本文關(guān)鍵詞：甜瓜CmLOX09對(duì)逆境脅迫、激素和信號(hào)物質(zhì)的響應(yīng)及對(duì)番茄的遺傳轉(zhuǎn)化　出處：《沈陽(yáng)農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 薄皮甜瓜 9-LOX 氫過氧化物裂解酶(HPL) 丙二烯氧化物合酶(AOS) 綠葉揮發(fā)物(GLVs) 茉莉酸(JA)

【摘要】：脂氧合酶(lipoxygenase,LOX)是植物體內(nèi)十八碳酸代謝途徑中的關(guān)鍵酶,催化脂類和多不飽和脂肪酸形成具有一定活性的產(chǎn)物,廣泛參與植物生長(zhǎng)發(fā)育、成熟衰老和香氣物質(zhì)的合成,并參與到逆境脅迫的防御反應(yīng)中。LOX通過催化含有(Z,Z)-1,4-戊二烯結(jié)構(gòu)的多不飽和脂肪酸進(jìn)一步形成氫過氧化物(HPOD),這些氫過氧化物為L(zhǎng)OX下游7條途徑提供底物并進(jìn)一步催化形成具有活性的化合物,統(tǒng)稱為氧脂素。目前LOX途徑中的氫過氧化物裂解酶(HPL)和丙二烯氧化物合酶(AOS)兩條支路已經(jīng)被證實(shí)能夠參與到植物逆境的防御反應(yīng)中。前期研究表明:5個(gè)甜瓜13-LOXs基因能夠響應(yīng)非生物脅迫及信號(hào)類物質(zhì)的處理,但尚不明確9-LOX是否參與對(duì)生物和非生物脅迫的防御反應(yīng)中,對(duì)CmLOX09的啟動(dòng)子分析顯示其含有對(duì)逆境、激素和信號(hào)類物質(zhì)的響應(yīng)元件。因此,本文以4葉1心的薄皮甜瓜幼苗為試材,研究了機(jī)械損傷;激素:生長(zhǎng)素(IAA)和赤霉素(GA3);信號(hào)類物質(zhì):脫落酸(ABA)、水楊酸(SA)、過氧化氫(H202)以及茉莉酸甲酯(MeJA);生物脅迫:接種白粉病菌等脅迫處理對(duì)CmLOX09及其下游CmHPL和CmAOS表達(dá)量的影響,此外,測(cè)定了 MeJA處理后甜瓜葉片中綠葉揮發(fā)物(GLVs)含量的變化以及接種白粉病菌后不同發(fā)病級(jí)別甜瓜葉片中茉莉酸(JA)含量的變化。主要結(jié)果如下:1.利用Plant CARE軟件對(duì)CmLOX09啟動(dòng)子分析預(yù)測(cè)顯示:其中含有多種響應(yīng)元件如光響應(yīng)元件(3-AF1 binding site、Box 4、ACE、AT1-motif 等)、激素響應(yīng)元件(p-box element)、信號(hào)類物質(zhì)響應(yīng)元件(ABRE element和TCA-element)、真菌脅迫響應(yīng)元件(Box-W1 element)和逆境響應(yīng)元件(TC-rich Repeats)等,這些結(jié)果表明:CmLOX09可能能夠響應(yīng)激素、信號(hào)類物質(zhì)、真菌脅迫及其他逆境脅迫。2.利用實(shí)時(shí)熒光定量PCR分析CmLOX09、CmHPL和CmAOS在逆境、激素及信號(hào)類物質(zhì)處理下的表達(dá)模式,結(jié)果表明:CmLOX09、CmHPL和CmAOS受機(jī)械損傷所誘導(dǎo);CmLOX09受IAA和GA3誘導(dǎo),然而CmHPL和CmAOS對(duì)IAA和GA3的響應(yīng)有所不同;CmLOX09、CmHPL和CmAOS同時(shí)受H202和MeJA的誘導(dǎo),相反,受ABA和SA的抑制;CmLOX09、CmHPL和CmAOS同時(shí)受白粉病菌的誘導(dǎo)。3.MeJA處理后6h、72h和168h甜瓜葉片中2-hexynol和2-hexenal的含量極顯著高于對(duì)照,表明CmLOX09可能通過HPL途徑產(chǎn)生的C6醇醛類物質(zhì)來響應(yīng)MeJA的處理。4.接種白粉病菌后,1級(jí)和3級(jí)甜瓜發(fā)病葉片中JA的含量極顯著高于未發(fā)病植株(0級(jí)),表明CmLOX09可能通過AOS途徑產(chǎn)生的JA來響應(yīng)白粉病菌的侵染。5.利用Gateway法成功構(gòu)建了 CmLOX09超表達(dá)載體;應(yīng)用農(nóng)桿菌介導(dǎo)法將甜瓜CmLOX09轉(zhuǎn)入到番茄植株基因組中;利用PCR、實(shí)時(shí)熒光定量PCR、Western blot印記分析、活體熒光成像儀以及Basta篩選對(duì)T0代轉(zhuǎn)基因植株進(jìn)行了鑒定。綜上所述:CmLOX09在防御反應(yīng)中起到積極的作用。CmLOX09可能通過HPL途徑產(chǎn)生的C6醛醇類物質(zhì)來響應(yīng)MeJA,通過AOS途徑產(chǎn)生的JA來響應(yīng)白粉病菌的侵染。通過農(nóng)桿菌介導(dǎo)法獲得了兩株CmLOX09超表達(dá)的番茄轉(zhuǎn)基因植株,該研究為后續(xù)通過轉(zhuǎn)基因植株研究CmLOX09在防御反應(yīng)中的具體功能奠定基礎(chǔ)。
[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.

【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S652;Q943.2

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1 鞠立君;甜瓜CmLOX09對(duì)逆境脅迫、激素和信號(hào)物質(zhì)的響應(yīng)及對(duì)番茄的遺傳轉(zhuǎn)化[D];沈陽(yáng)農(nóng)業(yè)大學(xué);2017年

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本文編號(hào)：1391814