【作者】 牛庆霖；

【导师】 冯殿齐； 孙明高；

【作者基本信息】 山东农业大学 ， 林木遗传育种， 2011， 硕士

【摘要】 杏(Armeniaca vulgaris Lam.)系蔷薇科(Rosaceac)杏属植物。杏原产于我国,种质资源极为丰富,是中国栽培历史最久的果树之一。但由于春季低温和晚霜,常使花芽、花,甚至幼果受冻,产量和经济效益受到极大的损失,因此选育抗寒性强的品种有重要意义。近十几年来,国内外迅速发展的转基因技术已经在育种中得到了广泛的应用,并且已经取得了长足发展,转基因技术已经成为改良品种最有前途的技术方法之一。花粉管导入法在农作物上取得了令人鼓舞的成绩,但是在果树上应用的研究却极少,由于果树育种周期长,不断地摸索试验快速有效的育种方法是很有意义的。本研究对杏进行了抗寒基因转化试验研究,并成功将沙冬青抗寒基因AmEBP1通过花粉管成功转化入岱玉杏T0代中,并对组培苗进行生理指标测定,其主要研究内容如下:1.岱玉杏×凯特杏杂交亲和,坐果率达到57%,果实横径、纵经、侧径可达4.081cm,4.403cm,4.412cm,均高于自然授粉的岱玉杏,表现出现杂种优势。不同处理导入DNA对转化率有一定影响,切除1/2柱头滴注外源DNA能提高转化率,但对坐果率有一定影响;花粉基因处理的坐果率较正常花粉坐果率下降9.5%;2.采用不同浓度DNA导入对转化率有一定的影响,当DNA浓度分别为200μg/mL、500μg/mL、800μg/mL时,坐果率分别为21.5%、26.4%、12.7%,坐果率没有随着外源DNA浓度高低而逐渐升高或降低,而是以500μg/mL的浓度相对比较高,3个DNA浓度处理的坐果率远低于对照;导入DNA浓度在500μg/mL时,切除花柱1/2时转基因成功率最高,在第7天时能达到75%,60天后为30%,但基因导入后随时间的延长,阳性比率仍然出现下降的趋势,说明基因丢失仍然存在。3.采用改良的WPM培养基,对岱玉杏不同胚发育时期培养结果得出,在改良WPM培养基上,PF值高于0.3时才能形成愈伤,附加激素BA在0.6 mg/mL-0.8 mg/mL时,愈伤诱导率最高,在PF=0.7时成苗率最高;成熟胚在MS和改良WPM上均能萌动,但有叶小、卷曲、畸形、或玻璃化,根芽生长不良的情况出现。4.在对岱玉杏,大果杏,凯特杏,金太阳4个品种成熟胚培养结果表明,低温处理的时间和4个品种杏的萌发呈现正相关,随着低温处理时间的延长,4个品种杏的萌动迹象越发明显,成苗率都有提升。大果杏在处理60天后,大田出苗率基本可以达到100﹪,是最易成苗的品种。岱玉杏无论种胚腐烂率很高,在60﹪以上。5.本研究得到岱玉杏转基因苗木。16株经过PCR检测阳性,Southern杂交检测有11个分别出现1-3条杂交带,另一个转基因植株中只出现了微弱的杂交带,证明AmEBP1基因已经整合到T0代组培苗中。6.通过对转基因株系与未转基因株系组培苗抗寒生理对比,转基因株系(组培苗)LT50为-4.37℃<对照-4.12℃;SOD、POD活性与可溶性蛋白含量在-5℃到-10℃之前达到最大值,总体均有先升后降的趋势,并有转基因高于对照组的;游离脯氨酸含量总体上均呈现“上升”的趋势;随着温度的降低MDA含量增加,-5℃之后转基因植株增幅速率明显低于对照。7.试验饲喂转基因杏果后,鸡群与羊群没有明显病症,测量血常规得出对照组与转基因组血液中红细胞数、白细胞数、血红蛋白量、红细胞压积、淋巴细胞比率,其指标值均无显著差异(P>0.05),并未对动物造成病害。更多还原

【Abstract】 Apricot department rosaceae apricot genus, Apricot is one own fruit trees of China, it distributing east-north of our country. Because of poignant chance of the temperature in early spring, the bud, flower and even young fruit will be hurt by the low temperature, and the production of the apricot fruit will be lower.So it is very important to breeding frost-resistant strong varieties .Last ten years it has made rapid development in transgenic technology.It has been widely used in fruit breeding at home and abroad. Transgenic has become one of the most promising methods to improve fruit varieties.Now to import the DNA by pollen tube it has been successfully used in ccrops, but rarely in fruit, which is reason to the long breeding cycle in fruit.So it is very meaningful to find a effective methods in breeding. My study use Daiyu apricot transfer the cold hardiness gene (AmEBP1) from Sand Holly by pollen tube and then determin the physiological indexes of the seedling cultivation. We have gain results followed.1.After Haidai jade apricot♀cross Kate♂and transferred with outer DNA, fruit-bearing rate reached 57%, horizontal diameter,vertical diameter and side diameter got 4.081 cm, can reach 4.403 cm, 4.412 cm,which are higher than Tarzan haidai jade apricot in the natural pollination., There heterosis performance appears.We gained the highest rate of conversion of DNA by cutting the 1/2 tube of flower,But there were certain influence to fruit-bearing rate.So take the method of cutting the 1/2 instead. Fruit-bearing rate by pollen gene treatment were less down 9.5% than normal pollen fruit-bearing rate;2.Using different concentrations of DNA conversion rate have certain effect, when DNA concentration for 200μg/mL respectively/mL, 500μg/mL, 800μg/mL, fruit-bearing rate for targeting were 21.5%,26.4%, 12.7%. Fruit-bearing rate was not as exogenous DNA concentration levels and gradually raise or lower, but the most with 500μg/mL and cutting the 1/2 tube of flower.which can achieve 75% in the first seven days, 30% 60 days later. But there were still that gene lost trend still exists after gene transgenic. 3.Take the modified WPM media for the different embryos period, The results show that it can form callus only when PF>0.3,and the callus induction get the most by PF=0.7 and 6-BA=0.6 mg/ml. Mature embryos can budding on the the modified WPM and MS medias.But there appears little leaves, curly, deformity, or vitrification, stressing the situation. poor growth situations.4. The results show that low processing time and the germination of 4 species apricot was positively correlated. With the extension of treatment time, signs of germination about the 4 varieties of apricot are more obvious, seedling rate has increase. In dealing with Daguo apricot 60 days, the field emergence almost can reach 100%,which is the the most vulnerable to seedling specie.Taishan Daiyu has a high rate of embryo rot either under 4℃or 0℃,and it has the low rate of seedling. That shows embryo of Taishan Daiyu is dysplasia and perishable.5. We get some transgenic seedlings of Taishan Daiyu apricot in this study ,16 tested positive for PCR.And Southern hybridization with digoingenin labeled DNA probe results showed 11 tissue culture had 1-3 respectively.It showed that exogenous AmEBPI gene were successfully transformed into T0 tissue culture of Taishan Daiyu.6. By the cold resistance contrast detection between transgenic plants and non-transgenic plants,it showed that LT50 of the transgenic plants was -4.37℃<CK-4.12℃,and the activity of SOD,POD could reach the max range from -5℃to -10℃,which was the same to SP. Trend of indicators were first overall and then falling and the transgenic ones were higher than the contrast. The content of Pro showed a general up trend; As the temperature decreased the content of MDA increased and the growth rate of transgenic plants was significantly lower than contrast.7. Test feeding genetically modified apricots, chickens and flocks have no obvious symptoms, measuring blood tests that control group and transgenic group, the number of the the indexes such as RBC, WBC, HGB, HCT, LYM, were no significant difference (P > 0.05), which did not cause disease to animals.更多还原