甜瓜CmLOX09对逆境胁迫、激素和信号物质的响应及对番茄的遗传转化

发布时间：2018-01-07 08:36

  本文关键词：甜瓜CmLOX09对逆境胁迫、激素和信号物质的响应及对番茄的遗传转化　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 薄皮甜瓜 9-LOX 氢过氧化物裂解酶(HPL) 丙二烯氧化物合酶(AOS) 绿叶挥发物(GLVs) 茉莉酸(JA)

【摘要】：脂氧合酶(lipoxygenase,LOX)是植物体内十八碳酸代谢途径中的关键酶,催化脂类和多不饱和脂肪酸形成具有一定活性的产物,广泛参与植物生长发育、成熟衰老和香气物质的合成,并参与到逆境胁迫的防御反应中。LOX通过催化含有(Z,Z)-1,4-戊二烯结构的多不饱和脂肪酸进一步形成氢过氧化物(HPOD),这些氢过氧化物为LOX下游7条途径提供底物并进一步催化形成具有活性的化合物,统称为氧脂素。目前LOX途径中的氢过氧化物裂解酶(HPL)和丙二烯氧化物合酶(AOS)两条支路已经被证实能够参与到植物逆境的防御反应中。前期研究表明:5个甜瓜13-LOXs基因能够响应非生物胁迫及信号类物质的处理,但尚不明确9-LOX是否参与对生物和非生物胁迫的防御反应中,对CmLOX09的启动子分析显示其含有对逆境、激素和信号类物质的响应元件。因此,本文以4叶1心的薄皮甜瓜幼苗为试材,研究了机械损伤;激素:生长素(IAA)和赤霉素(GA3);信号类物质:脱落酸(ABA)、水杨酸(SA)、过氧化氢(H202)以及茉莉酸甲酯(MeJA);生物胁迫:接种白粉病菌等胁迫处理对CmLOX09及其下游CmHPL和CmAOS表达量的影响,此外,测定了 MeJA处理后甜瓜叶片中绿叶挥发物(GLVs)含量的变化以及接种白粉病菌后不同发病级别甜瓜叶片中茉莉酸(JA)含量的变化。主要结果如下:1.利用Plant CARE软件对CmLOX09启动子分析预测显示:其中含有多种响应元件如光响应元件(3-AF1 binding site、Box 4、ACE、AT1-motif 等)、激素响应元件(p-box element)、信号类物质响应元件(ABRE element和TCA-element)、真菌胁迫响应元件(Box-W1 element)和逆境响应元件(TC-rich Repeats)等,这些结果表明:CmLOX09可能能够响应激素、信号类物质、真菌胁迫及其他逆境胁迫。2.利用实时荧光定量PCR分析CmLOX09、CmHPL和CmAOS在逆境、激素及信号类物质处理下的表达模式,结果表明:CmLOX09、CmHPL和CmAOS受机械损伤所诱导;CmLOX09受IAA和GA3诱导,然而CmHPL和CmAOS对IAA和GA3的响应有所不同;CmLOX09、CmHPL和CmAOS同时受H202和MeJA的诱导,相反,受ABA和SA的抑制;CmLOX09、CmHPL和CmAOS同时受白粉病菌的诱导。3.MeJA处理后6h、72h和168h甜瓜叶片中2-hexynol和2-hexenal的含量极显著高于对照,表明CmLOX09可能通过HPL途径产生的C6醇醛类物质来响应MeJA的处理。4.接种白粉病菌后,1级和3级甜瓜发病叶片中JA的含量极显著高于未发病植株(0级),表明CmLOX09可能通过AOS途径产生的JA来响应白粉病菌的侵染。5.利用Gateway法成功构建了 CmLOX09超表达载体;应用农杆菌介导法将甜瓜CmLOX09转入到番茄植株基因组中;利用PCR、实时荧光定量PCR、Western blot印记分析、活体荧光成像仪以及Basta筛选对T0代转基因植株进行了鉴定。综上所述:CmLOX09在防御反应中起到积极的作用。CmLOX09可能通过HPL途径产生的C6醛醇类物质来响应MeJA,通过AOS途径产生的JA来响应白粉病菌的侵染。通过农杆菌介导法获得了两株CmLOX09超表达的番茄转基因植株,该研究为后续通过转基因植株研究CmLOX09在防御反应中的具体功能奠定基础。
[Abstract]:Lipoxygenase (lipoxygenase, LOX) is a key enzyme in plants eighteen carbonate metabolic pathway, catalytic lipids and polyunsaturated fatty acids to form a product of a certain activity, widely involved in plant growth and development, synthesis and senescence of aroma substances, and to participate in the defense response of.LOX stress by catalytic (containing Z, Z) -1,4- diene structure of polyunsaturated fatty acids to form hydrogen peroxide (HPOD), hydrogen peroxide as these 7 pathways downstream of LOX substrates and further catalyzes the formation of compounds with activity, referred to as the oxylipin. At present in the LOX pathway of hydroperoxide lyase (HPL) and allene oxide synthase (AOS) two branches have been shown to participate in the defense response of plant adversity. Previous research showed that: 5 muskmelon 13-LOXs gene can response to abiotic stress and signal substances, But it is not clear whether 9-LOX is involved in biotic and abiotic stress defense response, the analysis showed that the CmLOX09 promoter contains adversity response element of hormone and signal substances. Therefore, this paper takes 4 leaves and 1 heart muskmelon seedlings as test materials, studied mechanical damage; hormones: auxin (IAA) and gibberellin (GA3); signal substances: abscisic acid (ABA), salicylic acid (SA), hydrogen peroxide (H202) and methyl jasmonate (MeJA); biological stress: inoculated ordrought effects of CmLOX09 and its downstream CmHPL and CmAOS expression in MeJA was determined after treatment muskmelon leaves green leaf volatiles (GLVs) content change and after inoculation with powdery mildew disease of muskmelon leaves in different levels of jasmonic acid (JA) content. The main results are as follows: 1. using Plant CARE software on CmLOX09 promoter prediction analysis showed that it contains many Response elements such as light responsive elements (3-AF1, binding site, Box 4, ACE, AT1-motif etc.), hormone response elements (p-box, element) signaling substances (ABRE element and TCA-element response element), fungi (Box-W1 element) stress response element and stress response element (TC-rich Repeats), these results suggest that CmLOX09 may in response to hormones, signaling substances, fungi and other adverse stress analysis of CmLOX09 using real-time fluorescence quantitative PCR.2. stress, CmHPL and CmAOS in the face of adversity, expression pattern, and hormone signaling substances under the treatment of the results show that: CmLOX09, CmHPL and CmAOS mechanical injury induced by IAA; CmLOX09 and GA3 induced CmHPL response, however and CmAOS of IAA and GA3 are different; CmLOX09, CmHPL and CmAOS induced by H202 and MeJA at the same time, on the contrary, inhibition of ABA and SA; CmLOX09, CmHPL and CmAOS and.3.MeJA after 6h treatment induced by powdery mildew The contents of 2-hexynol and 2-hexenal, 72h and 168h in the leaves of muskmelon were significantly higher than the control, C6 showed that alcohol aldehydes CmLOX09 may be produced by the HPL pathway in response to MeJA treatment.4. after inoculation with powdery mildew, the content of 1 and 3 the incidence of muskmelon leaf JA was significantly higher than that without plant disease (level 0). That CmLOX09 may be produced by way of AOS JA in response to.5. infection pathogen by using the Gateway method to construct CmLOX09 expression vector; application of Agrobacterium tumefaciens mediated transformation of Muskmelon CmLOX09 into tomato genome; using PCR, real-time fluorescence quantitative PCR analysis, Western blot mark, in vivo fluorescence imaging instrument and Basta screening the T0 transgenic plants were identified. Conclusion: CmLOX09 plays a role in the defense response of.CmLOX09 positive C6 aldehyde alcohols may be produced through the HPL pathway in response to MeJA, through the AOS pathway The JA produced to respond to the infection of powdery mildew. Two transgenic tomato plants with CmLOX09 overexpression were obtained by Agrobacterium mediated method. The study laid the foundation for further research on the specific functions of CmLOX09 in defense response through transgenic plants.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S652;Q943.2

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1 鞠立君;甜瓜CmLOX09对逆境胁迫、激素和信号物质的响应及对番茄的遗传转化[D];沈阳农业大学;2017年

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