【作者】 陈雁；

【导师】 孟金陵；

【作者基本信息】 华中农业大学 ， 发育生物学， 2002， 硕士

【摘要】 油菜是我国重要的油料作物，具有十分重要的经济意义。长江中下游地区作为我国油菜的主产区，4～5月油菜生育后期常遇高温多雨天气，菌核病发生严重，经济损失重大。来自于烟草的葡聚糖酶（Glucanase）基因，其编码产物能降解真菌的细胞壁结构成分葡聚糖。分子量为24kDa的植物蛋白AP24则属于细胞程序性死亡（PCD）的诱导蛋白渗透蛋白，对多种真菌也具明显抑制作用。将两基因分别与花椰菜花叶病毒35S启动子相连，根癌农杆菌介导法转化双低甘蓝型油菜品种6-203。对转基因T₂代和T₃代油菜经过田间两种方法的接种鉴定，即苗期离体叶接种和成株期茎杆插签，结合分子鉴定，筛选出外源转基因纯合的株系。 尝试新的转基因方法—活体转化法转化油菜，将自我调控叶片衰老系统P_SAG12-ipt融合基因以及gagal基因活体转化法分别转入华双3号，6-203。转基因获得的大量种子首先经卡那霉素（Km）筛选培养基快速筛选，而后继代培养，转入温室，移栽到大田。田间考察形态学性状，结合分子鉴定通过导入P_SAG12-ipt以延缓油菜功能叶片的衰老，延长持绿期；通过转入gagal基因旨在获得无花瓣油菜，通过降低油菜菌核病再次传播率达到抗病目的。 这三种转基因油菜的获得，将为抗病育种和抗衰老育种奠定基础。 本研究获得的主要结果如下： 1．油菜菌核病抗性的鉴定方法一定程度决定对油菜抗（耐）菌核病性的判断。通过两种接种方法的多次尝试和比较发现两种接种方法各有优缺点，综合起来作为判断油菜抗（耐）菌核病性的指标才是科学合理的。 2．尝试利用活体转化方法（in planta）转化油菜，证明该法在油菜上是可行的。此法不需经过组织培养，原位进行植物转化。转化效率可以满足基因转移及表达检测的需要。经PCR初步鉴定获得了转基因植株，并表达了预期性状。 3．转基因T₂代理应筛选出纯合株系。转基因抗病材料接种结果显示：在22个株系中发现一个株系有较强的抗病性，接种病斑直径比对照显著减小。初步估计是纯合株系，进一步的反转录PCR（RT-PCR）表明外源基因在植物体内发生转录。该株系T₃代又在两个时期的接种中表现很好，同样进行的RT-PCR和Northern blot都表明外源基因发生体内转录。 4．田间调查转基因抗衰老材料T₀代的单株，着重考察花期后每间隔一周株高和绿叶数的变化趋势，收获和进行考种，考察项目包括：茎粗，一次分枝数，二次分枝数，分枝部位，主枝荚果数，整枝荚果数，每果粒数，千粒重，单株产量，生物学产量等。结果发现，转基因植株生育后期叶片的持绿性显著延长；株高变化范围1.23～1.83m，单株荚果数（反应花总数量）变幅在161～605，千粒重变幅2.27～3.95g生物学产量21.4～104.229。T:代选到一个优良株系，在Tl代表现良好，叶片持绿期延长，有很强的营养生长优势，lr[j目PCR鉴定为阳性。T:代时该株系内每一单株无一例外的表现一致，包括生育期，I]一十片持绿时间等，证明为一个优良株系。5.转基因无花瓣油菜由于环境（光照，气温）等因素综合影响，表型很难稳定遗传。仅在T。代观察到无花瓣现象，但这些无花瓣材料往往发生许多不利性状的突变，诸如植株矮化，株型矮缩，花序无分枝，育性减弱:初花期雌蕊外露，盛花期花量极少;不能正常结果，套袋自交后少数能结实，但结实性低，多数是瘪粒。这些突变产生的原因可能是T一DNA插入植物基因组时，引起植物基因组的重排，导致不良睦状的产生。此外，外源基因插入的位点及插入拷贝数复杂性也影响其表达。更多还原

【Abstract】 Sclerotinia stem rot that caused by Sclerotinia sclerotiorum is the most devastating disease of rapeseed (Brassica napus L.) in China especially in Yangtze River Area and leads to great yield loss every year. Glucanase and AP24, which come from tobacco both, have broad spectrum of antifungal activity. AP24 and Glu were combined with CaMV35S promoter and were transferred into Brassica napus via Agrobacterium tumefaciens. We took two different inoculation methods during two different periods to estimate the resistance to Sclerotinia sclerotiorum of T2 and Ta progenies. That is the detached leaves inoculation and inoculation of stem by tooth sticks clinged with pathogene. The plants exhibited an increased resistance against the fungal attack compared with the no transgenic plants during two inoculations. The candidate plants were conducted with RT-PCR and Northern blot analysis. The result indicated the increase in the transcriptional level of foreign genes.A method for in situ transformation-in planta transformation was also preceded. We attempted this new transformation method on a variety of Brassica napus, Hua Shuang No.3, and collected 1,800 seed grains after treated with Agrobacterium tumefaciens containing the chimeric PsAGn-IPT gene. The harveated seeds were screened on MS medium with Kanamycine. The Km -resistant seedlings were transplanted into soil in a greenhouse then in the field and developed into 70 adult plants. So, 3.8% of in-planta seeds were grown to mature plants. PCR analysis showed that 16 among 70 plants had been integrated into the PsAGn-IPT. Several agronomy traits on the 16 TO plants were investigated including the plant height, green leaf number on main stems, flower number, biomass, seed yield and weight of seed grains etc. The highest green leaf number that the plant can reached of each plant varied much, from 15 to 56 among the 16 plants. The plant height ranged from 1.23 to 1.83m. The weight of seed grains varied from 2.27 to 3.95g/1000grains. The flower number ranged from 161 to 605. Biomass changed much from 21.4 to 104.22g. We chosed a nicer plant from TO generation, and the following field investigation also displayed favorable characters such as the green leaf number, biomass etc.With the same in planta transformation method we got the transgenic gagal plants. During the flowering time the apetalous flower can be found. But some bad characters were detected at the same time, which was depicted as following: the shorter plant shape, the fewer branch, little flower, less seed yield et al.更多还原