【作者】 张瑞萍；

【导师】 刘桂丰； 王玉成； 李成浩；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2009， 博士

【摘要】 水分胁迫可诱导许多基因的表达,脱水素基因（dehydrin gene,DHN）是其中之一,它属于LEA D-Ⅱ家族（late embryogenesis abundant proteins-Ⅱ）,对低温和外源ABA产生反应,或在干旱、盐以及胞外结冰的脱水胁迫植株中典型地积累,是植物遭受逆境胁迫时诱导产生的几种脱水应答蛋白质之一,脱水素富含亲水性氨基酸,具有高度亲水性,在水分亏缺下,脱水素可以作为核酸或胞质大分子的稳定剂,与膜脂结合从而阻止细胞内水分的过多流失,维持膜结构的水合保护体系,防止膜脂双分子层间距的减小,进而阻止膜融合以及生物膜结构的破坏。本文从柽柳中克隆出了具有优良抗逆能力的脱水素基因,研究了该基因在多种胁迫下的表达模式。同时,分别以白桦成熟合子胚和白桦叶片为外植体,应用农杆菌介导法,进行了白桦遗传转化研究,成功地将脱水素基因导入到白桦中,其中农杆菌介导的白桦成熟合子胚可直接诱导抗性芽形成,为白桦遗传转化研究提供了新的途径,具体研究结果如下:1.从柽柳cDNA文库中克隆得到ThDHN序列（FJ627947）,该基因全长1159bp,其中5’非编码区143 bp,3’非编码区401 bp,开放阅读框（ORF）为615 bp,编码204个氨基酸。预计该基因产物分子量为22.76kD,理论等电点为5.61。该基因序列中部有一个S片段,其后还有2个重复的K片段,属于SKn型。柽柳脱水素与已有物种的同源性在36.6%～86.7%之间,与柑桔属的脱水素蛋白（AAP56259）和咖啡的脱水素蛋白（ABC68275）的同源性最高,均为86.7%,与裸子植物欧洲赤松的脱水素蛋白（ACJ37787）的同源性最低,仅为36.6%。2.ThDHN基因可以被NaCl、ABA、冷和重金属隔（Cd）等胁迫所诱导,呈上调表达,其中,ABA诱导处理能够明显促进ThDHN基因在柽柳中的表达;而干旱胁迫则抑制了该基因在柽柳中的表达,我们推测柽柳脱水素基因依赖于ABA而表达。3.建立了白桦成熟合子胚高效不定芽诱导组培体系。白桦成熟合子胚经自来水冲洗约5-7天后,在培养基2.0 mg·L^-1 6-BA+0.2 mg·L^-1 NAA培养8～9 d后即可直接诱导不定芽形成,大大缩短了不定芽诱导周期,并且该培养基的不定芽诱导率为93.3%,平均不定芽数8.7个,不定芽的生根率为100%。4.筛选出头孢霉素为白桦遗传转化体系的抑菌抗生素,使用浓度为200 mg·L^-1;白桦遗传转化的选择类抗生素为卡那霉素,其使用浓度为20 mg·L^-1;应用农杆菌介导的遗传转化法,白桦成熟合子胚受侵染后可直接诱导不定芽再生,获得转化植株的周期短,省去了愈伤组织诱导和愈伤组织分化出芽阶段,是白桦遗传转化研究的新途径。5.应用PCR技术、PCR-Southem杂交和Northern杂交技术对脱水素基因在白桦基因组织中的存在和表达情况进行鉴定,PCR鉴定结果表明,阳性对照和供试转基因株系均获得了615bp的目的条带,而阴性对照则没有。PCR-Southern杂交结果证实,阳性对照和转基因株系均呈阳性,而阴性对照则没有。这初步表明,柽柳脱水素基因已经整合到白桦基因组中。进一步对其进行Northern杂交,转基因株系均呈阳性,而非转基因对照呈阴性,这表明,柽柳脱水素基因已经在转录水平上表达。6.筛选0.3%NaCl(约51.28 mmol·L^-1)为白桦组培苗耐盐压力值。在该盐浓度下,除转基因株系BTD7以外,所有转基因株系的主根数均多于非转基因株系,说明在同样盐胁迫条件下,盐胁迫对白桦转基因株系生长的影响小于非转基因株系,转基因白桦表现出较好的耐盐性。7.在PEG6000浓度为2.5%～7.5%的生根培养条件下,转基因株系的主根数明显多于非转基因株系,转基因白桦的抗旱性得到提高。更多还原

【Abstract】 Many genes can be induced by drought stress.Dehydrin gene（dehydrin gene,DHN） is one of them,it belongs to LEA D-Ⅱfamily group（late embryogenesis abundant proteins）, which can be induced by low temperature exogenous ABA,drought,high salinity where cellular dehydration occurs.Dehydrins are one of the proteins induced by water-deficit,due to its high hydrophilic,it can combine with the lecithoid membrane thereby to prevent the water lose of the interior cell,maintain the hydration protective system of the membrane structure, avoid the decrease of the distance between the double molecular layer of the lecithoid membrane,farther to prevent the fuse of the membrane and the wreck of the biology membrane structure.In the present,we cloned ThDHN from Tamarix hispida,and studied the expression mode of ThDHN under stress.Nextly ThDHN gene was introduced into Betula playphylla through zygotic embryos and birch leaves respectively.Then transgenic birch were obtained successfully the Agrobacterium-mediated transformation of zygotic embryos can induced directly resistance bud formation,as well as a higher efficiency for genetic transformation,which is a new way to genetic transformation to B.playphylla.The results are as follows:1.ThDHN（FJ627947） from cDNA library ThDHN was analyzed by bioinformatics,the gene has a total length of 1159bp,the 5’non-coding region is 143 bp,3’non-coding region is 401 bp,the ORF（open reading frame） is 615 bp,encoding 204 amino acids.Its expected molecular weight is 22.76kD,theoretical isoelectric point is 5.61.The gene sequence has one S-fragment,and two K-fragments,belongs to SKn type.The homology of ThDHN is 36.6%～86.7%,and it has a near relationship with Citrus dehydration protein（AAP56259） and Coffea canephora dehydrin-like protein（ABC68275）,the homology value is 86.7%,and it has a distant relationship with Pinus sylvestris dehydration protein（ACJ37787）,the homology is only 36.6%.2.The expression pattern was studied by real-time PCR technology,results showed that, ThDHN gene may induced by NaCl,ABA,cold and heavy metal septum（Cd） stress,it was upregulated, particularly,ABA treatment can improved significantly the expression of ThDHN in T.hispida;while drought stress can suppressed the expression of ThDHN,the relative expression levels is lower than that of control,we speculated that the expression of ThDHN dependent on the participation of ABA.3.Mature zygotic embryos of Betula platyphylla Suk.as explants were disinfected and then used to inducing adventitious buds.Under WPM+2.0 mg·L^-1 6-BA+0.2 mg·L^-1 NAA conditions,the incision adventitious buds formed,and the rate of adventitious buds differentiation is 93.3%,with induction of adventitious buds is 8.7;Under WPM+4.0 mg·L^-1 TDZ condition,the rate is 90.2%,and the induction number is 7.6.All these results indicate that WPM+2.0 mg L^-1 6-BA+0.2 mg·L^-1 NAA is better to differentiation of adventitious buds, and NAA can enhance the rooting of adventitious shoots,and the optimum concentration is 0.2 mg·L^-1.4.Cephalosporin sodium is the optimized antibacterial antibiotics,it’s effective concentration is 200 mg·L^-1;kanamycin is the optimized antibiotics for the transformant selection,it’s effective concentration is 20 mg·L^-1;At the same time,after infected by Agrobacterium,the zygotic embryos can induced adventitious bud directly,and the cycle of transformants is short,eliminating the stage of callus induction and callus differentiation,this provides a new way to B.platyphylla.5.PCR identification,PCR-Southern blotting and Northern blotting were used to true transformants,and 7 independent plant lines were confirmed by PCR,All of these plantets produced the expected amplification product of 615 bp,while the non-transgenic plant lines is not;PCR-Southern blotting and Northern blotting confirmed that indicating that the transgenic lines were true transformants.This result indicated preliminary that the ThDHN gene had integrated into birch,and expression in transcription level.6.Salt-stress tests was done within non-transgenetic plants,0.3%NaCl(51.28mmol·L^-1) is screeninged for the salt stress value.At that time,plants grow slowly,and the number of roots is less.Then,at this pressure,the main roots of 7 transgenic lines and non-transgenic lines was measured,results showed that,with the exception of BTD7,all of the main roots of transgenic lines were more than non-transgeneticlines,indicating that the damage of salt stress on transgenic lines is lighter than that on non-transgeneticlines,transgenic birch showed good salt tolerance.7.Under drought stress,2.5%-7.5%PEG6000 is screeninged for the drought stress value. After treated with PEG6000,the number of main root in all transgenic lines is more than nontransgenic lines,suggesting that the drought-resistance of transgenix birch is enhanced.更多还原