【作者】 单世华；

【导师】 庄伟建；

【作者基本信息】 福建农林大学 ， 作物遗传育种， 2003， 博士

【摘要】 花生是重要的油料作物和经济作物，黄曲霉病是影响花生生产、加工和贸易的重要病害，在我国南方花生产区黄曲霉病尤其严重。目前在花生及其近缘种尚未发现高抗乃至免疫黄曲霉病的种质，单纯通过常规育种无法解决该问题，而通过基因工程技术将外源抗病基因转入花生栽培种则应具有一定的可行性。本研究即是以农杆菌LBA4404为介导将几丁质酶和β-1，3-葡聚糖酶双价基因转入花生栽培种泉花10号和金花1012，以提高其对黄曲霉病的抗性。研究结果表明： 胚轴带部分子叶外植体在MS+5mg／L BA培养基有利于丛生芽潜伏茎诱导和伸长；减小子叶体积可延缓丛生芽伸长速度，促使其尽早吸收培养基中养分，且对芽诱导数量无影响。此后幼茎叶节在高浓度BA的MS培养基可产生大量丛生芽。胚轴外植体体胚诱导效果受种子收后贮藏时间的制约，新收获并干燥的花生种子体胚诱导率低，收后贮藏4～6月种子体胚诱导数量和速度均优于新收获种子；MS+40mg／L 2，4-D+0.5mg／L KT培养基既可诱导愈伤大量产生又能诱导体胚分化，将分化体胚直接转入MS+5mg／L BA诱导芽伸长可缩短试验周期约10d。MS+5mg／L BA+2mg／L NAA和MS+5mg／L BA+3mg／L NAA均可诱导胚小叶外植体产生愈伤和大量不定芽，后者产生的愈伤和芽相对较多，但褐化稍重。 研究表明，200mg／L Km可作为花生芽伸长生长的最低抑制浓度，50mg／L Km为外植体生根的最低抑制浓度。不同花生外植体遗传转化结果表明：通过丛生芽再生途径利用Km筛选得到21株转化植株，其中泉花10号11株，平均转化率1.6％，金花1012为10株，平均转化率1.5％。PCR检测和PCR-Southern杂交证明目的基因已整合入花生基因组，目前得到数粒T₁代种子进行抢救性繁殖，有待于分离情况测定和抗病性鉴定。花生丛生芽再生途径的基因转化以预培养0、1d效果较好，其中预培养1d结合外植体 福建农林大学博士学位论文划伤转化更佳;菌液侵染时间以10¹5min相对利于外源基因转化。胚状体和胚小叶再生途径基因转化目前仅分别得到数十粒抗性芽，需进一步诱导或伸长培养鉴定，二者均以10^ZOmin侵染时间较好，共培养均以72hr利于得到抗性芽，并且胚轴外植体基因转化时在体胚诱导伸长阶段进行Km筛选对提高抗性芽数量有利。子叶外植体转化褐化现象较严重，种子的新鲜程度和品种类型对转化有一定影响，目前仅得到1株抗性植株需进一步鉴定。研究发现，所使用的两品种间在不同的转化处理中也有一定差异。针对原有携带质粒载体的农杆菌菌株对抗生素不敏感的特点进行了菌株转换，质粒载体转入新的农杆菌菌株后，在含适宜浓度抗生素的固/液体YEP培养基皆完全被抑制，从而被成功用于本研究。 为比较研究外源基因转化情况及抗性植株生长状况，选择烟草品种翠碧1号进行相同基因转化试验，PCR检测得到10株左右阳性植株，转化率为34⁷0%。研究发现，3^smin侵染时间较好，工程菌液浓度以OD600为0.5转化率最高。目前转化植株已得到种子，对后代的分离情况和抗病性鉴定也随后进行。更多还原

【Abstract】 Peanut is one of the important oil and economic crop. A flavus infection, an worldwide problem, affects the production, processing and trading of peanut, and is serious in South China because of the special climate. As no germplasm highly resistant or immune has been found out so far, the problem can not be solved by traditional breeding method, but it could possibly be solved by transforming antifungal gene to peanut cultivars. Based on this method, the study has transformed the binary antifungle genes, Chitinase and P -1,3-Glucnase gene, mediated with A. tumefaciens into peanut cultivars, Quanhua No. 10 and Jinhua 1012, to improve their anti-invasion potential. The results were showed as below.The regeneration system of four kinds of peanut explants were partially modified and complemented in this research, including advantitious shoots(As), somatic embryogenesis(Se), embryo leaflet(El) and cotyledon adventitious shoots(Cs). MS medium containing 5mg/L BA was demonstrated again as the relative optimum medium to induce shoots differentiation and growth. It has some merit to delay shoots elongation when minifing the remnant cotyledon in A.s explants preparation, for it made the explants earlier to assimilate the nutrition in MS medium without ill effect on shoots induction number. And, many adventitious shoots were seen differentiating from the leaflets sampleing from the above shoots in MS with high BA. There was close relationship between Se induction efficiency and peanut post-harvest time. The result showed that the indued buds number and velocity from Se explants of peanut seed stored for about 4 to 6 months were more than that of newly mature seed harvested. Much callus and many somatic embryos could be induced and differentiated in MS medium containing 40mg/L 2,4-d and 0.5mg/L KT, and the regeneration period could be shrunk 10d earlier for somatic embryos when being transferred to MS+5mg/L medium to induce buds elongation directly. MS medium containing both 5mg/L BA+2mg/L NAA or 5mg/L BA+3mg/L NAA could induce much callus and adventitious buds from El explants, and the latter medium could induce much more callus and buds than the former, but with more degree of browning.Km-resistance trial showed that buds elongation could be inhibited to the maximal extend in 200mg/L of Km in medium, and roots could not grow out in50mg/L of Km. The transgenic research studies were made with four kinds of explants as mentioned above, results showed that 21 positive transformants were got assayed by PCR method for As transgenic method, 11 plants in Quanhua No. 10 and 10 in Jinhua No. 1012. It was verified that the target genes had been integrated into the Genomic DNA of transformants with PCR-Southern blot method. Now, several descendant seeds from the TO transformants are being propagated in culture medium to be assayed inheritance and diverse of the target genes, and the resistance to Aflavus. It was not good to invade the explants more than 15 minutes with A. tumefaciens liquid. In addition, no or only Id pre-cultivation could improve transformation efficiency, but 2d pre-cultivation didn’t show such result. Transformants have not yet been got through Se and El transformation ways but several scores of Km-resistance buds have been obtained and needed following induction and assay, and the better transformation efficiency was seen with 10~20min invasion in A. tumefaciens liquid and 72hr co-cultivation after it. Especially, it was found that more transformants would be got when Km screening more carried in buds elongation stage in Se transformation way. Although labouring transformtion trials were conducted in Cs method, only 1 Km-resistance explant was got, and the most limited factor was browning problem except for seed vigor and cultivars difference. In addition, there were some difference between the two used peanut cultivars during transformation. The A. tumefaciens strains was exchanged as the former strain was insensitive to biotics.In order to observe the similarity and compare the different transformat更多还原