【作者】 王英；

【导师】 师校欣；

【作者基本信息】 河北农业大学 ， 果树生物技术， 2009， 硕士

【摘要】 本研究以苹果(Malus domestica Borkh)主栽品种嘠拉(Gala)和富士(Fuji)组培苗为试材,研究了叶片离体再生和叶盘法遗传转化的若干影响因素,优化了苹果叶片离体再生不定芽系统和遗传转化系统,获得了携带β-1,3-葡聚糖酶基因的富士转化株系,并对嘎拉和富士转化株系进行了PCR,PCR-Sourthern和RT-PCR检测,成功获得了有目的基因表达的嘎拉和富士转化株系。进行接种苹果轮纹抗病检测鉴定时,转化株的β-1,3-葡聚糖酶、PAL和PPO活性较非转化株高,具有较强的抗病性。试验结果表明:1.富士的不定芽再生培养基中直接加入10.0～15.0mg/L SNP可以显著提高其再生效率。2.农杆菌在不加入抗生素的培养基中进行二次悬浮时,部分农杆菌会发生质粒丢失现象,随悬浮培养时间的延长,丢失率显著增高;质粒丢失程度与目的基因、启动子和农杆菌菌株的组合有关;同时,在0.5 h～6.0 h悬浮时间范围内,质粒丢失主要与菌液浓度有关,而与悬浮时间关系不大;直接吸取一悬液或离心后分别悬浮于细菌培养液和植物培养液等不同的悬浮培养方式下质粒稳定性没有差别。3.研究了影响农杆菌介导遗传转化的各种因素,优化了遗传转化途径:外植体在蔗糖浓度为60～90g/L的分化培养基上预培养1~2d;农杆菌二次悬浮后OD600值0.3～0.4时在摇床上晃动侵染8min;干燥共培养2d后进行脱菌、选择、筛选。4.共有12个富士转化株系通过了Kan抗性检测,具体包括三个方面:①转化株在附加Kan50mg/L的继代培养基上能够连续增殖继代,生长正常。②转化株叶片在附加或未加Kan50mg/L的再生培养基上的再生能力无明显差异。③转化株在附加Kan50mg/L的生根培养基中能正常生根。5.通过Kan抗性筛选的12个富士株系经PCR检测有11个株系呈现阳性,再经PCR-Southern blot检测,证明17个嘎拉转化株系和11个富士转化株系的检测结果全部呈现阳性,证明β-1,3-葡聚糖酶基因已经整合到嘎拉和富士植株基因组中。RT-PCR检测显示,β-1,3-葡聚糖酶基因在17个嘎拉转化株系都有不同量的表达,而只在3个富士转化株系中有少量表达。6.苹果轮纹病菌在24～28℃较适宜菌丝体生长,24℃时分生孢子囊和分生孢子发生最多;培养基pH5.0以及接种后在黑光灯下培养有利于分生孢子囊和分生孢子产生;菌丝体生长到一定程度后,刮除菌丝体对分生孢子囊和分生孢子的发生有显著促进作用。7.对部分嘎拉转化株系接种轮纹病菌,对与抗病性相关的生理指标进行检测,结果表明:转化株系的β-1,3-葡聚糖酶活性、PAL和PPO活性均高于非转化株,表现较强抗病性。更多还原

【Abstract】 In this study, the disease-resistant geneβ-1,3-glucanase was transferred into apple cultivar Gala and Fuji via Agrobacterium tumefaciens strain EHA 105. The adventitious bud regeneration system and genetic transformation system had been modified by studying the effect of explant types and status on the efficiency of regeneration and transformation. Transgenic plants were obtained and examined by PCR, PCR-Sourthern and RT-PCR. After inoculated Apple Ring Spot,the vigor ofβ-1,3-Glucanase, PAL and PPO in transformed plants were higher than untransformed plants, which proved that the transgenic plants were more disease-resistant. The result showed:1. Adding 10.0～15.0mg/L SNP into culture medium could improve the regeneration rate significantly.2. Some agrobacterium strains were plasmid instability when cultivated the second time under condition of liquid culture without presenting of antibiotic during genetic transformation via agrobacterium-mediated system, the losing increased along with the culture timing. No correlation was found between the losing and one single factor of target genes, promoters, and agrobacterium strains, but there was a correlation between the losing and combination of target genes, promoters, and agrobacterium strains. The losing was mainly related to the concentration of plasmids but liquid culture timing during the culture of 0.5 h~6.0 h. No difference was found in terms of the plasmid losing when liquid culture was done by taken solution directly from previous culture or centrifugate, and no matter bacteria culture or MS3. By comparing the factors on agrobacterium-mediated gene transformation, it suggests the optimal protocol for gene transformation in apple would be: at first, the explants should be pre-cultured for one day or two on the inducing medium with 60～90mg/L saccharose; the bacterium concentration for inoculation should be cultivated twice in the medium without presenting of antibiotic, the appropriate bacterium concentration was OD600=0.3～0.4 around, and the co-cultivation time is about 8 minutes, co-cultured on the filter paper less for two days, and then, the explants were transferred onto the inducing medium. 4. There were twelve transgenic Fuji plantlets passed the kanamycin-resistant test. The kanamycin-resistant test of transgenic plantlets was based on the three main aspects:①The transgenic plantlets were able to be subcultured on media supplemented with 50mg/L kanamycin, continuously.②The leaf pieces of the transgenic plantlets showed as same regeneration ability on media containing 50mg/L kanamycin as on media without kanamycin.③The transgenic plantles were able to root normally on media supplemented with 50mg/L kanamycin.5. Choose the plantlets which pass the kanamycin-resistant test for tested by PCR, the result showed that eleven transgenic plantlets showed specific positive band as same as the plasmid control.And also made PCR-Southern blot for further tests, which proved that seventeen Gala and eleven Fuji also showed specific positive band as same as the plasmid, The result indicated that the Glu gene has been integrated into the gala apple genome. Then through RT-PCR, it proved that the Glu gene has been expressed more or less in seventeen Gala and expressed a little in three Fuji.6. The mycelia of Apple Ring Spot grew well under 24～28℃condition, and could generated conidiophores most. Adjusted culture medium pH to 5.0 and incubated under black light after inoculation could benefit to the conidiophore’s generation. The removing of mycelia after their growth to some extent had significant promoting effect on the conidiophore’s generation.7. After inoculated Apple Ring Spot, the vigor ofβ-1, 3-Glucanase, PAL and PPO in transformed plants were higher than untransformed plants, which proved that the transgenic plants were more disease-resistant.更多还原