【作者】 周厚成；

【导师】 郭蔼光；

【作者基本信息】 西北农林科技大学 ， 生物化学及分子生物学， 2005， 硕士

【摘要】 草莓（Fragaria　ananassa　Duch.）是无性繁殖的多年生草本植物,易受多种病毒的侵染,病毒严重危害草莓生产。草莓镶脉病毒（Strawberry　vein　banding　virus,SVBV）是草莓病毒病害中对生产造成严重损失的四种主要潜隐性病毒之一。由于目前尚无有效的化学药剂防治草莓病毒病,抗病毒基因工程为防治草莓病毒病提供了一条技术途径。　本研究以SVBV 和不同品种的草莓试管苗为试材,取得以下研究结果:　1.利用SVBV 保守的CP 基因区设计特异引物,对SVBV 进行了PCR 检测,建立了SVBV的PCR 检测技术体系。并克隆了特异扩增产物,测序结果表明该片段长574bp,扩增片段序列与已报道的SVBV　CP 基因序列（序列号:Nc₀01725）的核苷酸同源性分别为89.2%,氨基酸同源性为96.3%;SVBV 多个分离物序列分析表明不同分离物CP 基因间存在分子变异。已将该CP 基因片段序列登录GenBank,登记号为AY862389。　2.以‘幸香’、‘丰香’、‘安娜’三个品种为试材,以MS 为基本培养基附加不同激素,研究不同基因型和激素配比对诱导叶片产生愈伤组织和再生不定芽的影响,建立了草莓叶片高效离体再生体系,为草莓的遗传转化研究与应用奠定了基础。结果表明,对于某一特定的基因型,外源激素种类及配比对诱导叶片产生愈伤组织和再生不定芽有明显的影响。供试三个品种中,TDZ 诱导效果均比6-BA 好。‘幸香’在TDZ　8.0　mg/L+IAA　1.0　mg/L 的诱导下,不定芽的再生频率达到86%,平均每个外植体再生不定芽数为2.4 个;在6-BA　1.0　mg/L+IAA　1.5　mg/L 的诱导下,不定芽的再生频率达到42%,平均每个外植体再生不定芽数为1.8 个。‘丰香’在TDZ　2.0　mg/L 的诱导下,不定芽的再生频率达到82%,平均每个外植体再生不定芽数为2.1 个;而在6-BA 的作用下,无不定芽的产生。‘安娜’只有在TDZ　2.0　mg/L 的诱导下有不定芽的产生,再生率为7%,平均每个外植体再生不定芽数为1.0 个,其余处理均无不定芽生成。丛状再生芽转移到MS+IBA0.1mg/L 的培养基恢复生长,并在1/2MS+IBA0.1mg/L 生根成苗。　3.在建立‘幸香’叶片不定芽再生体系的基础上,利用根癌农杆菌介导的遗传转化法进行‘幸香’遗传转化研究,通过抗生素抗性实验和对GUS 基因瞬时表达率分析,研究转化体系的最佳实验参数。结果表明:Km　40　mg/L 可以完全抑制‘幸香’草莓叶盘的再生,可以作为转化植株的筛选浓度,200mg/L　Cef 作为抑制农杆菌生长的浓度;预培养时间、侵染时间、共培养时间和AS 等对转化效率都有一定影响,预培养0～2d、农杆菌侵染时间3～5min、共培养72h 和添加25～100μmol/L　AS　GUS 瞬时表达率最高。经Km筛选,GUS 组织化学染色和PCR 检测证明共得到3 个转基因株系。更多还原

【Abstract】 Strawberry（Fragaria ananassa Duch.）is a kind of perennial herb and susceptible to virus disease for the vegetative propagation. Strawberry vein banding virus （SVBV） is one of four kinds of strawberry latent virus. Many negative effects such as strain degeneration, yield and quality reductions may be caused in commercial strawberry cultivars infected with SVBV. No effective chemical agent to control strawbery virus disease, plant genetic engineering technology including high effective plant regeneration, plant genetic transformation, has been used in the studies of improvement of virus disease resistance in strawberry. Strawberry leaves SVBV-infected and seeding leaves in vitro of strawberry cultivars ‘Toyonoka’、‘Sachinoka’and ‘Anna’were used as materials for strudying the detection of SVBV、cloning CP gene、high-frequency regeneration, genetic transformation systetem etc. The main results were as follows: 1. Total DNA, extracted from SVBV-infected strawberries leaves using CTAB method, was used as template to amplify by PCR. PCR amplification conditions and programs were optimized. About 600 bp fragment of SVBV was obtained based on optimum system. On the other hand, the total DNA was diluted to density gradient and amplified by PCR. The results showed that it can detect the virus in 2.5μg leaf tissue. PCR method is a reliable method of detecting SVBV. The PCR specific fragment was recycled, cloned into pMD18-T vector and sequenced. Compared with SVBV isolate that reported （GenBank accession number: Nc₀01725）, it showed that the sequence of coat protein gene of this isolate is 89.2% and 96.3% identity at the sequence levels of nucleotide and deduced amino acid. GenBank accession number of the specific fragment sequence is AY862389. 2. The present primary strawberry cultivars ‘Toyonoka’、‘Sachinoka’and ‘Anna’were used as experimental materials. To establish a reliable, rapid and effective in vitro regeneration system and to carry out the strawberry genetic transformation successfully, the factors affecting adventitious shoots regeneration were studied. The results indicated that Genotype was the main factor influencing regeneration system of strawberry in vitro, but the frequencies and amounts of shoot organogenesis were significantly affected by the hormone concentrations for a given genotype. Compared with 6-BA and TDZ, TDZ had a better effect on inducing shoot regeneration. The regeneration frequency of cultivar ‘Sachinoka’reached the highest level of 86% in MS+TDZ8.0mg/L+IAA0.1mg/L and 42% in MS+6-BA1.0mg/L+IAA1.5 mg/L medium, while cultivar ‘Toyonoka’obtained the highest level of 82% in MS+TDZ2.0mg/L+IAA0.1mg/L medium. and cultivar ‘Anna’obtained the highest level of 7% in MS+TDZ2.0mg/L+IAA0.1mg/L medium. The corresponding numbers of shoots regeneration were 2.4,1.8,2.1 and 1.0 per leaf disc, respectively. To cultivar ‘Toyonoka’and ‘Anna’, 6-BA can’t induce shoot regeneration. While using the rooting medium of 1/2MS+IBA0.1mg/L,the rooting rate achieved nearly 100%. 3. Studying effects of different antibiotic on the regeneration of strawberry leaves in vitro and using transient expression of GUS gene, the optimal conditions for Agrobacterium-mediated transformation of shrawberry leaf discs was investigated. On medium containing 40 mg/L kanamycin, the shoot regeneration capacity of cultivar ‘Sachinoka’was inhibited. The growth of Agrobactium was inhibited completely with 200 mg/L cefotaxine supplemented in the medium. As a result, 20 mg/L of kanamycin and 200 mg/L cefotaxine of were established as the concentration effective for selection of shoots and inibition of Agrobactium growth. Histochemical GUS assay showed that the transient expression rate was affected by pre-culture time, infected time, co-culture time and the concentration of adding acetosyringone. Pre-culture 0-2 d, infection 3 to 5 min,co-culture 72h and adding 25-100μmol/L acetosyringone contributed to the hoghest level of transient expression. The strawberry transgenic plants were developed with kanamycin resistance selection. Five transgenic lines were detected by GUS and PCR assays, and three of them show the positive band same as NPTⅡgene　and　CaMV35S.更多还原