【作者】 刘凯；

【导师】 易干军； 王仁才；

【作者基本信息】 湖南农业大学 ， 果树学， 2012， 博士

【摘要】 香蕉(Musa SPP.)是重要的热带水果之一,也是世界上继水稻、小麦和玉米之后的第四大粮食作物。在我国华南地区,种植香蕉已成为农民的主要经济来源,在热区经济和农村社会发展中发挥着越来越重要的作用。香蕉对低温非常敏感,周期性的寒害给我国香蕉产业造成了巨大的经济损失。培育抗寒品种是解决香蕉寒害的重要途径。香蕉的主栽品种大多为三倍体,难以通过传统育种方式培育抗寒品种,而转基因技术为培育抗寒品种提供了很好的途径。拟南芥CBF1是COR(cold regulated)冷应答基因的转录激活因子,其发现为基因工程改良植物抗逆性提供了一条重要的途径。该类转录因子能与下游COR基因启动子上的核心元件CRT/DRE特异结合,促进下游一系列基因的表达,激活植物体内的多种耐逆性机制,从而提高转基因植株耐低温、干早和高盐等非生物胁迫的能力。在此基础之上,很多学者进行了拟南芥CBF1(AtCBF1)基因转化各种植物的研究,并取得了理想的抗寒效果。本研究利用农杆菌介导,转化由花椰菜花叶病毒35S启动子启动的AtCBF1基因到东莞大蕉(Musa spp.ABB group)和夫人指蕉(Musa spp.AA group)的胚性悬浮细胞(ECS)中,经体细胞再生途径,成功获得转基因植株。并对转基因植株的抗寒性进行了检测。构建了转基因大蕉的SSH-cDNA文库,以筛选与抗寒性相关的内源基因。这些研究结果对香蕉的抗寒育种具有重要的理论和实际意义。本研究获得的主要研究结果如下：1.根据GenBank中公布的AtCBFl基因cDNA全序列,设计特异引物从野生型拟南芥中扩增出目的基因的全序列,经比对与CBF1基因(NM118681)的同源性为100%。利用质粒PBI121及pCAMBIA1301构建其植物表达载体p1301-CBF1并转化农杆菌菌株EHA105,制备工程菌以转化香蕉。2.以大蕉(Musa spp.ABB group)和夫人指蕉(Musa spp.AA group)的未成熟雄花为外植体,诱导愈伤组织和胚性愈伤组织,对胚性愈伤组织进行液体培养成功地诱导出了这两个品种的ECSs。然后,建立了ECSs途径的再生体系。3.利用农杆菌EHA105介导大蕉(Musa spp.ABB group)和夫人指蕉(Musa spp.AA group)的ECSs,侵染和共培养之后,在含有5mg/L潮霉素(Hyg)和400mg/L头孢菌素(Cef)的MS培养基中进行体胚筛选和萌发。萌发的抗性胚生根后,获得的抗性植株。4.经GUS组织化学染色和PCR扩增检测,获得了大蕉6个转基因株系,共53株,10个株系的转基因夫人指蕉,共102株,证明了AtCBF1基因已整合至香蕉的基因组内。形态学观察发现,转基因大蕉和夫人指蕉植株均表现出矮化、叶片增厚及叶色深绿等表型。5.利用RT-PCR和qRT-PCR对外源基因AtCBF1的表达进行定性和定量检测,结果验证了AtCBF1基因在转基因大蕉和夫人指蕉中均获得了表达,同时qRT-PCR的结果表明,AtCBF1基因的表达水平在各转基因株系中存在差异。6.在7℃低温下处理5天,对2个转基因大蕉株系(T1和T3)和2个转基因夫人指蕉株系(L1和L4)进行抗寒性相关的生理生化指标测定。结果显示,转基因大蕉和夫人指蕉的离子渗漏率、丙二醛(MDA)含量均要低于对照植株；而转基因大蕉的SOD活性高于对照植株,脯氨酸含量、可溶性糖含量和叶片的相对含水量在转基因大蕉和夫人指蕉中均高于对照植株。7.各20株转基因大蕉和夫人指蕉置于4℃低温分别处理5天和3天后,观察其冷害症状。转基因大蕉没有出现冷害症状,而对照已出现了萎焉脱水症状,但均没有发现冷害致死现象。转基因夫人指蕉低温处理后叶片稍微卷曲,但没有表现出冷害症状,而对照植株出现严重脱水,25℃恢复生长3天后,高达90%对照植株死亡,而转基因植株无死亡的现象。这些结果直观显示,过量表达AtCBF1基因的转基因大蕉和夫人指蕉的抗寒性均获得了提高。8.本研究构建了转基因大蕉的SSH-cDNA文库,从文库中筛选到了Clp基因和乌头酸水合酶基因,这两个基因均与抗逆性有关。利用qRT-PCR对Clp基因的表达进行验证发现,Clp基因在转基因植株内表现上调。因此推测,AtCBF1蛋白可能调控了内源基因Clp和乌头酸水合酶基因等的表达,提高了转基因植株的抗寒性,需要进一步的验证。更多还原

【Abstract】 Banana（Musa spp.） is one of important tropical fruits, and also the fouth largest food crops after rice, maize and wheat in the world.Planting banana is a main source of income for persant in Southern China.which plays more and more important role in tropical economy and rural development.Banana is very sensitive to low-temperature and periodic cold injury cause huge financial losses to banana industry in China.At present, breeding cold tolerance varieties is an important approach to solve banana cold injury.The leading cultivars of banana are mostly triploid, which lead to difficultly obtain cold tolerance varities through tranditional breeding. However, using transgenic technology provides a good way for cultivating cold tolerance varities. Arabidopsis CBF1is a transcription factor which activates downstream COR （cold regulated） genes and provides an important pathway for improving plant stress resistance by genetic engineering.This transcription factor can combine cis-element （CRT/DRE） of promoter of downstream COR genes to induce expression and activate various mechanisms of stress resistance in plants for improving tolerance to low-temperature, drought, high salt stress and so on. On the basic of this, there were many scholars who introduced AtCBF1gene into various kinds of plants and initiated many cold resistant varieties.AtCBFl driven by a cauliflower mosaic virus35S promoter was introduced into banana plants by Agrobacterium-mediated transformation of ECSs of Dongguandajiao（Musa spp.ABB group） and Ladys’ fingers.（Musa spp.AA group）. Transgenic plants were obtained through somatic embryo regeneration approach. Cold tolerance of transgenic plants were detected in this thesis. At the same time,We constructed SSH-cDNA library of transgenic Dajiao plant to screen endogenous genes relative to cold tolerance in Dajiao banana.These results could supply theoretical and pratical significance for cold resistance breeding of banana. The main results were as follows:1. The target gene was amplified from wild type Arabidopsis with specific primers through cDNA complete sequence of AtCBF1gene published in GenBank. which shared100%homology with CBF1gene（NM₁18681）.Plant expression vector （p1301-CBFl）of AtCBF1gene were built by plasmid PBI121and pCAMBIA1301.which was used to transform Agrobacterium strain EHA105. and Engineering bacterium was in preparation for transforming ECSs of banana.2.The immature male flowers of Musa ABB cv.Dongguandajiao and Musa AA cv.Lady’s fingers were used as explants to induce calluses and embryogenic calluses.After that, ECSs were successfully induced in liquid culture by embryogenic calluses.Then, regeneration system of ECSs were established.3. After ECSs of banana cultivar Dajiao （ABB） and Lady’fingers（AA） were infected and co-cultured with EHA105harboring p1301-CBF1plant expression vector, resistant somatic embryos were screened and germinated in MS medium including5mg/L hygromycin（Hyg）and400mg/L cefotaxime（Cef）. Afterwards.the resistant embryos were rooted in rooting medium and resistant banana plants were obtained.4. The obtained resistant plants were identified by GUS histochemical staining and PCR amplification.Six transgenic lines of Dajiao and ten transgenic lines of Lady’s fingers had been acquired with number of fifty-three and one hundred and three plants, respectively,which confirmed that AtCBF1gene had been introduced into genome of bananas. Morphological observation displayed that all transgenic plants existed retardation in their growth, showing a kind of dwarfing.and their leaves were thicker and dark green.5. Determinations of qualitative and quantitative of AtCBF1gene expression were performed by RT-PCR and qRT-PCR, respectively.Results showed that AtCBFl gene had been expressed in transgenic Dajiao and Lady’s fingers plants and expression levels of AtCBFl gene existed difference among different lines.6. Two transgenic lines（T1and T3lines） of Dajiao and Lady’fingers（L1and L4lines） were treated under7℃for five days for determintion of physiological and biochemical indexes in relation to cold tolerance.Results showed that ion leakage rate and malondialdehyde（MDA） contents in transgenic Dajiao and Lady’fingers plants were lower than non-transgenic plants.However, SOD activity in transgenic Dajiao plants was higher than non-transgenic plants and free proline, soluble sugar and relative water contents were higher in comparison with non-transgenic plants.7. Each twenty transgenic plants of Dajiao and Lady’s fingers were treated under4℃for five days and three days,respectively.After that.Transgenic dajiao plants did not display any cold injury symptom, but non-transgenic plants existed dehydration with wilt symptom.However, all plants incuding non-transgenic plants did not show death under cold injury.After treatment,leaves of transgenic Lady’s fingers plants showed a little curly,but no cold injury. Ho wever,leaves of non-transgenic plants displayed severe dehydration and up to90%of non-transgenic plants died at25℃for three days’ recovering growth but there was no death found in transgenic plants.These results directly showed that over-expression of AtCBFl gene in transgenic Dajiao and Lady’s fingers plants could improve cold tolerance.8. SSH-cDNA library of transgenic Dajiao plants was constructed in this study. Clp gene and aconitate hydratase gene were screened from cDNA library,which were relative to stress resistance.The expression confirmation of Clp gene in transgenic Dajiao plants was performed qRT-PCR and showed up-regulation in transgenic plants. Therefore speculated that AtCBFl protein might regulate and control the expression of endogenous Clp gene, aconitate hydratase gene and so on to improve cold tolerance of transgenic plants, but needed to be further validated.更多还原