【作者】 王三红；

【导师】 章镇； 姚泉洪；

【作者基本信息】 南京农业大学 ， 果树学， 2007， 博士

【摘要】 植物基因工程的发展和应用为植物遗传改良开拓了新的途径.相对于传统的育种方法,利用植物基因工程育种周期短、目标性更强,可以定向改良某些遗传性状,为果树提供了新的育种途径。但目前果树的遗传转化大多还局限于单个基因的遗传转化,多基因遗传转化对于果树综合性状改良具有重要意义。口蹄疫病毒(FMDV)和一些小核糖核酸病毒中,在多聚蛋白相互连接处有一段16～20个氨基酸的寡肽——2A序列.在共翻译时,2A序列能够独立地在自身的C末端发生断裂而不需要其他蛋白酶和细胞因子的参与,如果在其上、下游连接其他的异源蛋白时也照样能独立行使功能。在植物中,2A同样也具有这种自身在C末端发生剪切的功能.本研究将甜菜碱合成代谢相关基因通过2A序列融合在一个开放阅读框内,将融合基因转化酵母和烟草,证明了2A融合甜菜碱合成酶基因在微生物酵母和植物中的有效性.然后以2A序列融合抗逆转录因子DREB基因,铁载体蛋白IRT基因和rolC基因转化八棱海棠,研究了2A序列融合多基因在果树遗传转化上的应用,试验内容主要包括:1.克隆了盐地碱篷(Suaeda salsa)甜菜碱合成酶基因SsCMO和SsBADH,并构建了以口蹄疫病毒2A序列融合SsCMO和SsBADH基因于一个开放阅读框(ORF)的中间载体。根据辽宁碱蓬(Suaeda liaotungensis)甜菜碱合成酶CMO和BADH序列设计引物,克隆盐地碱蓬甜菜碱合成酶基因SsCMO和SsBADH,在Genbank上的登录号分别为:DQ656523和DQ641924.盐地碱篷SsCMO基因编码区长度为1329 bp,编码442个氨基酸,核苷酸和氨基酸序列与发表的辽宁碱蓬CMO基因(SlCMO)核苷酸和氨基酸序列相同;SsBADH编码区长度为1506 bp,编码501个氨基酸,其核苷酸序列和辽宁碱蓬BADH基因(SIBADH)的核苷酸序列有99%的同源性,氨基酸序列相同。依次将口蹄疫病毒FMDV的2A序列、SsCMO基因和SsBADH基因连接入改造的pBluscript KS(+)多克隆位点,构建了融合甜菜碱合成酶基因于一个开放阅读框(ORF)的中间载体pBKSMC2AB.2.构建了以2A序列融合甜菜碱合成酶基因的酵母表达载体并转化毕赤酵母Pichiapastoris GS115,研究融合基因在酵母中的表达及转融合基因酵母对环境胁迫的抗性.构建甜菜碱合成酶融合基因的酵母表达载体,转化毕赤酵母Pichia pastorisGS115.测定了转融合基因的重组酵母与转空质粒的酵母CMO和BADH活性、甜菜碱含量的差异.0.5%甲醇诱导96h,在表达量高的重组酵母中,CMO和BADH的活性是对照转质粒空载体pPIC3酵母(YPIC3)的45倍和44倍;转融合基因的重组酵母甜菜碱含量比对照YPIC3高28～35倍.SDS-PAGE分析表明,在重组酵母A764和A765中比对照YPIC3多出45 KDa和54 KDa的两条蛋白带,这与SsCOM和SsBADH基因编码蛋白质预测分子量的大小吻合.试验结果说明在转甜菜碱合成酶融合基因酵母中表达了多聚蛋白且多聚蛋白“剪切”出各自功能的蛋白.转甜菜碱融合基因的酵母提高了对盐、高温和甲醇的抗性.3.构建了以2A序列融合甜菜碱合成酶基因的植物表达载体,转化农杆菌LBA4404中,通过农杆菌介导法将甜菜碱合成酶融合基因转化烟草和八棱海棠.研究了不同浓度潮霉素对八棱海棠再生的影响和以潮霉素为筛选标记的八棱海棠及烟草的遗传转化.以潮霉素为筛选标记将融合基因转化八棱海棠,试验没有获得抗性植株,以潮霉素为筛选标记将融合基因转化烟草获得了转基因植株.分析表明,转融合基因的烟草甜菜碱含量显著高于非转基因烟草,并且耐盐性得到显著提高.4.分别从水稻、番茄和发根农杆菌中克隆了DREB、IRT和rolC基因,以口蹄疫病毒2A序列融合DREB、IRT和rolC基因于一个ORF内形成融合基因Rirol,构建三价融合基因Rirol植物表达载体,并通过农杆菌介导法将三价融合基因Rirol转化八棱海棠.试验研究了不同农杆菌菌株转化八棱海棠的效率,获得了抗卡那霉素的八棱海棠.经GUS检测、PCR和Southern杂交分析证明融合基因成功导入到八棱海棠基因组中.对转基因八棱海棠相关性状分析表明,转基因八棱海棠比对照具有较强的耐盐性,在表型上表现出节间缩短,分枝性强,侧根发达等特点.证明了外源融合基因在转基因植株中得到表达,也证明了利用2A序列进行多基因转化八棱海棠的可行性,为苹果等果树多基因转化提供了新的思路和途径.更多还原

【Abstract】 Apple（Malus×domestica Borkh） is one of the most important fruit trees in the world and in China.However,drought,soil salinity and iron deficiency,which widely exist in many apple growth areas of China,adversely affect apple productivity and quality.To deal with the problem,enhancing cultivation management or breeding new cultivars high-resistant to abiotie stress are required.On the other hand,dwarf cultivation is easy for tree management and beneficial to early fruit setting,high productivity and good fruit quality.Therefore,it is important for apple cultivation to breed rootstocks with dwarf and high resistance to stress.Conventional apple breeding is constrained by their extensive reproductive cycle with long juvenile periods,complex reproductive biology,and high degree of heterozygosity.As alternative,genetic engineering offers the possibilities introduce new characteistics into existing commercial cultivars.However,until now all genetic modifications in apple are almost single gene transformation.Multiple genes transformation is necessary for improving integration characteristics of apple.In the foot-and-mouth disease virus（FMDV） and some other picomaviruses the oligopeptide（-20 amino acids） 2A region of the polyprotein mediates cleavage at its own C terminus to release it from the 2B region.Dissociation occurs at the carboxy terminus of the 2A sequence without any requirement for cytosolic factors.2A is functional when replacing its up- and down- stream genes and is also functional in plants.In this experiment,the genes encoding glycine betaine synthase were cloned from a halophyte,Suaeda salsa and fused in a single open reading frame.The fused genes were transformed to P.pastoris GS115,Nicotiana tabacum and Malus robusta,respectively,and the expression of the fused genes in P.pastoris and plants as well as the function of 2A were analyzed.The genes DREB（encoding dehydreation responsive element binding protein）,IRTl（encoding iron-regulated cation transporter） and rolC（encoding cytokinin-β-glucosidase） were cloned from Oryza sativa,Lycopersicon esculentum and Agrobacterium rhizogenes,respectively. The trivalent gene Rirol was synthesized by fusing DREB,IRTl and rolC with two copies of 2A in a single open reading frame and transformed to Malus robusta mediated by Agrobacterium tumefaciens.The main results are presented as follows:1.According to the homologous sequence of encoding two enzymes of betaine synthesis in Suaeda liaotungensis,SsCMO and SsBADH were cloned by PCR in Suaeda salsa.The nucleotide sequences of SaCMO and SsBADH show 100%and 99%homolog with SICMO and SlBADH,respectively.The deduced amino acid sequence of SsCMO and SsBADH are identical with that of SlCMO and SlBADH,respectively.To construct SsCMO and SsBADH plant co-expression vector,the SsCMO,SsBADH genes were fused into a single open reading frame（ORF） with a copy of the FMDV 2A protein gene placed between the two genes.A transitional plasmid pBKSMC2AB was constructed by introducing 2A sequence,SsCMO,SsBADH to MCS of pBKSM（modified from pBluscript KS（+）） successively.2.The fused genes ’SsCMO-2A-SsBADH’ was placed under the control of the alcohol oxidase（AOXl） promoter in pPIC3B and the vector was then linearized and transformed into P.pastoris GS115.The expression of the fused genes linked by the 2A sequence in P. pastoris and the ability of recombinant yeasts to tolerate environmental stress were studied. The results showed that the activities of enzymes CMO and BADH were 26.5 and 28 U·mg^-1 protein in recombinant yeasts,which were 45-fold and 44-fold higher than those in control GS115,respectively.SDS-PAGE analysis of proteins expressed in recombinant and control yeasts showed that compared with the control recombinant yeasts,A764,A765 expressed two additional proteins of 45KDa and 54KDa,the size of which matched the predicted size of CMO and BADH.The content of glycine betaine in recombinant yeasts was 28-to 34-fold higher than that in control GS115 induced with 0.5%methanol for 96 h. Recombinant yeasts were more resistant to salt,methanol and high temperature stress than control.It was concluded that the genes fused by FMDV 2A region were expressed in Pichia pastoris successfully and the polyprotein was ’cleaved’ to each functional protein. The yeasts transformed the fused genes which were more resistant to salt,methanol and high temperature stress than control as result of glycine betaine synthesis genes introduced.3.The plant expression vector harboured the fused genes ’SsCMO-2A-SsBADH’ under the control of the super promoter was constructed and transformed to tobacco and Malus robusta mediated by Agrobacterium tumefaciens.Transgenic Malus robusta was not obtained by transformation via hygromycin resistant selectable marker.Tobacco transformed with the fused genes increased the content of glycine betaine and was improved the tolerance to salt stress.4.DREB,IRT1 and rolC genes were fused with two copies of FMDV 2A region and transformed to Malus robusta mediated by Agrobacterium tumefaciens.Some kanamycin-resistant plants were obtained by selection.The fused genes were proved to integrate the genome of some Km-resistant plants of Malus robusta by GUS staining,PCR and Southern bolting.The transgenic plants showed higher resistance to salt stress, shortened internodes and more branching of shoots and roots.The study primarily proved that cotransforming multiple genes fused by FMDV 2A region may be a new way introducing multiple genes into fruit trees.更多还原