【作者】 邵长文；

【导师】 张兴国；

【作者基本信息】 西南农业大学 ， 蔬菜学， 2004， 硕士

【摘要】 黄瓜（Cucumis sativus L．）原产于热带和亚热带区域，是一种重要喜温类蔬菜，生长适宜温度为25-32℃，低温界限为10-12℃，10℃以下生长停止，5℃有受害的危险，经过低温锻炼的幼苗能忍受短时间1-2℃的低温。由于种属内没有抗寒的资源材料，显著提高黄瓜的抗寒性就不能通过有性杂交转育，只能利用基因工程手段导入其他物种的抗寒相关基因，从而来打破植物遗传群体中抗寒种质资源缺乏的限制，为黄瓜抗寒育种提供新的种质资源。 冷诱导启动子RD29A受干旱和低温诱导而产生表达活性；cor15a基因在拟南芥菜抗寒中起重要的作用，CBF3冷调控转录因子基因能促进大量冷诱导基因在在常温下的表达。本试验将冷诱导启动子RD29A分别控制下的拟南芥菜cbf3冷调控转录因子基因和cor15a抗寒基因构建植物表达载体，分别构建成携有RD29A-cor15a-Tnos表达盒的植物表达载体pVCT2016和携有RD29A-cor15a-Tnos和RD29A-CBF3-Tnos串联双表达盒的植物表达载体pRD29A-CBF3。通过农杆菌介导，导入黄瓜基因组，创制出耐寒黄瓜新材料。 1 植物表达载体的构建 1．1 表达载体pVCT2016的构建 克隆载体pSAU1001经限制性内切酶XbaⅠ酶切后经DNA聚合酶Ⅰ Klenow片段补平粘末端，再用SacⅠ酶酶切，电泳，从1％的琼脂糖凝胶中回收纯化426bp小片段；表达载体pRD29A-GUS-T经SmaⅠ和SacⅠ双酶切后回收13550bp大片段；用T₄连接酶连接两个片断，转化大肠杆菌，在kan抗性的LB平板上筛选出阳性克隆，进行colony PCR鉴定和酶切鉴定。提取质粒pVCT2016，用冻融法导入农杆菌菌株LBA4404（pAL4404）中。 1．2 表达载体pRD29A-CBF3的构建：西南农业大学硕士学位论文 为了构建表达载体pRD29A一BF3，我们又构建了两个中间载体pBs.RD29A一cBF3和pGEM一肋29A一CBF3.克隆载体pVCTZolZ经月认班11趁必oRI双酶切后切成2635bp和1949bp两片断，回收小片段汪几”dlll尼co孙双酶切pBS n Ks+，乙醇沉淀去除12bP小片断，回收2949bP大片段，T4连接酶连接，构建成pBs-RD29A一CBF3;pBs一劝29A一cBF3经刀“成月叮clal双酶切后，回收1974bp小片段，pGEM·7zf经刀“优月叮c如I双酶切后，回收2988bP大片段，T4连接酶连接，转化大肠杆菌，构建成PGEM.RD29A￡BF3.pGEM.RD29A-cBF3用五七。RI酶切成2983bP和1978bP两片断，电泳回收1978bP小片断，P vCT2016经五七口Iu酶切后成线状DNA片段，T4连接酶连接，转化大肠杆菌和农杆菌，构建成植物表达载体pRD29A一BF3.2植物表达载体对黄瓜子叶的遗传转化研究2.1黄瓜子叶的遗传转化再生体系的优化 在前人工作的基础之上‘I，’·“‘，’·‘·’·”，对黄瓜子叶的遗传转化的培养基进行了优化，获得了优化的培养基配方，提高了外植体的芽分化率，克服了培养过程中出现的玻璃化，褐化现象。以2天龄的黄瓜子叶为外植体，以MS（1960年）为基本培养基，通过加入不同浓度和不同组合的BA、ABA和ZT，筛选到了试管苗分化最适培养基（璐附加BA Zmg/L+ZT lmg/L+超A0.smg/L+3%蔗糖+0.7%琼脂的固体培养基）、不定芽生长培养基（MS附加3%蔗糖和0.7%琼脂的固体培养基）和根分化生长最适培养基（l/ZMS附加3%蔗糖+0.，%琼脂的固体培养基）. 在最适培养基上研究了黄瓜子叶外植体对卡那霉素的敏感性、预培养天数、共培养时间及感菌时间等因素的影响，建立了黄瓜高频转基因再生体系，即取1天龄的黄瓜无菌子叶在不定芽分化最适培养基OCM（MS加BAZmg/L+ZTlmg/L+ABAO，sm以L+3%蔗糖+0.7%琼脂的固体培养基）上预培养1²天，投入到活化的农杆菌LBA4404（pRD29A一CBF3+P AL44O4）菌液中浸染15分钟，在ocM上暗培养2天，在附加Carb 300mg/L的ocM培养基上延迟筛选培养一周后，转入筛选培养基（戊M+Kan80m岁L+c arb ZOOmg/L）中筛选抗性愈伤和抗性芽，每两周更换一次培养基，抗性芽转入不定芽生长培养基中培养1²周，然后转入生根培养基中诱导生根，共获得具有Kan抗性的黄瓜再生芽14个，共获得9株抗性再生植株。2.2转基因黄瓜的分子鉴定 .取Kan抗性的黄瓜植株叶片，用CTAB法提取的基因组DNA作为PcR扩增的模板，以Tnos终止子的引物P’;和coR15A基因5’端引物P0:为成对引物进行PcR扩增。结果表明，有4株再生植株的基因组DNA能扩增出426bP的条带.同时，以P一RD29A启动子的5’引物P，与cBF3基因上的3’端引物P。，为成对引物进行PcR扩增，有两株再生植株的的基因组DNA能扩增出1638bP的条带，两对引物的扩增产物均与阳性质粒对照处于同一位置，同时，为了排除PcR中文摘要检测假阳性的可能，进行了转荃因植株的Southern杂交验证。杂交结果证明两株转基因植株中其中一株植株的目的基因拷贝数为2个，另一株为一个拷贝，证明目的基因已经导入黄瓜基因组中。更多还原

【Abstract】 Cucumber (Cucumis sativus i.)i originated in tropics and sub-tropics ,is a kind of vegetable which grows well only under temperature at about 25-32℃, their cold temperature is at the range of 10-12℃, they will stop growth when the temperature is under 10℃,and will be injuried by the temperature at about 5℃, seedlings can endure 1-2℃ cold temperature for steak after cold acclimation. There is no cold resistance material in the species, so, prominently improving the cold endurance can not be generated through general genetic hybrid breeding, the only method can be used is the gene engineering method through which other species’ cold-regulated genes and cold-induced transcriptional factors(genes) can be transferred into cucumber, promote abundant of genes expression at the level of transcription and break down the constraint of plasmgerm deficientin the genetic group, provide, a new plasmgerm for cucumber chilling-tolerance breeding.P- RD29A is a kind of promoter which can be induced into expression by dehydration, plant hormone ABA, and low-temperature. Cor15a gene takes an important role in cold resistance of Arabidopsis thaliana, cold-induced transcriptional factors genes CBF3 can promote the expression of cold-regulated genes. In this paper, two plant expression vectors concerning chilling tolerance were constructed. pVCT2016 consists of RD29A-corl5a-Tnos expression box,pRD29A-CBF3 consists of RD29A-corl5a-Tnos and RD29A-CBF3-Tnos tandem expression boxes. Through Agrobacterium-mediated transformation, two expression boxes were transformed into cucumber genomics, and a new chilling tolerance cucumber was created.1. The construction of plant expression vector 1.1 The construction of expression vector pVCT2016:After digestion of clone vector pSAU1001 with restriction endonuclease Xba I, filled in the cohesive end with DNA polymerase I Klenow fragment, then, digested with Sad, after electrophoresis, the band of 426bps in 1% argose was collected, and the fragment was purified; the expression vector pRD29A-GUS-T was digestion with SmaI and SacI, and 13550bps fragment was collected and purified, and the two fragment was ligated with T4 DNA ligase, the ligated product was transformed into E coli strain XL1-BLUE, select the white colony in plate of LB including Kanamycin 50mg/L,and the positive colony was detected, and further identification of restriction endonuclease digestion performed. Exact the pVCT2016 from XL1-Blue, and the plasmid was transformed into LBA4404(pAL4404),and the plant expression vector was constructed.1.2 The construction of expression vector pRD29A-CBF3:In order to construct plant expression vector pRD29A-CBF3, two middle vectors was constructed. After double digestion of clone vector pVCT2012 with restriction endonucleases HindIII and EcoRI, two fragments of 2635bps and 1949bps produced and the band of 1949bps was cut from argarose and purified , pBS II KS+ digested with Hindlll and EcoRl, precipitation with ethanol, 12bp fragment was discarded, and 2849bp fragment was purified, ligated the two fragments with T4 DNA ligase, transformed into XL1-Blue, the middle vector pBS-RD29A-CBF3 was constructed. After pBS-RD29A-CBF3 was digested with BamHI/Cla I, purified the 1974bp fragment, pGEM-7Zf digested with BamHI/Cla I, purified the 2988bp fragment, ligated the two fragments with T4 DNA ligase, transformed into XL1-Blue, the middle vector pGEM-RD29A-CBF3 was constructed. pGEM-RD29A-CBF3 was digested into 2983bp and 1978bp two fragments with EcoRl, and the 1978bp fragment was purified, pVCT2016 was digested into linear DNA with EcoRI, and subsequent ligation was performed, the two fragments was ligated and transformed into Escherichia coli(XL1-BIue) and Agrobacterium tumefaciens (LBA4404/pAL4404) , and the plant expression vector pRD29A-CBF3 was constructed.2.Study on transformation of plant expression vectors into cucumber cotyledons2.1 Optimization of regeneration system on cucumber cotyledons genetic transformationOn the base of other people’s anterior work, the cucum更多还原