【作者】 尚霄丽；

【导师】 冯建灿；

【作者基本信息】 河南农业大学 ， 森林培育， 2010， 博士

【摘要】 猕猴桃(Actinidia)果实风味独特,营养丰富,被誉为“水果之王”。枣(Zizyphus jujuba Mill.)是我国第一大干果树种。猕猴桃属雌雄异株,遗传背景复杂；枣花蕾小、自然坐果率低、胚败育率高等因素,传统杂交育种方法存在周期长、效率低等问题,获得优质多抗的猕猴桃、枣新品种困难重重。基因工程的发展为猕猴桃、枣的育种工作开辟了一条新的途径。本研究以河南特异红果肉猕猴桃资源中华猕猴桃‘伏牛95-2’(Actinidia chinensis’Funiu95-2’)为材料,建立了中华猕猴桃‘伏牛95-2’高效再生体系、遗传转化体系,且获得了转抗菌肽D基因的转基因植株,PCR检测、Southern杂交检测证明外源基因已整合到猕猴桃基因组中。灰枣遗传转化研究,本试验以灰枣(Zizyphus jujuba ’Huizao’)为材料建立了灰枣组培快繁体系、叶片直接再生体系及对灰枣遗传转化进行了初步探讨。主要结果如下：1.以中华猕猴桃‘伏牛95-2’叶片为外植体,探讨了不同植物生长调节物质对叶片再生不定芽增殖及生根的影响,建立了高效的再生体系。结果表明,叶片外植体在MS+ZT1.0mg/L+ NAA 0.3mg/L培养基上,不定芽分化率最高为87.5%；在MS+ 6-BA 2.0 mg/L+NAA0.5mg/L培养基中,不定芽增殖系数最高为4.2；适宜不定芽生根培养基为1/2MS+NAA2.0mg/L,生根率为100%。2.以中华猕猴桃‘伏牛95-2’叶片为外植体,研究了抗生素对外植体生长的影响。结果表明,中华猕猴桃‘伏牛95-2’对卡那霉素较敏感,浓度20mg/L时愈伤组织及不定芽分化率均为0,20mg/L为中华猕猴桃‘伏牛95-2’叶片遗传转化时筛选最佳浓度；生根培养基中附加30mg/L卡那霉素时,则完全抑制根的形成。头孢霉素和羧苄青霉素对中华猕猴桃‘伏牛95-2’叶片分化均有抑制作用,但头孢霉素对叶片分化的抑制作用比羧苄青霉素小；结合遗传转化中的抑菌试验,选用400mg/L头孢霉素为中华猕猴桃‘伏牛95-2’遗传转化中适宜抑菌抗生素及浓度。3.以中华猕猴桃‘伏牛95-2’叶片作为根癌农杆菌介导的受体材料,建立了高效遗传转化体系。结果表明,叶片预培养3天,菌液浓度OD600值为0.3,真空渗入方式侵染10 min,共培养4天条件下,GUS基因瞬时表达率最高,达到92.2%。对转基因抗性植株进行PCR检测和GUS组织化学染色,初步证明外源基因已整合到中华猕猴桃‘伏牛95-2’的基因组中。4.以中华猕猴桃‘伏牛95-2’叶片为外植体进行遗传转化,对共培养后抗性芽的筛选方式进行了研究,并对获得的转抗菌肽D基因的抗性植株进行了分子生物检测。结果表明,逐步提高卡那霉素选择压力与保持卡那霉素选择压力不变相比,抗性芽获得率提高了28.4%；部分抗性植株经PCR、PCR-Southern、Southern杂交检测,外源基因已整合至猕猴桃基因组中。5.以灰枣一年生枣头茎段为材料,对启动培养时抗生素对污染的抑制,不同植物生长调节物质对茎段启动、不定芽增殖及生根的影响进行了研究。结果表明,大田采样时,以70%酒精灭菌30s,再以0.1%HgCl2灭菌10～15min后污染率依然高达97.8%；培养基中添加150mg/L或200mg/L头孢霉素可使污染率降为0；茎段接种于MS+KT 2.0 mg/L+NAAO.lmg/L培养基中获得的粗壮枣头较多。茎段萌发的腋芽接种于Ms+KT2.0 mg/L+NAA0.3 mg/L培养基中,枣苗长势壮、叶片大、叶深绿；而接种于Ms+6-BA2.0 mg/L+NAA0.3 mg/L培养基中,组培苗较弱,增殖系数较高为2.7。将继代增殖的再生芽接种在生根培养基上诱导生根,最佳的生根培养基为1/2MS+NAA0.5mg/L,培养40d时生根率达95.56%。6.以灰枣组培苗叶片为材料,研究了不同叶片来源、碳源、叶片放置方式、暗培养时间、乙烯抑制剂等处理对叶片再生的影响。结果表明,叶片直接再生不定芽的适宜培养基为WPM +TDZ 0.5mg/L+NAA0.2mg/L；最佳叶片来源为灰枣组培苗中部平展,大而厚叶片；近叶柄处再生率比近叶尖处高59.74%；以远轴面接触培养基叶片再生率较高为69.61%；蔗糖浓度为40g/L可降低再生芽的玻璃化程度；叶片暗培养时间以10d叶片再生率较高为81.6%；0.5mg/L AgNO3和0.1 mg/L的STS均提高了灰枣叶片再生率,再生率分别为82.25%,91.26%。7.以灰枣叶片为受体,研究了卡那霉素对叶片再生的影响、头孢霉素的抑菌效果及不同共培养时间对转化率的影响。结果表明,当灰枣叶片再生培养基中添加40mg/L卡那霉素能抑制不定芽的分化,40mg/L为灰枣遗传转化中的临界浓度；不同头孢霉素抑菌浓度效果差异明显,高浓度的头孢(大于等于500mg/L)才能抑制农杆菌的生长；灰枣遗传转化中,共培养时间应较短2d能获得较高转化率21.1%；对获得的灰枣转化植株叶片进行组织化学染色分析发现,叶片叶脉为蓝色,初步确定外源基因已转入灰枣基因组中。更多还原

【Abstract】 Kiwifurit (Actinidia) has high nutritional value and special flavor. It is known as the king of fruit. Jujube (Zizyphus jujuba Mill.) is the first major dry fruit tree in China. However, kiwifruit is a dioecious species with a complicated genetic background, and jujube has smaller buds, lower natural fruit setting rate and higher embryo abortion rate. Conventional breeding was resulted in slow progress owing to long growth and reproductive cycle. Modern biotechnology creates a new path for kiwifruit and jujube breeding.In this paper, differential red flesh Kiwifurit in Henan was used as experimental materials.An efficient regeneration and genetic transformation system were developed and transgenic plants expressing antibacterial peptide D gene were obtained.The PCR and Southern blot analysis showed that the APD foreign gene had been integrated into kiwifruit genomes. In our study, leaves were used as explants for genetic transformation of Zizyphus jujuba’Huizao’, and an efficient regeneration and genetic transformation system were established. The main results were as follow:1. Using leaves of Actinidia chinensis’Funiu 95-2’as explants, influences of different plant growth regulators on regeneration, proliferation and rooting of adventitious bud were considered, and an efficient regeneration system was developed. The results indicated that frequency (87.5%) of adventitious shoot induction was obtained when leaves were incubated on MS medium supplemented with 1.0 mg/L ZT and 0.3 mg/L NAA. The best result for shoot proliferation was obtained in MS medium supplemented with 2.0 mg/L 6-BA plus 0.5 mg/L NAA, on which the highest multiplication coefficient was 4.2.1/2 MS medium with 2.0 mg/LNAA was suitable for rooting, and the rate of rooting was 100%.2. Using leaves of Actinidia chinensis’Funiu 95-2’as explants, effects of antibiotics on explant growth were studied. The results showed both of differentiation rate of callus and adventitious shoots were zero when 20mg/L kanamycin was added to media, so the optimal concentration kanamycin was 20mg/L in the genetic transformation of leaf of Actinidia chinensis. Roots were not induced from media containing 30mg/L Kanamycin. Cefotaxime and Carbenicillin had negative effects on adventitious shoot differentiation, while inhabitation of Carbenicillin was stronger than that of Cefotaxime. According to our experiment results,400mg/L Cefotaxime was an optimum antibiotics to genetic transformation by Agrobacteriumin of Actinidia chinensis’Funiu 95-2’.3. The results of genetic transformation of leaves of Acitnidia chinensis’Funiu 95-2’showed that the highest transient expression frequency (92.2%) was obtained when leaf explants were pre-cultured for 3 days, and then infected for 10 min with Agrobacterium tumefaciens (OD600= 0.3) by vacuum infiltration, and finally co-cultured for 4 days. PCR detection and GUS histochemical staining initially proved that the foreign gene had been integrated into the Acitnidia chinensis genome.4. The selection protocl of resistant shoots after co-cultured was investigated, and the transgenic plants with APD gene were detected by molecular biology. The results showed frequency of resistant shoot regeneration in election media with gradually increased kanamycin concentration was 28.4% higher than that in the stable pressure. PCR and Southern blot analysis of partial Kan-resistant plants indicated that the foreign gene had been integrated into kiwifruit genomes.5. Using vegetative shoots of jujube as materials, the influences of antibiotics on controlling contamination and different plant growth regulators on propagation and rooting were studied. The results showed that contamination rate remained as high as 97.8% when explants were firstly sterilized in 70% alcohol for 30s and then in 0.1% mercury dichloride for 10-15 min. Contamination rate of explants was reduced to zero when media was supplemented with 150mg/L or 200mg/L cefotaxime. The similar result was obtained with streptomycin, while streptomycin could inhibit developing of axillary buds. In the initial culture phase, strong vegetative shoots of jujube were obtained from the MS media containing 2.0 mg/L KT and 0.1mg/L NAA. In the subculture phase, the plantlets with fully expanded and dark green leaves grew stronger in MS media including 2.0 mg/L KT plus 0.3 mg/L NAA.However, the plantlets were weaker when sub-cultured in MS medium supplemented 2.0 mg/L 6-BA and 0.3 mg/L NAA, and the multiplication coefficient was 2.7. The half MS media containing 0.5mg/L NAA was optimal for rooting with 95.56% rooting rate. 6. The effects of basal medium, leaf maturity, leaf position on the shoot, orientation of explant contacting medium, sucrose concentration, days of dark incubation, and ethylene inhibitors on leaf regeneration were investigated. The results indicated that WPM supplemented with 0.5mg/L TDZ plus 0.2mg/L NAA was effective for leaf explant regeneration of’Huizao’. The optimal explant was young, and thick expanded leaves from the intermediate shoot. More adventitious buds developed at the petiole portion of the midrib and surrounding area, and regeneration capacity increased with 59.74% from the tip towards the base of leaf. The frequency of regeneration of’Huizao’was 16.81% higher when the abaxial surface of the explants was in contact with the medium compared that the adaxial surface of the explants was in contact with the medium. When sucrose concentration was 40g/L in the medium, the hyperhydric degree of adventitious buds was reduced. A 10-day culture in the dark could increase the frequency of regeneration with 81.6%. The addition of 0.5 mg/L AgNO3 and 0.1mg/LSTS to the medium could pomote leaf regeneration, and the frequency of regeneration of leaf explants was 82.25%,91.26% respectively.7. Influence of kanamycin on regeneration of leaves, cefotaxime on inhibiting Agrobacterium, and days of co-culture on transformation frequency were investigated. The results showed that the differentiation rate of adventitious bud was zero when kanamycin concentration in the medium was increased to 40mg/L, so it was the critical concentration in the genetic transformation of’Huizao’. The effects of different concentrations of cefotaxime were divers, and higher concentration (500mg/L) of cefotaxime could inhibit Agrobacterium growth. The transformation rate of Z.jujuba’Huizao’was 21.1% after 2 days of co-culture.The veins of transgenic plantlets were stained blue after histochemical assay, which preliminarily proved that the foreign gene had been integrated into the genomes of’Huizao’更多还原