【摘要】：為闡明添加生物炭對煙草類胡蘿卜素代謝的影響機制,本試驗以煙草品種K326為材料,在大田試驗中設(shè)置不同的生物炭處理,從煙株發(fā)育過程中葉片類胡蘿卜素積累動態(tài)、類胡蘿卜素代謝途徑關(guān)鍵基因表達和烤后樣中性致香物質(zhì)三個方面進行了系統(tǒng)研究。在此基礎(chǔ)上,以類胡蘿卜素合成途徑中的關(guān)鍵分支點酶LCY-b為切入點,通過同源克隆的方法獲得煙草品種K326 LCY-b基因全長序列,利用in-fusion方法成功構(gòu)建了LCY-b基因過表達載體,采用農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化煙草植株,得到LCY-b過表達陽性植株共6株。將T0代陽性植株進行大田種植,進一步探究LCY-b基因?qū)煵蓊惡}卜素代謝及烤后樣中性致香物質(zhì)的影響,為煙草類胡蘿卜素代謝的分子調(diào)控奠定了基礎(chǔ)。主要研究結(jié)果如下:1.不同生物炭肥施用量對煙草類胡蘿卜素代謝影響的分析結(jié)果表明:隨著煙葉成熟,類胡蘿卜素合成相關(guān)基因(PSY、ZDS、LCY-b)表達逐漸減弱,降解相關(guān)基因(CCD1)表達逐漸增強,與打頂后類胡蘿卜素含量逐漸下降的結(jié)果一致。煙葉成熟后期,添加生物炭的處理類胡蘿卜素積累量較多,其中T1(300 kg·hm-2)處理的類胡蘿卜素各組分含量在打頂后一直保持在較高水平。RT-PCR分析結(jié)果顯示:T1(300 kg·hm-2)和T2(600 kg·hm-2)處理中類胡蘿卜素合成及降解相關(guān)基因的表達量相對較強,說明適量添加生物炭肥對煙葉類胡蘿卜素的合成和降解均有明顯的促進作用,整體上減緩了類胡蘿卜素含量下降趨勢,提高了成熟期煙葉類胡蘿卜素積累量。煙葉調(diào)制后,T1處理的類胡蘿卜素降解產(chǎn)物含量最高,未添加生物炭肥的處理含量最低。以上結(jié)果說明,適量添加生物炭能夠增強類胡蘿卜素代謝相關(guān)酶活性,有利于成熟期煙葉類胡蘿卜素的積累及調(diào)制后中性致香物質(zhì)的形成。2.采用同源克隆的方法獲得煙草LCY-b基因全長序列1557 bp,并對其進行了詳細的生物信息學(xué)分析。序列分析表明:K326 LCY-b基因包含一個1503 bp的開放讀碼框(ORF),編碼500個氨基酸。預(yù)測蛋白質(zhì)分子量為56.05 KDa,理論等電點(p I)為6.68。蛋白二級結(jié)構(gòu)預(yù)測結(jié)果顯示以α螺旋和無規(guī)則卷曲為主。3.利用in-fusion方法成功構(gòu)建LCY-b過表達載體,采用農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化煙草植株,獲得LCY-b過表達陽性植株。分子檢測證明LCY-b基因在煙草基因組中成功實現(xiàn)了整合和轉(zhuǎn)錄。過表達陽性植株的q PCR檢測結(jié)果表明:部分轉(zhuǎn)基因陽性植株LCY-b基因的表達量顯著高于K326對照。相應(yīng)地,類胡蘿卜素組分中的β-胡蘿卜素、新黃質(zhì)、紫黃質(zhì)所占比例增加,而葉黃素所占比例有所降低。對烤后樣類胡蘿卜降解產(chǎn)物的分析結(jié)果顯示:LCY-b過表達株系中的β-胡蘿卜素降解產(chǎn)物(β-二氫大馬酮、β-環(huán)檸檬醛、香葉基丙酮、二氫獼猴桃內(nèi)酯等)顯著高于K326未轉(zhuǎn)基因?qū)φ?而葉黃素降解產(chǎn)物(巨豆三烯酮)則與K326相當(dāng)或低于K326對照。研究結(jié)果說明在煙草植株中過表達LCY-b基因可以改變類胡蘿卜素組分比例,影響烤后樣中性致香物質(zhì)的含量及組分。
[Abstract]:In order to elucidate the mechanism of the effect of biochar addition on carotenoid metabolism in tobacco, a tobacco variety K326 was used as the material in this experiment. Different biochar treatments were set up in the field to study the dynamics of carotenoid accumulation in tobacco leaves, the expression of key genes involved in carotenoid metabolism pathway and the neutral aroma-inducing substances in tobacco leaves. On this basis, the full-length sequence of LCY-b gene of tobacco variety K326 was obtained by homologous cloning, and the LCY-b gene overexpression vector was successfully constructed by in-fusion method. The tobacco plants were transformed by Agrobacterium tumefaciens mediated transformation. Six plants were over-expressed with LCY-b gene. T0 positive plants were planted in the field to further explore the effects of LCY-b gene on carotenoid metabolism and neutral aroma-causing substances in tobacco, which laid the foundation for the molecular regulation of carotenoid metabolism in tobacco. The main results were as follows: 1. Different application of biochar fertilizer on tobacco carotenoids. The results showed that the expression of PSY, ZDS and LCY-b decreased gradually with the maturation of tobacco leaves, and the expression of CCD1 increased gradually, which was consistent with the decrease of carotenoid content after topping. The results of RT-PCR analysis showed that the expression of genes related to carotenoid synthesis and degradation in T1 (300 kg The synthesis and degradation of carotenoids were promoted obviously, and the decreasing trend of carotenoids content was slowed down, and the accumulation of carotenoids in mature tobacco leaves was increased. After curing, the content of carotenoids degradation products in T1 treatment was the highest, while that in the treatment without biochar fertilizer was the lowest. Carbon can enhance the activity of carotenoid metabolism-related enzymes, which is beneficial to the accumulation of carotenoids in mature tobacco leaves and the formation of neutral aroma-inducing substances. 2. The full-length sequence of LCY-b gene in tobacco was obtained by homologous cloning method and its bioinformatics analysis was carried out in detail. The predicted protein molecular weight was 56.05 KDa and the theoretical isoelectric point (PI) was 6.68. The predicted secondary structure of the protein was mainly alpha helix and irregular curl. 3. LCY-b overexpression vector was successfully constructed by in-fusion method and transformed into tobacco plants by Agrobacterium tumefaciens. LCY-b gene was successfully integrated and transcribed in tobacco genome by molecular detection. The results of q-PCR showed that the expression of LCY-b gene in some transgenic plants was significantly higher than that in K326 control. The proportion of violaxanthin increased, but the proportion of lutein decreased. The analysis of the degradation products of carotenoids after baking showed that the degradation products of beta-carotene (beta-dihydro-damascone, beta-cyclic citral, geranyl acetone, dihydro-kiwifruit lactone, etc.) in LCY-b overexpression lines were significantly higher than those in K326 untransformed control. The results showed that over-expression of LCY-b gene in tobacco plants could alter the proportion of carotenoids and affect the content and components of neutral aroma-causing substances.
【學(xué)位授予單位】：河南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S572

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1 張嘉煒;生物炭對煙草類胡蘿卜素代謝的影響及LCY-b基因過表達調(diào)控研究[D];河南農(nóng)業(yè)大學(xué);2016年

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