猕猴桃叶片再生体系的建立和转抗菌肽D基因对保护酶活性的影响

发布时间：2018-05-11 20:07

本文选题：猕猴桃 + 组织培养　；参考：《河南农业大学》2016年硕士论文

【摘要】：猕猴桃(Actinidia chinensis Planch.)是世界上一种重要的水果,该属有54个种,21个变种,共约75个分类单元,其中中华猕猴桃(A.chinensis Planch.var.chinensis,果肉绿色)和美味猕猴桃(A.chinensis var.deliciosa,果肉金黄色)是栽培最广泛的的两个商业种类。而软枣猕猴桃(Actinidia arguta Planch)目前仅有零星栽培,因其果面无毛,风味独特,是一种具有发展前景的果树资源。其中‘天源红’软枣猕猴桃果皮、果肉和果心全为红色,酸甜适口,光洁无毛,可带皮直接食用,具有重要推广经济价值。为了建立‘天源红’软枣猕猴桃快繁体系,本试验以组培苗叶片及茎尖为材料,探讨了植物生长调节剂对叶片愈伤再生的影响。猕猴桃细菌性溃疡病,是一种毁灭性细菌病害,对世界猕猴桃产业的发展造成了严重的威胁。目前尚无有效方法抑制猕猴桃溃疡病害的发生,而采取基因工程技术培育抗病性强的品种是避免溃疡病带来损失的有效方法之一。本研究以转抗菌肽D的中华猕猴桃为材料,诱导生根和移栽获得了大量的猕猴桃盆栽苗,通过喷施猕猴桃细菌性溃疡病病原菌,分析猕猴桃喷施菌液前后相关酶活性的变化,预测转基因植株的抗病性变化。主要研究结果如下:(1)不同浓度的6-BA和NAA对‘天源红’软枣猕猴桃茎段增殖的影响分析结果表明,茎段增殖倍数随着6-BA的浓度增加而增大,在6-BA浓度为3.0 mg/L,NAA浓度为0.5 mg/L时,茎段增殖倍数达到最大,为3.29±0.03。(2)不同浓度的ZT和6-BA对叶片形成愈伤的结果表明,6-BA和ZT对愈伤形成的影响有很大的不同。6-BA浓度从0.5 mg/L到3.0 mg/L,叶片均不能形成愈伤组织,褐化率高,而ZT能明显诱导愈伤组织的形成,且褐化率较低。(3)不同浓度的ZT和IAA对叶片再生率的影响结果表明,当ZT浓度为2.5 mg/L,IAA浓度为0.5 mg/L时,叶片再生率达到最高,为45.00%。(4)不同时间的暗培养对叶片再生率的影响。以MS+ZT 2.5mg/L+IAA 0.5 mg/L为基本培养基,分别进行15 d,20 d,25 d,30 d的暗处理,结果表明,当叶片进行30d的暗处理,叶片再生率最高,为81.67%。(5)以转抗菌肽D的中华猕猴桃抗性芽为材料,通过诱导生根处理和移栽获得了大量的猕猴桃盆栽苗,生根结果显示,使用1.0 g/L的IBA处理15 s生根效果最好,形成的愈伤较少,且根系较多。(6)转基因猕猴桃的PCR和RT-PCR检测结果表明,外源抗菌肽D基因已经整合到猕猴桃基因组中,并在猕猴桃基因组中进行转录,且不同株系间的外源基因的表达量有差异。(7)对喷施菌液前后猕猴桃苗叶片中的过氧化氢酶(CAT)、过氧化物酶(POD)和苯丙氨酸解氨酶(PAL)的活性分析结果表明,转抗菌肽D基因在猕猴桃基因组中的过量表达对猕猴桃叶片CAT、POD、PAL活性变化影响不明显。但是在喷施病原微生物(菌液浓度为1.2×107~108,OD值为0.2左右)的条件下,转基因植株的CAT和POD活性比对照植株明显降低,PAL活性比对照植株明显升高。
[Abstract]:Actinidia chinensis Planch.Actinidia A. chinensis Planch.var. chinensis (green pulp) and A. chinensis var. deliciosa are the two most widely cultivated commercial species. Actinidia arguta Planche is only cultivated sporadically, because of its hairless surface and unique flavor, Actinidia arguta Planche is a promising fruit resource. Among them, 'Tianyanhong' soft jujube kiwifruit peel, pulp and heart are all red, sweet and sour palatability, smooth and clean hairless, can be eaten directly with skin, has important economic value. In order to establish the rapid propagation system of Actinidia chinensis, the effects of plant growth regulators on leaf callus regeneration were studied. Kiwifruit bacterial canker is a kind of destructive bacterial disease, which poses a serious threat to the development of kiwifruit industry in the world. At present, there is no effective method to inhibit the occurrence of ulcer disease in kiwifruit. Genetic engineering is one of the effective methods to avoid the damage caused by canker disease. In this study, a large number of kiwifruit plantlets were obtained by rooting and transplanting with antimicrobial peptide D, and the changes of enzyme activity before and after spraying kiwifruit bacterial canker were analyzed. The change of disease resistance of transgenic plants was predicted. The main results were as follows: (1) the effects of different concentrations of 6-BA and NAA on the growth of Actinidia chinensis stem segments were analyzed. The results showed that the multiplication ratio of stem segments increased with the increase of 6-BA concentration, when the concentration of 6-BA was 3.0 mg / L, the concentration of NAA was 0.5 mg/L. The effects of ZT and 6-BA on callus formation of leaves were significantly different from 0.5 mg/L to 3.0 mg / L in different concentrations of ZT and ZT. All leaves could not form callus and the browning rate was high. The results showed that when ZT concentration was 2.5 mg 路L ~ (-1) IAA concentration was 0.5 mg/L, leaf regeneration rate was the highest. The effect of dark culture at different time on leaf regeneration rate. MS ZT 2.5mg/L IAA 0.5 mg/L was used as the basic medium, respectively, for 15 days, 20 days and 25 days for 30 days. The results showed that when leaves were treated with dark for 30 days, the regeneration rate of leaves was the highest, which was 81.67. 5) the resistant buds of Actinidia chinensis transferred with antimicrobial peptide D were used as materials. A large number of potted kiwifruit seedlings were obtained by inducing rooting and transplanting. The rooting results showed that IBA of 1.0 g / L for 15 s had the best rooting effect and less callus formation. The results of PCR and RT-PCR analysis of transgenic kiwifruit showed that the exogenous antimicrobial peptide D gene had been integrated into the genome of kiwifruit and transcribed in the genome of kiwifruit. The activity of catalase (CAT), peroxidase (POD) and phenylalanine ammonia-lyase (pal) in leaves of kiwifruit seedlings were analyzed before and after spraying with different strains. The overexpression of transgenic antimicrobial peptide D gene in the genomes of kiwifruit had no significant effect on the change of the activity of POD pal in the leaves of kiwifruit (Actinidia deliciosa L.). However, the CAT and POD activities of transgenic plants were significantly lower than those of control plants under the condition of spraying pathogenic microorganisms (the OD value was about 1.2 脳 10 ~ (7) C ~ (10 ~ 8) O _ (2), which was significantly higher than that of the control plants.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S663.4

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