【作者】 胡钟东；

【导师】 章镇；

【作者基本信息】 南京农业大学 ， 果树学， 2007， 博士

【摘要】 砂梨（Pyrus pyrifolia Nakai）原产于我国中部、南部和西部各省,适于在温暖多雨地区栽培,是原产于中国的四个梨栽培种之一,具有耐热、抗旱、抗火疫病等优良性状。但其果实大多贮藏性差。如何提高果实耐贮性是解决好砂梨产业化的关键问题。目前的研究主要是针对砂梨果实的生理特点,通过改善果实的贮藏条件和采用一些乙烯抑制剂来延长砂梨果实的货架期,不能解决根本问题。我们以‘若光’梨为材料,研究适于‘若光’梨叶片离体再生的培养基的种类以及激素配比,克隆‘若光’果实中与成熟相关的基因并构建其RNA干扰载体,将构建的载体分别进行农杆菌介导的梨、番茄、烟草转化,研究干扰基因在植物体内的表达特性,为将来获得耐贮砂梨新种质提供了基础。1、为了获得‘若光’梨高效的叶片离体再生体系,和为‘若光’梨的耐贮基因工程研究提供条件,以‘若光’梨叶片为材料,研究了不同激素的种类与浓度组合对砂梨叶片离体再生的影响。结果发现,以NN₆₉为基本培养基,附加TDZ 1.0 mg/L+IBA 0.15 mg/L+AgNO₃ 0.1 mg/L处理‘若光’梨叶片,暗培养21d后,再置于光照条件下培养30d,‘若光’梨叶片不定芽再生频率达到了63.2%。2、根据已登录的砂梨ACO3（GenBank Accession No.AB042107）序列设计并合成一对特异引物,以‘若光’梨叶片总DNA为模板,利用染色体步移技术扩增基因的上游调控序列。回收该片段并克隆到pMD19-T载体上,测序后去除接头和基因编码序列,得到一段长715bp的上游序列。登陆PLACE数据库在线进行植物DNA顺式作用元件预测与分析,起始位点的上游89bp处有一个TATA-Box核心启动子元件。另外,序列还存在多个特异调控基因表达的元件。初步判断其为砂梨果实中ACC氧化酶基因的诱导型启动子。3、以‘若光’梨成熟果实为材料,提取总RNA。在ACC氧化酶基因（ACO）mRNA序列同源性较高区域,用Primer premier5软件设计一对特异引物,利用RT-PCR克隆得到了401bp长的片段。经过测序和核苷酸、氨基酸比对,发现与登录的ACC氧化酶基因（ACO3）同源性达到99%。据此判断,得到的片段为砂梨的ACC氧化酶基因序列。根据序列设计两对分别加了不同酶切位点的正、反基因的特异上下游引物,扩增出ACO的正、反义基因。将ACO反义基因、与副球菌中类胡萝卜素合成有关的间隔基因YYT（GenBank with accession number AY345165）以及ACO正义基因三个片段串联在一起,并克隆到T载体。经测序鉴定正确后,双酶酶切下目标片断并插入到植物表达载体中,构建成能够表达ACC氧化酶基因的双链RNA的植物载体pYF028。4、为了研究ACO干扰基因在植物体中的表达特性,通过农杆菌介导法转化番茄子叶,将侵染的520个外植体先经MS+ZT 1mg/L+IAA 0.5 mg/L+Km 50 mg/L+250 mg/LCef+蔗糖30 g/L+琼脂7 g/L（pH 5.8）筛选培养基,初步筛选出Km抗性芽。将筛选出的抗性芽再经过2次继代培养,共得到34个抗性株系。将抗性株系转到生根培养基MS+IAA 0.5 mg/L+Km 50 mg/L+Cef250mg/L+蔗糖30 g/L+琼脂7g/L（pH5.8）中生根,经过30d的生根培养,共得到了6个生根株系。经过GUS染色、PCR、RT-PCR检测验证。初步证实,干扰基因已经整合到番茄的基因组中。进一步进行其乙烯发生量的检测,结果发现,转干扰基因番茄植株的乙烯释放量显著高于对照。5、以‘若光’梨成熟果实为材料,提取总RNA并反转录成cDNA。根据脂氧合酶氨基酸保守区设计1对简并引物,采用RT-PCR克隆到1段长827bp的cDNA序列。经Blast比对和DNAstar软件聚类分析,结果表明,由该序列推导出的氨基酸序列含有脂氧合酶氨基酸保守结构域,与马铃薯脂氧合酶基因的氨基酸序列相似性达到77.5%。判断为砂梨脂氧合酶基因片段,命名为LOX1,并将序列登录到GenBank,登录号为EF215448。根据RNAi载体的构建原则,选择LOX1中一开放阅读框设计携带酶切位点的特异引物,通过PCR扩增LOX1正、反向基因片段,再与YYT间隔区串连,插入植物表达载体pYF7713中的相应位置。构建了干扰砂梨LOX基因表达的RNAi植物双元表达载体pYL028。6、为了研究干扰基因在植物体中的表达特性,我们将构建好的‘若光’梨LOX1干扰表达载体（pYL028）,通过农杆菌介导法转化烟草叶片;将侵染的113个外植体先经MS+6-BA 2 mg/L+NAA 0.1 mg/L+Km 50 mg/L+Cb 250 mg/L+蔗糖30 g/L+琼脂7 g/L（pH 5.8）的筛选培养基培养,初步筛选出Km抗性芽。抗性芽再经2次继代培养,共得到16个抗性株系。将抗性株系转到1/2MS+IBA 0.1 mg/L+Cb 250 mg/L+Km 50 mg/L+蔗糖30 g/L+琼脂7 g/L（pH 5.8）的生根培养基中生根,经过30d的生根培养,共得到了5个生根株系。经过GUS染色、PCR、RT-PCR检测验证,初步证实,干扰基因已经整合到烟草的基因组中。进一步检测脂氧合酶及相关酶的活性,结果显示,在低温胁迫条件下,转基因植株与未转基因的对照植株相比,LOX酶的活性被抑制37.8%;SOD、CAT和POD酶活性显著升高。MDA的含量显著降低。7、根据脂氧合酶（lipoxygenase,LOX）氨基酸保守区设计一对简并引物,采用RT-PCR技术从‘若光’梨成熟果实中克隆到一段eDNA特异片段,转化大肠杆菌DH5α感受态细胞,挑单克隆测序,得到了两段长度均为827bp,编码275个氨基酸残基的核苷酸序列,经BLAST分析,由序列推导的氨基酸序列含脂氧合酶基因氨基酸保守结构域,据此推断得到的片段为砂梨脂氧合酶基因家族中的2个新成员,分别命名为LOX2、LOX3,并登录GenBank（EF215449,EF215450）。经DNAStar软件分析,结果显示砂梨果实中表达的LOX2基因可能与果实种子发育有关,而LOX1和LOX3基因为砂梨果实胁迫条件下诱导表达的基因。更多还原

【Abstract】 Sand pear（Pyrus pyrifolia Nakai） originated in central,southern and western of China, and it is suitable for cultivation in the warm and wet area with some advantages such as heat-resisting,combats drought,the fire blight-resistant and so on.But its fruit storage ability is poor,which brings a problem in pear industrialization:how to improve the storage ability of pear fruit.The present research is mainly concentrated on elongating the fruit shelf life according to the physiological characteristic of the sand pear fruit by improving the storage conditions for fruit and using some ethylene inhibitors,but these methods can not solve the problem ultimately.In this paper,the different kinds of culture medium,the proportion and kinds of hormones in regeneration of ’Wakahikari’ pear leaf in vivo were studied.The ripeness related genes were cloned from fruit and their RNAi expression vectors were constructed.The expression characteristics of interference genes were studied through Agrobacterium mediated gene transformation of pear,tomato and tobacco,which make it possible to obtain a new pear germ plasm with good storage ability in the future.1、In order to obtain high regeneration frequency and provide conditions for gene transformation of pear（Pyrus pyrifolia ’Wakahikari’）,the effects of different hormonal combinations of varieties and concentrations on regeneration of pear leaves in vitro were studied.The results showed that ’Wakahikari’ leaves had 63.2%regeneration frequency in NN₆₉ medium with TDZ 1.0mg/L,IBA 0.15mg/L,AgNO₃ 0.1mg/L,cultured for 30 days in light after 21 days in dark.2、A pair of specific primer were designed and synthesized according to the AC03 sequence （GenBank with accession number AB042107） from pear（Pyrus pyrifolia Nakai）.The upstream regulatory sequence was amplified from genomic DNA of pear（Pyrus pyrifolia ’Wakahikari’） leaves by Genomewalker,the DNA fragment was subcloned into pMD19-T vector.A 715bp fragment was obtained and sequenced.A core promoter element frame of TATA-Box at 89bp upstream of initial site was detected by landing the PLACE database and predicting cis-acting element of plant DNA online.In addition,substructure analysis showed that there were some specific elements related to regulating gene expression,thus it is likely an inducible promoter of ACC oxidase gene in pear fruit.3、The total RNA was isolated from mature fruit of pear（Pyrus Pyrifolia ’Wakahikari’）.A pair of specific primer was designed by software of Primer premier 5 according to the mRNA conserved region sequence of ACC oxidase gene.A 401 bp fragment was obtained by RT-PCR.The cDNA sequence was 99%homologous to ACC oxidase gene（AC03） of Pyrus pyrifolia by sequencing.It showed that it was ACC oxidase gene of sand pear.Two pairs of specific primers with different sites of restricted enzyme were designed according to the sequence.And antisense and sense ACC oxidase gene were amplified,then ligated with the YYT gene,which was related to carotenoid synthesis in Subcocci as interval sequence.The fusion gene was digested by two restricted enzymes and cloned into plant expression vector to construct the recombinant plasmid pYF028,which could express dsRNA ofACC oxidase gene.4、To study the expression characteristics of ACO interference gene in plant,ACO interference gene was transfered into tomato by Agrobacterium mediated system.Kmr buds were screened from 520 explants cultured on selective medium with MS+ZT 1mg/L+ IAA 0.5mg/L+Km 50mg/L+Cef 250mg/L+sucrose 30g/L+agar 7g/L,and gained 34 Kmr plants after two subcultures.Six plants with roots were obtained on MS+IAA 0.5mg/L+Km 50mg/L+Cef 250mg/L+sucrose 30g/L+agar 7g/L for 30d.By GUS detection,PCR and RT-PCR analysis,it indicated that the ACO interference gene was transferred and integrated into the genome of tomato.The result showed that the release quantity of ethylene in transgentic tomato was significantly higher than the untransgentic control after detecting the release of ethylene.5、The total RNA was isolated from mature fruit of sand pear（Pyrus pyrifolia ’Wakahikari’）.A specific fragment with predicted size was amplified from cDNA of Pyrus pyrifolia by RT-PCR with degenerate oligonuelectide primers designed from the conserved domain of other plant lipoxygenases.The cDNA fragment was 827bp.Amino acid sequence revealed that the cDNA fragment had conserved domain of lipoxygenase and was 77.5%homologus with potato through blasting and clustering analysis.It is likely that the cDNA fragment was lipoxygenase gene of Pyrus pyrifolia named as LOX1.The cDNA sequence of this clone was deposited in GenBank with accession number EF215448.The specific primers with restricted enemzy site were designed from an open read frame（ORF） of the cDNA sequences according to the principles of construction of its corresponding RNAi vector.Two fragments were amplified by PCR,one is postive orientation,the other is reverse orientation,ligated with YYT interval region.The fusion fragment was introduced into plasmid of pYF7713,it was successful that the recombinant plasmid pYL028 was constructed,which could express dsRNA of pear LOX gene.6、To study the expression characteristic of interference gene in plant,we transfered LOX1 interference gene into tobacco through Agrobacterium mediated system.Km^r buds from 113 explants cultured on selective medium with MS+6-BA 2mg/L+NAA 0.1mg/L+Km 50mg/L+Cb 250mg/L+sucrose 30g/L+agar 7g/L with pH 5.8 wre screened out,and 16 Km^r plants were obtained after two subcultures.Five plants with roots were obtained on I/2MS+IBA 0.5mg/L+Km 50mg/L+Cef 250mg/L+sucrose 30g/L+agar 7g/L with pH 5.8 for 30d.By GUS detection and PCR and RT-PCR analysis,it showed that the LOX1 interference gene was transferred and integrated into the genome of tobacco.It was found that 37.8%of LOX activity was inhibited in transgenic plant compared with control under low temperature stress,but the activities of SOD,CAT and POD were enhanced and the content of MDA was significantly lower than that of the control.7、A specific fragment of the predidicted size was amplified from eDNA of mature fruit in Pyrus pyrifolia by RT-PCR with degenerate oligonuclectide primers designed according to the conserved domain of other plant lipoxygenases.Two 827bp fragments coding 275 amino acids were obtained and sequenced.The amino acid sequences revealed that the cDNA fragments had lipoxygenase conserved domain by BLAST online.We deduced that they were another two fresh members from lipoxygenase gene family in Pyrus pyrifolia Nakai.We named them LOX2 and LOX3.The cDNA sequences were deposited in GenBank with accession number EF215449 and EF215450.The results showed that LOX2 gene expressing in pear（Pyrus pyrifolia Nakai） fruit was related to seed development in fruit,the LOX1 and LOX3 expression in fruit was induced by stress.更多还原