硕士学位论文.北京:中国农业科学院,2009.
20.任立群,赵志涛,孙波.石蜡包埋组织切片原位核酸杂交研究[J].白求恩医科大学学报,2001,27(3):235~236.
21.舒朝忠,罗进勇.实时逆转录PCR[J].国外医学.临床生物化学与检验学分册. 2001,22(5): 249~251.
22.宋敏,胡建民,何孔旺.PCR定量方法概述.上海畜牧兽医通讯,2005,2:18~19.
23.苏建家.原位杂交原理及操作方法的体会[J].广西医学院学报. 1992, 3(9): 86~87.
24.孙梅,刘红林.原位杂交技术及其在动物基因定位上的应用进展[M].黄牛杂志. 2000, 6(26):39~43.
25.藤崎宪治.昆虫におけな分散性多型性の适应的意义と进化.冈山大农学报(日), 1994, 83: 113~132.
26.巫国瑞,黄次伟,陶林勇,冯炳灿,陈福云,等.影响褐飞虱猖獗和为害的因素[J].生态学报,1984,4(2):157~165.
27.王冬燕,审小卫,郭线茹.小麦生长中后期2种麦蚜垂直分布机生态位的研究[J].河南农业科学,2006,10:56~58.
28.王健,吴振延.外源激素对褐飞虱翅型分化的影响[J].昆虫学报,1998,41(4):371~374.
29.王凯,王海燕,李宕.蚜虫翅型分化研究进展[J].安徽农业科学,2008,36(20):8671~8672,8748.
30.王群,杜建光.褐飞虱翅型分化遗传规律的研究[J].昆虫学报,1997,40(4):343~347.
31.王荣富,程遐年.稻飞虱翅型分化的调节控制[J].安徽农业大学学报,1993,23(4):496~499.
32.杨益众.影响禾谷缢管蚜翅型分化的因子初探[J].江苏农业研究.2000,21(1):56~59.
33.阳成波,印遇龙,龚建华.实时定量PCR研究进展及其应用[J],中国预防兽医学报,2003,25(5):395.
34.俞晓平,吕仲贤,巫国瑞,陶林勇,张志涛.褐飞虱的前飞翅型分化的研究[J].昆虫学报,1997,Vol.40,Suppl:128~133.
35.于国龙.实时荧光定量PCR在医学遗传学方面的应用[J].国外医学.遗传学分册. 2003, 26(3): 125~129.
36.张蓓.实时荧光定量PCR的研究进展及其应用[J].国外医学.临床生物化学与检验学分册. 2003,24(6): 327~329.
37.张晶,张惠文,张成刚.实时荧光定量PCR及其在微生物生态学中的应用[J].生态学报, 2005, 25(6): 1445~1450.
38.张广学,钟铁森.中国经济昆虫志,第二十五册同翅目蚜虫类(一).北京:科技出版社,1983.
39.赵惠燕,汪世泽.孤雌胎生棉蚜胚胎学观察[J].昆虫知识,1992,1:19~21.
40.邹运鼎,陈基诚,王士槐.稻株营养物质与褐飞虱翅型分化的关系[J].昆虫学报,1982,25(2): 220~222.
41. Adams A E, Johnson D I, Longnecker R M, Sloat B F, Pringle J R. Cdc42 and Cdc43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae [J]. Cell Biology.1990; 111(1):131~42.
42. Alexandra R K, Stella Fanok. Cell Culture-Taqman PCR Assay for Evaluation of Cryptosporidium parvum Disinfection [J]. Applied and Environmental Microbiology. 2003, 69: 2505-2511.
43. Ardue M L. In situ Hybridization.In: Hames B D & Higgins S J, et al. Nucleicaeid. Hybridization, A practica approach.Oxford: IRL press.1985:170.
44. Bao W, Thullberg M, Zhang H, Onischenko A, Stromblad S. Cell attachment to the extracellular matrix induces proteasomal degradation of p21(CIP1)via Cdc42/Rac1 signaling [J].Molecular Cell Biology. 2002, 22(13): 4587~97.
45. Barro P J. The role of temperature, photoperiod, crowding and plant quality on the production of alate viviparous females of the bird cherry-oat aphid, Rhopalosiphum padi. EntomologiaExperimentalis et Applicata.1992, 65(3): 205~214.
46. Braendle C, Davis G K, Brisson J A, Stern D L. Wing dimorphism in aphids.Heredity. 2006, 97, 192~199.
47. Bulter K, Zorn A M, Gurdon J B. Nonradioactive in situ hybridization to Xenopus tissue sections.Methods, 2001, 23:303~312.
48. Bubner B et al., Use of real-time PCR for determining copy number and zygosity in transgenic plants. Plant Cell Reports, 2004, 23: p. 263~271.
49. Buongiorno-Nardelli M, Amaldi F. Autoradiographic detection of molecular hybrids between rRNA and DNA in tissue sections [J].Nature, 1969, 225: 946~947.
50. Carter K C,Tanejia K L,Lawrenee J B. Diserete nuelear domains of poly(A) RNA and their relationship to the functional organization of the nueleus[J]. Cell Biology, 1991, 115:1191~1202.
51. Christiansen-Weniger P and Hardie J. Wing development in parasitized male and female Sitobion fragariae [J]. Physiological Entomology, 1998. 23(3): p. 208~213.
52. Chuang T H,Hahn K M,Lee J D.The small GTPasea Cdc42 initiates an apoptotic signaling pathway in Jurkat T lymphocytes[J]. Molecular Cell Biology, 1997, 8(9):1687~1689.
53. Cloutier C, Perron J M. Polymorphism and sensitivity to a juvenile hormone analogue in the potato aphid, Macrosiphum euphorbiae (Homoptera: Aphididae). Entomologia Experimentalis et Applicata. 1975, 18:457~464.
54. Correia K M, Conlon R A.Whole-mount in situ hybridization to mouse embryos.Methods, 2001,
23:335~338.
55. Dirks R W, Daniel K C, Raap A K.RNAs radiate from gene to cytoplasm as revealed by fluorescence in situ hybridization [J].Cell.Science, 1995, 108:2565~2572.
56. Dixon A F G. Biology of Aphids.Edward Arnold, 1973, 5~16.
57. Dixon A F G. Aphid Ecology. Chapman & Hall, London, 1998.
58. Dixon A F G, Agarwala BK. Ladybird-induced life-history changes in aphids. Proceedings of the Royal Society London Series B, 1999, 266, 1549~1553.
59. Ecott L H.Methodological considerations in the utilization of in situ hybridization. In:Valentino K,Ebetwine J,Barchas J. In situ hybridization. Application to neurobiology. New York:Oxford University press,1986:3.
60. Einspanier R, Lutz B, Rief S.Tracing residual recombinant feed molecules during digestion and rumen bacterial diversity in cattle fed transgene maize.European Food Research and Technology, 2004, 218(3): 269~273.
61. Elellis R A.New techniques in gene product analysis.Arch pathol Lab Med,1987,111:620
62. Erickson J W, Cerione R A.Multiple roles for Cdc42 in cell regulation. Current Opinion in Cell Biology.2001; 13(2):153~157.
63. Gall G, Pardue M L. Formation and detection of RNA DNA hybrid molecules in cytological preparations. Proc Nat Acad Science USA.1969,63:378~381.
64. Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R C M. An overview of real-timequantitative PCR: applications to quantify cytokine gene expression. Methods.2001, 25: 386~401.
65. Hardie J. Juvenile hormone mimics the photoperiodic apterization of the alate gynopara and virginopara of the aphid. Aphid fabae. Physiol. Entomology. 1980. 385~396.
66. Hardie J. Juvenile hormone and photoperiodically controlled polymorphism in Aphids Fabae: prenatal effects on presumptive oviparae. Insect physiol. 1981,Vol. 27, No.4:257~265.
67. Hardie J. Juvenile hormone and photoperiodically controlled polymorphism in Aphids Fabae: postnateal effects on presurnptime Gynoparae. Insect physiol. 1981,Vol. 27, No.5:347~355.
68. Hales D F. Juvenile hormone and aphid polymorphism. In: Phase and caste determination in insects. Endocrine Aspects (Ed. by Luscher M), Pergamon Press, Oxford, 1976, pp: 105~115.
69. Hart M J,Shinjo K,Hall A,Evans T, Cerione R A.Identification of the human Platelet GTPase activating protein for the CDC42Hs protein[J]. Biological Chemistry. 1991:266(31):20840~20848.
70. Harden N, Lee J, Loh H Y, Ong Y M, Tan I, Leung T, Manser E, and Lim L A. Drosophila homolog of the Rac- and Cdc42-activated serine/threonine kinase PAK is a potential focal adhesion and focal complex protein that colocalizes with dynamic actin structures [J]. Molecular Cell Biology, 1996; 16: 1896~1908.
71. Harrewijn P.Aphids,Their Biology,Natural Enemies and Control,1987:255~266.
72. Heid C A, Stevens J, Livak K J. Real time quantitative PCR [J]. Genome Research. 1996, 6(10): 986-994.
73. Hille R, Lambers D. Polymorphism in Ahididae. A. Rev. Entomology. 1966, 11, 47~48.
74. Huang S, Deerinek T J, Elliaman M H,Spector D L.In vivo analysis of the stability and transport of nuelear poly(A)RNA [J].Cell Biology,1994.126:877~899.
75. Huang S, Speetor D L.Intron-dependent recruitment of pre-mRNA splicing factors to sites of transcription [J]. Cell Biology, 1996. 133:719~732.
76. Iwanaga K and Tojo S. Effects of juvenile hormone and rearing density on wing dimorphism and o?cyte development in the brown planthopper, Nilaparvata lugens [J]. Journal of Insect Physiology, 1986. 32(6): p. 585~590.
77. James C K, Keith L P. Transmission of plant viruses by aphid vectors [J]. Molecular Plant Pathology. 2004, 5(5): 505~511.
78. Jimenez-Gareia, Speetor D J.In vivo evidence that transcription and splicing are coordinated by a reeruiting mechanism.Cell, 1993.73:47~59.
79. Johnson B. Wing polymorphism in aphids II. Interaction between aphids. Entomologia Experimentalis et Applicata, 1965, 8, 49~64.
80. Johnson B. Wing polymorphism in aphids III. The influence of the host plant Entomologia Experimentalis et Applicata, 1966a, 9, 213~222.
81. Johnson B. Wing polymorphism in aphids IV. The effect of temperature and photoperiod. Entomologia Experimentalis et Applicata, 1966b, 9, 301~313.
82. Johnson D I. Cdc42: An essential Rho-type GTPase controlling eukaryotic cell polarity. Microbiol and Molecular Biology Reviews. 1999, 63(1):54~105.
83. Kawada K.Polymorphism andmorph determination[C]//Minks A K.
84. Kidd NAC, Tozer DJ. Host plant and crowding effects in the induction of alatae in the large pine aphid. Cinara pinea. Entomology. Appl, 1984, 35:37~42.
85. Kisimoto R. Effect of crowding during the larval period on the detemination of the wing-form of an adult plant-thopper. Nature (London). 1956,178:641~642.
86. Kennedy J S, Stroyan H L G. Biology of Aphids. Annual Review of Entomology. 1959,4, 139~160.
87. Kuner G, Weisser W W.The interplay between density-and trait-mediated effects in predator-prey interactions: a case study in aphid wing polymorphism. Oecologia. 2003, 135, 304~312.
88. Lamb K P,white D F. Effect of temperature,stawation and crowding on the production of alate young by the cabbage aphid(Brevicoryne brassicae). Entomology,exp. Appl,1966,9:174~184.
89. Latham V M, Kislwaskis E H, Singer R H, Ross A F.Beta-actin mRNA localization is regulated by signal transduction mechanisma [J].Cell Biology, 1994, 126:1211~1219.
90. Lees A D. Clonal polymorphism in aphids. In Insect Polymorphism (Ed.by Kennedy J S). Symposium NO.1.Royal Entomological Society of London. 1961, 68~78.
91. Lees A D. The control of polymophism in apids. Adv. Insect Physiology, 1966, 3, 207~277.
92. Lehmann U. Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods, 2001. 25(4): p. 409~418.
93. Livak K J. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods, 2001. 25(4): p. 402~408.
94. Luo Li qun, Lee A. Genghis Khan (Gek) as a putative effector for Drosophila Cdc42 and regulator of actin polymerization. Proc Natl Acad Sci U S A. 1997, 94 (24): 12963~12968.
95. Malumbres M, Pellicer A.Genomic analysis of metastasis reveals an essential role for RhoC [J]. http://www.bioscience.org./1998/v3/d/malumbre/d887~912. Htm.
96. Marcus J M. The development and evolution of crossveins in insect wings [J]. Anatomy. 2001, 199(Pt 1-2):211~216.
97. Moonen P, Boonstra J, Van D.Validation of a Light Cycler-based reverse transcription polymerase chain reaction for the detection of foot-and-mouth disease virus[J].Virol Methods, 2003, 113(1): 35~41.
98. Morris R G, Arends M J, Bishop P E. Sensitivity of digoxigenin and biotin labeled probes for detection of human papilloma virus by in situ hybridization [J]. Clin Pathol, 1990, 43:800~805.
99. Muller C B,Willams I S,Hardie J. The role of nutrition,crowding and interspecific interactions in the development of winged aphids. Ecological Entomol, 2001(26): 330~340.
100. Nijhout F I, Wheeler D E. Juvenile hormone and the physiological basis of insect polymorphism [Q]. Rer. Biology., 1982, 57: 109~133.
101. Riddiford L M.Cellular and molecular actions of juvenile hormone.I.General considerations and prementamorphic actions.Andvances in Insect Physiology. 1994, 24:213~274.
102. Schwartzberg Z G, Kunert G, Westerlund S A, Hoffmann K H and Weisser W W. Juvenile hormone titres and winged offsping production do not correlate in the pea aphid, Acyrthosiphon pisum[J].Journal of Insevt Physiology. 2008, 54:1332~1336.
103. Singer R H.Toward a rapid and sensitive in situ hybridization methodology using isotopic and non-isotopic probe.In:Valentino K,Ebefwine J,Barehas FJ,eds.In situ hybridization:Application to neurobiology.New York:Oxford University press,1986:71.
104. Sloggett J, Weisser W. Parasitoids induce production of the dispersal morph in the pea aphid, Acyrthosiphon pisum. OIKOS. 2002, 98, 323~333.
105. Smith M, Mackay P A.Genetic variation in male alary dimorphism in populations of pea aphid, Acyrthosiphon pisum. Entomologia Experimentaliset Applicata, 1989(51):125~132.
106. Southwood T R E. A hormonal theory of the mechanism of wing polymorphism in heteroptera. Proceedings of the Royal Entomological Society of London. Series A, General Entomology, 1961. 36(4-6): p. 63~66.
107. Sundell C, Singer R H. Requirement of mierofilmaets in sorting of actin messenger RNA [J]. Science, 1991.253:1275~1277.
108. Sutherland O R W. The role of the host plant in the prodction of winged forms by two strains of the pea aphid. Acyrthosiphon pisum [J]. Insect Physiology,1969, 15:2179~2201.
109. Sutherland O R W, Mittler T E. Influence of diet composition and crowding on wing production by the aphid Myzus persicae [J]. Journal of Insect Physiology, 1971. 17(2): p. 321~328.
110. Tauz D, Pfeifle C. A non-radioactive in situ hybridization method for the localization of specific FRNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma.1989.98:81~85.
111. Tobe S S,Ruegg R P,Stay B B,Baker F C,Miller C A, Schooley D A.Juvenile titer and regulation in the cockroach Diploptera punctata.Experientia.1985,41: 1028~1034.
112. Tsuji H,Kawada K. Development and degeneration of wing buds and indirect flight muscles in the pea aphid (Acyrtosiphon pisum (Harris)). Jap J Appl Entomol Zool, 1987(31): 247~252.
113. Van Aelst L, D’Souzar-Schorey C. Rho GTPases and signaling networks. Genes Dev, 1997, 11(18):2295~2322.
114. Walker M J.Real time and quantitative PCR applications to mechanism based toxicology [J]. Biochem Mol Toxicol, 2001, 15(3):121.
115. Watt A D,Dixon A F G. The role of cereal gonth stages and crowding in the induction of alatae in sitobion averac and comsequences for population growth. Ecol, 1981, Entl: 441~447.
116. Weis-White W S. The environmental conditions affecting the genetic mechanisms of wing production in the chrysanthemum aphid. Am. Nat,1984, 80:245~70.
117. Winer J, Jung C K S, Shackel I. Development and Validation of Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction for Monitoring Gene Expression in Cardiac Myocytesin Vitro. Analytical Biochemistry. 1999, 270(1): 41~49.
118. Wigglestorth. Insect polymorphism-a tentative synthesis. In: Kennedy J S (Ed.) Insect Polymorphism. Royal Entomological Society, London. 1961, 103~113.
119. White W S. The environmental conditions affecting the genetic mechanisms of wing production inthe chrysanthemum aphid. Am. Nat, 1981, 80: 245~270.
120. Xing Y, Johnoson C V, Dobner P R, Lawrence J B.Higher level organization of individual gene transeription and RNA splicing: Integration of nuelear strueture and function.Science, 1993, 259:1326~1330.
121. Yamada S. Dual Wing-form Determination Mechanism in the Brown Planthopper, Nilaparvata lugens STOL (Homoptera: Delphacidae). Applied entomology and zoology, 1991, 26(4), 590~592.
122. Yerle M, Pinton P A. Construction of a whole-genome radiation hybrid panel for high-recolution gene mapping in pigs [J]. Cytogenet Cell Genet, 1998. 82: 182~188.
123. Zera A J, Denno R F. Physiology and ecology of dispersal polymorphism in insects. Annual Review of Entomology. 1997, 42, 207~231.
124. Zera A J, Strambi C, Tiebel K C, Strambi A, Rankin M. Juvenile-hormoneand ecdysteroid titers during critical periods of wing morph determination in Gryllus rubens. Journal of Insect Physiology. 1989, 35, 501~511.