【作者】 牛庆霖；

【导师】 曹帮华； 冯殿齐；

【作者基本信息】 山东农业大学 ， 森林培育， 2014， 博士

【摘要】 中国杏种资源丰富分布广泛，由于北方春季有低温和晚霜，容易使杏花、幼果受冻，产量和经济效益受到极大损失，因此选育高抗寒杏具有重要的意义。传统的植物育种方法在改善植物耐寒性方面取得的成功非常有限，利用传统方法选育抗寒杏，育种周期长，且抗寒效果不显著，且杏树的抗寒研究多集中在抗寒生理方面。利用现代生物学技术对杏进行抗寒性选育，更具高效性和针对性，可以加速高寒杏新种质的选育进程。植物低温条件下，体内蛋白质的合成与其抗寒性密切相关。沙冬青ErbB3结合蛋白基因（AmEBP1）是沙冬青(Ammopiptanthus mongolicus Cheng f．)中与低温胁迫相关的基因，低温条件下，能够抑制植物细胞翻译因子（eIF2a）的磷酸化，调控eIF2a的活性，保证蛋白质的正常合成，从而提高植物抗寒能力。本研究以沙冬青总RNA反转录所得cDNA第一条链为模板克隆了AmEBP1基因编码区序列，通过酶切和连接反应构建载体并转化到杏幼胚中，经幼胚培养获得转基因植株，并对转基因植株的耐寒性能进行了研究，为培育抗寒杏新种质打下了基础。主要研究如下：1）以沙冬青总RNA为模板，RT-PCR方法克隆得到目的基因AmEBP1，PCR与测序结果表明，与GeneBank公布的AmEBP1的cDNA（DQ519359）序列长度一致;2）构建原核表达载体pET-22b-AmEBP1转化大肠杆菌，0℃时转化AmEBP1的大肠杆菌存活率是转化空载体的3倍，可以看出，表达了AmEBP1的大肠杆菌具有明显较强的抗寒性；3）大果、岱玉等6个早熟杏品种，不完全花比率差异显著，其中以大果杏不完全花比率最低，仅为11.62%，红荷包最高，高达80.91%，岱玉50.08%，金太阳27.22%，新世纪25.43％；座果率大果杏最高可达29.34％，其次是金太阳，岱玉坐果为7.23%，最低的为红荷包；4）以大果杏花期后30d幼胚为材料，建立了杏幼胚培养体系。WPM培养基作为大果杏的基本培养基，蔗糖10g·L-1，琼脂8g·L-1，pH调至5.8；幼胚诱导芽丛的培养基选择WPM+6-BA0.8mg·L-1+NAA0.2mg·L-1；芽团生根培养基宜选择WPM+NAA0.1mg·L-1+6-BA0.8mg·L-1+IBA0.2mg·L-1培养基；5）构建植物表达载体pCAMBIA2300-AmEBP1，利用花粉管通道与基因枪结合方法转化大果杏，转化后的幼胚材料先接种WPM+Kan50mg·L-1筛选后，转移到WPM+6-BA0.8mg·L-1+NAA0.2mg·L-1培养基上诱导分化扩繁，再将扩繁材料转移到生根培养基上培养成苗；6）将WPM+Kan50mg·L-1筛选后的材料进行PCR检测，得到6个阳性植株；进一步Southern检测得出此6株系分别出现1-3条杂交带，Southern结果说明AmEBP1基因已经整合到这6个株系的基因组DNA中；将单拷贝转基因株系1、6进行RT-PCR结果显示AmEBP1基因在转录水平上表达；且多带扩繁后目的基因能稳定存在芽切苗中；7）将转基因阳性株系1、6在分化培养基上诱导芽丛，再将芽丛切成带个芽的芽团PCR检测，剔除阴性，继续分化诱导到一定基数，在诱导生根成苗，进行下一步抗寒试验。抗寒试验结果显示：相同低温（-4和-8℃）条件下，转基因植株均比对照表现出较高的成活率；随着处理温度的降低，转基因株系REC与MDA含量始终低于对照植株，但可溶性蛋白含量明显高于对照；转基因植株的低温半致死温度（LT50）比未转基因对照低2.13℃；抗寒试验的其他酶活、可溶性物质的含量结果也说明，转AmEBP1大果杏有较强的抗寒性（比对照）。8）转基因大果杏根际土与周生植物总DNA中未发现目标基因；转基因株系和非转基因株系的土壤微生物优势菌群种类相同，数量无显著差异。更多还原

【Abstract】 There are many resources about apricot seeds which are widely distributed in China.According to the cold spring and late frost in the North，a seriously impact on the flower andyoung fruit，which has a great damage to the products and economic benefits. So it is verygreat important to the cold-resistance breeding on apricot. Traditional plant breedingapproaches have had limited success in improving the cold tolerance. It cost a long time tobreed cold-resistance apricot with traditional method，even had no obvious effect. Much moreresearch focus on the cold resistant physiology about the apricot. Using the modernbiological technology to improve the cold-tolerance of plants with higher efficency andspecility, can accelerate the breeding process.The cold-resistance about the plant, is quite closely related to the synthesis of proteins underlow temperature conditions.The gene encoding ErbB3binding protein of Ammopiptanthusmongolicus（AmEBP1）associated with cold stress, which could inhibit the plant translationfactor（eIF2a）out of phosphorylation. AmEBP1also regulated the activity of the eIF2a tomake sure protein synthesis, which improved the cold resistance ability of the plant. In thisresearch, we designed specific primers by the nucleotide acid sequence of Ammopiptanthusmongolicus in Genbank. The gene coding sequence of AmEBP1were cloned from the totalRNA of the Ammopiptanthus mongolicus, then put the AmEBP1transformed into Daguoapricot to cultivate transgenic plants by immature embryos tissue culture. The main studywere as follows:1Ammopiptanthus total RNA was extracted from its leaves and the cDNA ofAmmopiptanthus gene AmEBP1was amplified from its total RNA by RT-PCR method.2The prokaryotic expression vector pET-22b-AmEBP1was constructed,which had beentransformed into E. coli. The survival rate of transgenic E. coli was higher than that of thenontransformed of E. coli at low temperatures.3Daguo,Daiyu and the other6apicot plants had significant difference on incomplete flowerratio. Daguo had the lowest rate at11.62%, but Honghebao highest above80.91%, Daiyu50.08%，Jintaiyang27.22%，Xinshiji25.43%. Daguo had the highest rate of set rate of fruit about29.34％, the second higher was Jintaiyang, the lowest was Honghebao, and7.23%onDaiyu.4A tissue culture system about apricot was established taking the young Daguo embryo formaterial.WPM medium was choosed as the basal culture medium for the young embryo ofDaguo with sucrose10g L-1，agar8g L-1，and pH5.8. WPM+6-BA0.8mg L-1+NAA0.2mg L-1was the differentiation medium to produce sprout and induce callus for buds;the best rooting medium was WPM+NAA0.1mg L-1+6-BA0.8mg L-1+IBA0.2mg L-1;5The plant expression vectors pCAMBIA2300-AmEBP1was constructed,which wassucceed to transform into Daguo embryos by pollen tube pathway and embryonic callus byparticle gun.Then the transformed material firstly grew in the WPM+Kan50mg L-1to getresistant plants,which secondly grew in the differentiation medium WPM0.8mg L-1+NAA0.2mg L-1to induce buds group to PCR. The last ones were put ionto the rooting medium togrew transgenic lines.6In total,6PCR testing plants were obtained from the kanamycin resistant ones.The put the6plants identified by Southern hybridization，then6lines showed positive hybridization results.These results indicated that the AmEBP1gene has integrated into the genomic DNA oftransgenic Daguo. RT-PCR analysis on line1and6which had single copy which showed thatthe imported AmEBP1gene expressed at transcriptional level in transgenic plants.7Put the transgene-positive lines1and6induced in the differentiation medium to inducebuds, then PCR identified the group buds, removed the negative ones, continued to inducedifferentiation. When got a lage number of materials,grew in the certain base to rooting fornext cold test. Results of the cold resistance tests pointed out that in the low temperaturetreatments（-4and-8℃）, the transgenic plants exhibited higher survival rates than theuntransformed control plants．Under the lower temperature cold treatment，the REC and MDAof transgenic plants was obviously lower than that of untransformed control plants. But thesoluble protein of transgenic lines was greatly higher than that of untransformed control lines.The LT50of transgenic lines was remarkable lower than that of the untransformed controlplants by2.13℃. The soluble protein, soluble sugars, free proline and MDA determinationresults of transgenic plants also supported that the imported AmEBP1gene increased the coldresistance of the Daguo apricot．8There are no target genes founded in the DNA of the rhizosphere soil and rhizosphere plantsabout the transgenic lines; Initial test of rhizosphere soil microbial effects of transgenic plantshowed that transgenic and non-transgenic lines modified strains of microorganism are bacteria, fungi and actinomycetes. There are no significant differences in the number, neitherin the varieties of the tested soils.更多还原