【作者】 杜海莲；

【导师】 陈启锋；

【作者基本信息】 福建农林大学 ， 作物遗传育种， 2000， 博士

【摘要】 本研究首次将禽流感病毒（H₅N₁）基因h5n1a导入马铃薯获得表达，并优化了转化系统，为商品化生产转基因植物疫苗提供科学依据。 1、为了优化马铃薯（Solanum tuberosum L.）以块茎为受体的再生系统。着重对诱导分化和结薯培养基作了较系统的筛选。以MS为基本培养基，调整激素配比，得到M1、M2、M3、M4等4种培养基。比较试验结果以M4（MS＋2.0mg／L BA＋3.0mg／L ZT＋0.5mg／LNAA＋0.5mg／L GA₃）诱导块茎分化的效果最佳，丛芽分化频率达89.4％，分化时间提早约20天。同时筛选了诱导结薯的较优培养方法，获得使试管薯结薯数增加近1倍的壮苗和诱导结薯培养基组合（壮苗：1／2MS＋0.5mg／L BA＋0.5mg／L GA₃＋3％Suc；诱导结薯：MS＋2.0mg／LBA＋3.0mg／L ZT＋10％Suc），为获得高频转化奠定了基础。 2、构建了4个植物表达双元载体。p1301BG含有增强型绿色莹光蛋白基因egfp；p1301BΩG在egfp与35s启动子之间插入65bp的增强子序列Ω因子；p1301H5N1含有禽流感病毒基因h5n1a；p1301HBs含有乙肝表面抗原基因HBsAg。这4个载体均以35s为真核表达启动子，报告基因和目的基因以Tnos为终止子、植物筛选标记基因以35s PolyA加尾，融合成嵌合基因。原核抗性为Kan。分别用直接法导入农杆菌EHA105、LBA4404和AGL1。 3、初步比较了ZHFQ-2型火药式基因枪与农杆菌的转化效果，发现，该基因枪微弹分散性差，转化结果不稳定，对受体材料损伤较大，未能筛选到转化株；而农杆菌转化相对较稳定，并初步发现外源基因的瞬时表达大多集中在受伤和形成层部位。 4、对农杆菌转化条件的比较研究发现，菌株、菌体状态、稀 中文摘要 释度以及真空、激素和AS处理，均对转化有很大影响。对3个菌 株（LBA4404、EHA105和AGLI）的侵染性比较发现，用EHA105转化 马铃薯块茎，GUS瞬时表达效果更好；比较几种转化条件下的GUS 瞬时表达结果，筛选到可获得高且稳定的GUS瞬时表达的转化条 件：菌体用 Mg（MS＋0．sing／L GA3＋10umol／L AS）液体培养基稀释 4倍，与受体于28oC、100rpm振荡侵染20－30min，共培养时在分 化培养基上加 30卜 mol／L AS（乙酚丁香酮），16hr弱光照共培养 3d， 使GUS瞬时表达活性提高了3．4倍。 5、在进行抗生素基础抗性测定和转化体筛选时，比较马铃薯块茎 对Hgg和Kan两种筛选剂的敏感性，发现马铃薯块茎受体对Hnn更 敏感，因此选用Hgg为抗性筛选剂。在此基础上改进了筛选策略， 提出间段压力筛选法。该方法与常规的连续压力筛选法相比，大 大减低了基础抗性确定的主观性对转化效率的影响。使获得抗性 株的频率提高了82儿外源基因阳性率提高了2．4倍，获得转化株 的时间提早7－10天。 6、获得 HSNI转基因马铃薯株系 31个，并对其外源基因的整 合和表达做了系统检测。GUS表达活性结果表明，外源基因己插入 马铃薯基因组并得到表达，但株系间差异较大，说明外源基因整 合的随机性。用hsnla双引物扩增马铃薯基因组总DNA，得到与 目的基因大小一致的特异性片段。以DIG标记的hsnla探针进行 Southern杂交，进一步证实基因hsnla已整合到马铃薯基因组中， 且初步确定为单拷贝插入。 7、用HSNI 鼠单抗与马铃薯块茎蛋白粗提液进行抗原抗体反 应证实，在马铃薯中表达的外源基因具有抗原抗体反应活性，这 是禽流感病毒（HSNI）基因首次在植物中的表达。由于禽流感病 2 禽流感病毒（HSNI）基因（hjnla）在马铃薯中的转化与表达研究 毒m)是新病毒株，最近才得到其鼠的单克隆抗体，还末能 进行定量检测，以及动物免疫实验结果检测。 8、为了证实所建立的转化系统的有效性，本研究同时进行了 乙肝表面抗原＊BSAg)基因在马铃薯中的表达研究，并对结果做 了详细报道。在马铃薯中表达的乙肝表面抗原，经乙肝抗原检测 试剂合检测，具有较强的抗体结合活性，且初步测定表达的抗原 蛋白最高占马铃薯可溶性总蛋白的0．34“ 9、本试验虽然获得了预期结果，但对转基因马铃薯作为疫苗 的动物试验，以及遗传稳定性等试验等尚需时日，无法进行。虽 然我们同时进行了乙肝表面抗原基因HbsAg－s的转化试验，以检 验所设计的技术路线的有效性。但为了实现商品化的植物疫苗生 产目的，尚需进一步研究解决取材、方法、转化、筛选和检测等 一系更多还原

【Abstract】 Potato(So]anuni tuberosum L) plants were firstly transformed with the gene(h5nla) encoding an Avian Influena A (H5NI)Virus, and the gene was expressed in transgenic potato. Meanwhile, the transfer system was optimized in order to realize the aim of transgenic plant as low-cost vaccine production system. The results were as follows. 1. To optimize the regeneration system of potato using tuber disc as explants, it is necessary for the chosen media to have the high efficient regeneration shoots of tuber disc. Using MS as the basic medium and adjusting the hormone content and rate, four media were obtained which were Ml, M2, M3, M4. By comparing the regeneration rate of tuber discs on the four media, M4(MS-f-2.Omg/L BA+3.OmgIL ZT+0.5mgIL NAA+0.Smg/L GA3) was selected as the highest efficient one for shoots regeneration. And on M4 medium, ahead of time 20 days or so, shoots could be regeneraed from 89.4 percent tuber discs. Meanwhile care was taken to select more efficient media combination for making plants stronger and producing more tubers. The media were M7( I /2MS+0. 5mgIL BA+0. 5mg/L GA3+3%Suc) combinating with M14(MS?.Omg/L BA+3.Omg/L ZT+l0%Suc) , by which the number of tubers produced was twice as much as that on other media. The high efficient regeneration system is essential for high efficient transforma- tion system. 2. Four plant expression plasmids were constructed for transformation. P13O1BG was constructed for expressing green fluorescent protein gene(egfr); pl3OlB Q G was for enhanced 4 expression of eg4~, by inserting a 65bp enhancer( Q) between 35s promoter and gene egfp; p13OIH5NI was for expression of Avian Influena A (H5N1)Virus gene h5nla; pl3OlHBs was for expression a small subunit protein of Hepatitis B Surface Antigen(HBsAg). In the four plasmids, the CaMV35s promoter and nos terminator fragments were syncretized with reporter gene and targeted gene. The selectable maker gene terminated by polyA fragment for transformation of plants was hygromycin phosphotransferase gene which allows selection with hygromycin. And the selectable maker gene for plasmids was neomycin phosphotransferae gene, which allows selection with kanamycin. Agrobacterium strains LBA4404, EHAIO5 and AGL1 cells were transformed by the direct method with plasmids mentioned above prepared from Escherichia coil clones. 3. The transformation results mediated by gunpowder(ZHFQ-2), demonstrated?that the explants were seriously damaged during the transfer process, and no transfer plants were selected; By contrast, the results mediated by Agrobacterium were stable. 4. By comparing several conditions of transformation mediated by Agrobacterium, we found that the transfer efficiency was highly effected by strains, content of strain cells, hormone, and AS etc. Among the three strains (LBA4404, EH1O5 and AGL1), the invasiveness of the strain EHA 105 was the highest. The GUS transient activity under different transfer conditions showed that the following conditions selected were much more efficient for transformation: Agrobacteria that were resuspended and diluted with solution(MS+0.Smg/L GA3+AS I Oumol/L) four times, invaded potato tuber discs swirring at lOOrpmI28 0C for 20-30mm. Following co-cultivating, the explants were transfered to M4 medium with the addition of 30 u mo]IL AS更多还原