冰岛虞美人（Papaver nudicaule）不同品种耐寒性评价及其冷诱导基因PnDREB1的克隆

发布时间：2018-02-10 02:23

本文关键词： 冰岛虞美人低温耐性 Pn2DREB1 基因克隆表达分析　出处：《四川农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：本试验以冰岛虞美人不同品种为实验材料,研究人工模拟低温对植株叶片形态及生理、生化指标的影响,采用主成分分析法和隶属函数法对其耐寒性进行综合评价。同时根据其他植物中已知冷诱导转录因子DREBl的保守序列,设计引物,从冰岛虞美人克隆获得PnDREB1的全长序列,并对其进行生物信息学分析和低温诱导表达分析。获得以下主要研究结果：1冰岛虞美人.不同品种耐寒性评价以‘香槟气泡’、国产品种、 ‘仙境’、 ‘花匠’4个品种成熟植株为试验材料,研究低温环境对植株叶片形态、电导率、MDA等11个形态、生理、生化指标的影响。结果表明：(1)不同低温(3、0、-3、-6、-9℃)处理24h,4个品种冰岛虞美人相对电导率随温度降低呈上升趋势, ‘香槟气泡’、国产品种、‘仙境’、‘花匠’的低温半致死温度分别为-7.7℃、-5.2℃、-1.9℃、-4.5℃。(2)-3℃处理0、24、48、72h时,4个品种冰岛虞美人叶片均出现不同程度的冻害,72h后冻害指数分别为0.30、1.46、2.34、1.67；随低温胁迫时间的延长,植株MDA含量均呈先下降后上升的变化趋势；‘香槟气泡’可溶性蛋白含量先降后升,其他三种则先升后降；‘花匠’脯氨酸含量持续上升,其他三种则先升后降；‘香槟气泡’SOD活性持续上升,其他三种植株先升后降；4个品种冰岛虞美人叶片POD活性先升后降,CAT活性呈“上升-下降-上升”的变化趋势,APX活性先升后降,GR活性与ASA含量均先降后升。说明4个品种冰岛虞美人对低温均有一定的适应性,但不同品种对低温的适应性调节存在明显差异。(3)用主成分分析法和隶属函数法对MDA、可溶性蛋白、脯氨酸等9个生理、生化指标进行分析,得出4个品种冰岛虞美人耐寒性强弱顺序为：‘香槟气泡’国产品种‘花匠’‘仙境’,该结果与各品种低温半致死温度排序及冻害指数排序基本一致,可为冰岛虞美人耐寒指标确定、品种筛选提供科学依据。2.冰岛虞美人PnDREB1基因的克隆及序列分析通过RI-PCR法从冰岛虞美人品种‘香槟气泡’中分离出一个新的DREB1转录因子,命名为PnDREB1。该基因序列全长1035bp,无内含子,开放阅读框(ORF)长699bp,编码232个氨基酸残基,预测蛋白分子量为26.3kDa,含有1个由58个氨基酸组成的AP2/ERF结构域,且在N-端含有一个富含碱性氨基酸区域,推测为核定位信号(NLS):PKR(K)K(P)AGRxKFxETRHP,结构域下游含有DSV(A)WR序列,这两段多肽序列均属于DREB1/CBF基因家族的特征序列。该基因编码的蛋白质与荷花DREB1B、梅花DREB1B、菊花DREB1、沙梨DREB、拟南DREB1D等具有高度同源性。系统进化树分析显示,冰岛虞美人PnDREBl属于DREB/CBF转录因子家族中的A-1亚组,与菊花DREB1亲缘关系最近。生物信息学预测分析表明,冰岛虞美人PnDREB1基因编码的蛋白属于亲水性蛋白,无跨膜结构,无信号肽,可能位于叶绿体或细胞核中。二级结构及三级结构预测显示,该蛋白含有1个α-螺旋,3个β-折叠和多个β-转角。半定量RT-PCR分析表明,低温处理前,冰岛虞美人叶片内PnDREB1基因表达水平很低,几乎检测不到。-3℃处理后,PnDREB1转录水平随处理时间的延长呈先上升后下降的变化趋势,在处理8h时其相对表达量达到最大值。
[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.

【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S682.19

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