本文關(guān)鍵詞： 冰島虞美人 低溫耐性 Pn2DREB1 基因克隆 表達(dá)分析　出處：《四川農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：本試驗(yàn)以冰島虞美人不同品種為實(shí)驗(yàn)材料,研究人工模擬低溫對(duì)植株葉片形態(tài)及生理、生化指標(biāo)的影響,采用主成分分析法和隸屬函數(shù)法對(duì)其耐寒性進(jìn)行綜合評(píng)價(jià)。同時(shí)根據(jù)其他植物中已知冷誘導(dǎo)轉(zhuǎn)錄因子DREBl的保守序列,設(shè)計(jì)引物,從冰島虞美人克隆獲得PnDREB1的全長(zhǎng)序列,并對(duì)其進(jìn)行生物信息學(xué)分析和低溫誘導(dǎo)表達(dá)分析。獲得以下主要研究結(jié)果：1冰島虞美人.不同品種耐寒性評(píng)價(jià)以‘香檳氣泡’、國(guó)產(chǎn)品種、 ‘仙境’、 ‘花匠’4個(gè)品種成熟植株為試驗(yàn)材料,研究低溫環(huán)境對(duì)植株葉片形態(tài)、電導(dǎo)率、MDA等11個(gè)形態(tài)、生理、生化指標(biāo)的影響。結(jié)果表明：(1)不同低溫(3、0、-3、-6、-9℃)處理24h,4個(gè)品種冰島虞美人相對(duì)電導(dǎo)率隨溫度降低呈上升趨勢(shì), ‘香檳氣泡’、國(guó)產(chǎn)品種、‘仙境’、‘花匠’的低溫半致死溫度分別為-7.7℃、-5.2℃、-1.9℃、-4.5℃。(2)-3℃處理0、24、48、72h時(shí),4個(gè)品種冰島虞美人葉片均出現(xiàn)不同程度的凍害,72h后凍害指數(shù)分別為0.30、1.46、2.34、1.67；隨低溫脅迫時(shí)間的延長(zhǎng),植株MDA含量均呈先下降后上升的變化趨勢(shì)；‘香檳氣泡’可溶性蛋白含量先降后升,其他三種則先升后降；‘花匠’脯氨酸含量持續(xù)上升,其他三種則先升后降；‘香檳氣泡’SOD活性持續(xù)上升,其他三種植株先升后降；4個(gè)品種冰島虞美人葉片POD活性先升后降,CAT活性呈“上升-下降-上升”的變化趨勢(shì),APX活性先升后降,GR活性與ASA含量均先降后升。說明4個(gè)品種冰島虞美人對(duì)低溫均有一定的適應(yīng)性,但不同品種對(duì)低溫的適應(yīng)性調(diào)節(jié)存在明顯差異。(3)用主成分分析法和隸屬函數(shù)法對(duì)MDA、可溶性蛋白、脯氨酸等9個(gè)生理、生化指標(biāo)進(jìn)行分析,得出4個(gè)品種冰島虞美人耐寒性強(qiáng)弱順序?yàn)椋骸銠墯馀荨瘒?guó)產(chǎn)品種‘花匠’‘仙境’,該結(jié)果與各品種低溫半致死溫度排序及凍害指數(shù)排序基本一致,可為冰島虞美人耐寒指標(biāo)確定、品種篩選提供科學(xué)依據(jù)。2.冰島虞美人PnDREB1基因的克隆及序列分析通過RI-PCR法從冰島虞美人品種‘香檳氣泡’中分離出一個(gè)新的DREB1轉(zhuǎn)錄因子,命名為PnDREB1。該基因序列全長(zhǎng)1035bp,無內(nèi)含子,開放閱讀框(ORF)長(zhǎng)699bp,編碼232個(gè)氨基酸殘基,預(yù)測(cè)蛋白分子量為26.3kDa,含有1個(gè)由58個(gè)氨基酸組成的AP2/ERF結(jié)構(gòu)域,且在N-端含有一個(gè)富含堿性氨基酸區(qū)域,推測(cè)為核定位信號(hào)(NLS):PKR(K)K(P)AGRxKFxETRHP,結(jié)構(gòu)域下游含有DSV(A)WR序列,這兩段多肽序列均屬于DREB1/CBF基因家族的特征序列。該基因編碼的蛋白質(zhì)與荷花DREB1B、梅花DREB1B、菊花DREB1、沙梨DREB、擬南DREB1D等具有高度同源性。系統(tǒng)進(jìn)化樹分析顯示,冰島虞美人PnDREBl屬于DREB/CBF轉(zhuǎn)錄因子家族中的A-1亞組,與菊花DREB1親緣關(guān)系最近。生物信息學(xué)預(yù)測(cè)分析表明,冰島虞美人PnDREB1基因編碼的蛋白屬于親水性蛋白,無跨膜結(jié)構(gòu),無信號(hào)肽,可能位于葉綠體或細(xì)胞核中。二級(jí)結(jié)構(gòu)及三級(jí)結(jié)構(gòu)預(yù)測(cè)顯示,該蛋白含有1個(gè)α-螺旋,3個(gè)β-折疊和多個(gè)β-轉(zhuǎn)角。半定量RT-PCR分析表明,低溫處理前,冰島虞美人葉片內(nèi)PnDREB1基因表達(dá)水平很低,幾乎檢測(cè)不到。-3℃處理后,PnDREB1轉(zhuǎn)錄水平隨處理時(shí)間的延長(zhǎng)呈先上升后下降的變化趨勢(shì),在處理8h時(shí)其相對(duì)表達(dá)量達(dá)到最大值。
[Abstract]:The Iceland poppy cultivars as experimental materials, research on artificial low temperature on the morphology and physiology of plants, biochemical effects, using principal component analysis method and membership function method to evaluate the cold resistance. At the same time according to the conserved sequences in other plants known cold induced transcription factor DREBl were obtained the full-length sequence of PnDREB1 from the Iceland poppy and its cloning, bioinformatics analysis and low temperature induced expression analysis. The main results were as follows: 1. Different varieties of Iceland poppy cold resistance evaluation to "champagne bubble", the domestic varieties, "Wonderland", "gardener" mature 4 cultivars as experimental materials, electrical conductivity study on low temperature environment on leaf morphology, MDA, etc. 11 morphology, physiological, biochemical effects. The results showed that: (1) different temperature (3,0, -3, -6, -9 C) 24h, 4 The relative conductivity of poppy varieties in Iceland showed an increasing trend with the decrease of temperature, the "champagne bubble", the domestic varieties, "Wonderland", "gardener" semilethal temperature were -7.7 degrees, -5.2 degrees, -1.9 degrees, -4.5 degrees. (2) -3 treatment 0,24,48,72h, 4 varieties of Iceland poppy leaves were freezing different degrees of 72h after freezing injury index were 0.30,1.46,2.34,1.67; withthelowtemperaturestresstimeincreasing, MDA content in plant were decreased at first and then increased; the soluble protein content of champagne bubbles' rise after the first drop, the other three are at first and then decreased; the proline content of the gardener 'continued to rise, the other three first increased and then decreased; "champagne bubbles' SOD activity continued to rise, the other three plants decreased; Iceland poppy POD activity in leaves of 4 varieties increased first and then decreased, CAT activity showed a trend of" up down up " , APX activity first increased and then decreased, GR activity and ASA content were decreased and then increased. 4 varieties of Iceland poppy has certain adaptability to low temperature, but the adaptability of different varieties to low temperature regulation there is obvious difference. (3) analysis method and the method of membership function of MDA, soluble protein and proline by principal components. 9 physiological analysis, biochemical index, the 4 varieties of Iceland poppy cold resistance order: "champagne bubble" domestic varieties "gardener" Wonderland ", with the result of different varieties of semilethal temperature sorting and freezing index sorting is consistent, can be identified as the Iceland poppy cold index, variety screening provides cloning the scientific basis and sequence analysis of PnDREB1 gene of.2. Iceland poppy isolated a new DREB1 transcription factor from the Iceland poppy variety 'champagne bubbles' by RI-PCR method, named the PnDREB1. gene sequence The length of 1035BP, with no intron, open reading frame (ORF) length of 699bp, encoding 232 amino acid residues with a predicted molecular weight of 26.3kDa, containing 1 composed of 58 amino acids and contains a AP2/ERF domain, a region rich in basic amino acids at the N- end, presumably the nuclear localization signal (NLS). PKR (K) K (P) AGRxKFxETRHP, the downstream domain containing DSV (A) WR sequence, feature sequences of the two peptides sequences belong to the DREB1/CBF gene family. The protein with lotus DREB1B, the gene encoding DREB1B DREB1 chrysanthemum, plum, pear DREB, Arabidopsis DREB1D is highly homologous to the system evolution. The tree analysis showed that Iceland poppy PnDREBl belongs to the DREB/CBF family of transcription factors in the A-1 subgroup, DREB1 genetic relationship with chrysanthemum recently. Bioinformatics analysis showed that the Iceland poppy PnDREB1 gene encoding protein is a hydrophilic protein without transmembrane structure, no signal peptide That may be located in chloroplast or nucleus. Two level structure and three secondary structure prediction showed that the protein contains 1 alpha helix, 3 beta sheet and a plurality of p-turns. Semi quantitative RT-PCR analysis showed that the low temperature treatment before, Iceland poppy leaves PnDREB1 gene expression level is very low, almost not detected to.-3 treatment after prolonged first increased and then decreased the transcription level of PnDREB1 with the treatment time, when dealing with 8h the relative expression quantity reached the maximum value.

【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S682.19

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本文編號(hào)：1499432