【作者】 关心；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 果树学， 2010， 硕士

【摘要】 乙二醛氧化酶(Glyoxal oxidase, VpGLOX)基因,为课题组前期通过mRNA差异显示技术及RACE(rapid amplification of cDNA ends)技术,从中国野生葡萄高抗白粉病的华东葡萄‘白河-35-1’(Vitis pseudoreticulata W.T. Wang‘Baihe-35-1’)中克隆到与抗白粉病相关的新基因。本研究以中国野生华东葡萄2个后代——高感白粉病‘6-12-2’,高抗白粉病‘6-12-6’,叶片为试材,通过农杆菌介导的真空渗透法将VpGLOX进行瞬时表达,对该基因进行功能分析;同时以‘6-12-2’,‘6-12-4’,‘6-12-6’,‘商南-2’(Vitis pseudoreticulata W.T.‘Shangnan-2’),‘湖南-1’(Vitis pseudoreticulata W.T.‘Hunan-1’),‘佳利酿’(Vitis vinifera cv.‘Carignane’),‘白河-35-1’为材料,运用并比较了多种遗传转化方法,对转基因植株进行检测。取得的主要结果如下:1.以培养6 w的‘6-12-2’,‘6-12-6’葡萄试管苗叶片为材料进行转化,通过GUS (β-glucuronidase)染色检测瞬时转化效率,结果表明,GUS报告基因在顶端第3-6片叶都可以表达,不同叶片部位造成的差异不显著,而第3-4片叶离体培养生长状态最佳。对上述4种野生型及转基因型叶片分别接种白粉菌,于3,5,7,12 d记录其菌丝发生过程,其中,12 d时可以明显观察到两个转基因型叶片内菌丝生长受到抑制。应用real-time PCR技术检测VpGLOX的表达,‘6-12-2’表达量只在转基因型叶片中增加,而且很快回复到原来水平;‘6-12-6’转基因型叶片中出现了第二个表达高峰。2.以‘6-12-2’为材料,研究TDZ,NAA不同组合对叶片、叶柄不定芽再生的影响。叶片在1/2MS + 1.0 mg·L-1 TDZ + 0.15 mg·L-1 NAA培养基上再生出不定芽,再生率为1.11%;叶柄在1/2MS + 2.0 mg·L-1 TDZ + 0.15 mg·L-1 NAA培养基上再生出不定芽,再生率为1.67%。生根成苗培养基为1/2MS + 0.15 mg·L-1 IBA + 0.02 mg·L-1 NAA。3.以‘湖南-1’(♀),‘商南-2’(♂)的花蕾为材料,研究葡萄胚状体再生途径并进行遗传转化。‘商南-2’在NN69 + 1.0 mg·L-1 2,4-D + 2.0 mg·L-1 6-BA培养基上诱导愈伤量最高,为8.06%,但之后转接到不同浓度组合的6-BA,NOA,IAA诱导愈伤培养基上,均没有胚状体形成;对其进行遗传转化研究,检测其绿色荧光蛋白(Green Fluorescent Protein, GFP)瞬时表达,结果表明,在花药单核靠边期进行原位转化,及转移到分芽繁殖培养基上1 w进行转化,‘湖南-1’,‘商南-2’转化率均可达到93.33%以上。4.以‘6-12-2’,‘6-12-6’为材料,分别对其花序、果实进行原位转化,研究该方法应用于葡萄的转基因效率。提取其种子、果皮及实生苗叶片DNA,进行PCR检测,结果表明没有得到转基因植株。5.以‘6-12-2’,‘6-12-4’,‘6-12-6’,‘商南-2’,‘佳利酿’,‘白河-35-1’为材料,研究微茎尖及带芽茎段快繁成苗途径。最适条件有,C2D4B液体培养基上80 rpm震荡,光照培养,随后转移到添加GA 0.5 mg·L-1的C2D4B固体培养基上,遗传转化前不需经过黄化培养;离体节间丛生芽生长的最适培养基为1/2MS + 2.0 mg·L-1 TDZ + 0.5 mg·L-1 NAA。6.对遗传转化体系进行优化。农杆菌浓度OD600为0.4,侵染10 min,共培养3 d有利于获得最高遗传转化效率。7.经潮霉素筛选,对抗性植株经PCR和PCR-Southern blot检测,表明目的基因已整合到植株基因组中,共获得‘6-12-2’45个转基因株系;‘6-12-4’17个转基因株系;‘6-12-6’10个转基因株系;‘白河-35-1’3个转基因株系;‘佳利酿’4个转基因株系。8.对转基因植株‘6-12-2’1株,‘白河-35-1’1株,进行炼苗、移栽。更多还原

【Abstract】 Vitis pseudoreticulata glyoxal oxidase (VpGLOX) was previously isolated from the Chinese wild vine Vitis pseudoreticulata accession‘Baihe-35-1’during a screen for genes that are upregulated in response to infection with grapevine powdery mildew(Uncinula necator, PM). In the present study, a possible function of VpGLOX for defence against PM was investigated using Agrobacterium-mediated transient expression. After optimizing agro-infiltration, VpGLOX was transiently overexpressed in leaves of either PM-susceptible (accession‘6-12-2’) or PM-resistant (accession‘6-12-6’) plants;‘6-12-2’,‘6-12-4’,‘6-12-6’, which belongs to segregating populations derived from a Vitis pseudoreticulata‘Baihe-35-1’×Vitis vinifera cv.‘Carignane’cross, Vitis pseudoreticulata W.T.‘Shangnan-2’, Vitis pseudoreticulata W.T.‘Hunan-1’,‘Carignane’, and‘Baihe-35-1’were used for Agrobacterium-mediated genetic transformation by a variety of genetic transformation methods, and then the putative transformed grape lines were confirmed. The maily results were as follows:1. The efficiency of transfection was verified using aβ-glucuronidase (GUS) reporter and was found to comprise most leaf areas regardless of the initial leaf position on leaves of the third to the sixth leaves beneath the apex from 6-week old in vitro grown plants. The third and fourth leaves beneath the apex were most fit for infiltrated since they were fully expanded and vigorous. Upon infection with U. necator, clear differences were observed with respect to hyphal development between agro-infiltrated leaves and control groups of both, the susceptible and the resistant, genotypes. At 12 dpi, the overexpressed‘6-12-2’and‘6-12-6’presented sparse hyphae compared to the profuse fungal growth in control‘6-12-2’and‘6-12-6’. The expression of VpGLOX was followed by real-time PCR in both genotypes. Whereas‘6-12-2’expression was found to increase only in transfected leaves and remained transient,‘6-12-6’appeared a second peak later in transfected leaves.2. Regeneration systerm from leaf and petiole of‘6-12-2’and‘6-12-6’using TDZ and NAA was studied. The results were: adventitious bud from leaves of‘6-12-2’could regenerate on 1/2MS medium with 1.0 mg·L-1 TDZ and 0.15 mg·L-1 NAA, but little regenerating rate 1.11%;adventitious bud from petioles of‘6-12-6’could regenerate on 1/2MS medium with 2.0 mg·L-1 TDZ and 0.15 mg·L-1 NAA, regenerating rate of 1.67%. The medium fitted for roots inducing was 1/2MS + 0.15 mg·L-1 IBA + 0.02 mg·L-1 NAA.3. Somatic embryogenesis and plant regeneration from flowers of‘Hunan-1’and‘Shangnan-2’were studied.‘Shangnan-2’cultured on medium NN69 + 1.0 mg·L-1 2,4-D + 2.0 mg·L-1 6-BA were best for callus induction, with the induction rate of 8.06%. Then they were transferred to embryo proliferation medium supplementing combination of hormones, 6-BA,NOA and IAA, which turned out to be 0% formation of the somatic embryos. Transient-expression of green fluorescent protein (GFP) gene in transgenic flowers at different developmental stages showed that floweral dip at anthers’uninucleate pollen stage, or infection after 1 week on somatic embryos formation and proliferation, the trangenic rate could be over 93.33%.4. The study on in-planta Agrobacterium-mediated transgenic efficiency on the flowers and fruits of‘6-12-2’and‘6-12-6’were performed. The DNA from seeds, peels and seedling leaves were extracted. But none of them could be detected the putative VpGLOX by PCR analysis.5. In this study, shoot apical meristem explants of‘6-12-2’,‘6-12-4’,‘6-12-6’,‘Shangnan-2’and‘Baihe-35-1’were used for Agrobacterium-mediated genetic transformation. For the micropropagation shoots,which was determined should be in liquid C2D4B medium at 80 rpm constant orbital shaking. Then cultures were placed on fresh solidified C2D4B medium with 0.5 mg·L-1 GA. Besides, it did not need to be subjected to a dark growth phase before transformation. For the in vitro internode explants, which could grow well on 1/2MS medium with 2.0 mg·L-1 TDZ and 0.5 mg·L-1 NAA.6. The highest transformation frequency was obtained when explants were infected for 10 min with the concentration of Agrobacterium tunefaciens OD (600 nm) reached a value of 0.4, and then co-cultivated for 3 d.7. Polymerase chain reaction (PCR) and PCR-Southern blot analyses were used to confirm putative transformed grape lines. Up to forty-five micropropagation shoots of‘6-12-2’, seventeen micropropagation shoots of‘6-12-4’, ten micropropagation shoots of‘6-12-6’, three micropropagation shoots of‘Baihe-35-1’, four micropropagation shoots of‘Carignane’, produced stable transgenic plants.8. One of‘6-12-2’and one of‘Baihe-35-1’have been domesticated and transplanted.更多还原