【作者】 曹军；

【导师】 庄东红；

【作者基本信息】 汕头大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 土壤盐渍化是影响农业生产和生态环境的严重问题。据联合国环境组织报道全球约有50％的耕地受到盐胁迫危害。我国也有大面积的盐碱地，仅海岸带、滩涂地就达一亿多亩，并且有逐年增加的趋势。面对人口不断增加、耕地日趋减少和淡水资源严重不足的现实，如何利用大面积的盐碱地和丰富的海水资源，这是人类迫切需要解决的重大课题。随着生物技术的发展，人们寄希望于基因工程育种，即通过直接导入耐盐基因，使转基因作物获得耐盐性，实现在盐碱地或海滩上用海水浇灌种植农作物的梦想。 本论文研究选用已在模式植物中证实能增强耐盐性的mtlD（mannitol-1-phosphate dehydrogenase）基因及生长在海滩上的具有酬盐碱等特性的盐生植物-秋茄（Kandelia candel L. Druce）和海边月见草（Oenothera littaralis Schlect）的总DNA作为目的基因，通过根癌农杆菌介导法和花粉管通道法转化优良栽培花生品种（Arachis hypogaea L. cv. Shanyou 523），以期获得转基因耐盐花生。本研究是国内外首次进行单个耐盐基因和盐生植物总DNA转化花生的研究。主要结果如下： 1．建立了根癌农杆菌介导mtLD基因导入化生的遗传转化体系。整个遗传转化过程主要分为预培养、共培养、诱芽、促芽、生根及移栽六个步骤。取萌发7～8 d的花生叶片，切取中段为外植体，在诱芽培养基（MS+0.8 mg／L NAA+3 mg／L 6-BA+2 mg／L AgNO₃+6 mg／L GLutamine+30 g／L Sucrose+0.7％Agar，pH 5.8）上预培养3d，然后在农杆菌菌液中浸泡5 min，放在诱芽培养基上共培养2 d，再用含有500 mg／L羧苄青霉素的无菌蒸馏水冲洗后，置于添加了500 mg／L羧苄青霉素和80 mg／L新霉素的诱芽培养基上进行选择培养，直到长出幼芽（大约5 w）。转入添加了500mg／L羧苄青霉素和80 mg／L新霉素的促芽培养基（MS+3 mg／L6-BA+2 mg／L AgNO₃+30 g／L Sucrose+0.7％ Agar，pH 5.8）中培养，3 w后将长出的小苗转移至生根培养基（MS+0.8 mg／L NAA+2 mg／L AgNO₃+30 g／L Sucrose+0.7％Agar，pH 5.8），大约2 w后将长出强壮根系的植株移栽到沙土中。 2．通过根癌农杆菌介导mtlD基因转化花生共获得57个再生植株。提取这些植株的叶片DNA进行PCR反应，然后对PCR产物进行Southern杂交分析。结果有3株转化植株在PCR反应中能产生1.1 kb大小的预期DNA片段，而且该片段可与mtlD汕头大学硕士学位论文基因探针进行特异性杂交，基因己整合进花生基因组中而l泪性对照植株没有此预期DNA片段产生，表明。t1D本次实验的转化率为5.3%。花粉管通道介导。t1D基因转化花生的研究。在2001年秋季获得“汕油523”T.，代转化植株共29株，I>CR检测到2株阳性柏株，均为100卜g/m1的DNA处理浓度和花警管注射法处理，转化率为5.9%。由这2株}‘日性植株共繁殖了22株T，代植株，捉取它们fl勺l矛}·，}t)N八分别进行}’CtZ反应，少1又、Jj，Ct丈产物进行Southern杂交分析的结果，其中一株的后代（共9株）尸C尺产物都没有产生预期大小的DNA片段，而另一株的后代（共13株）中有4株在PCI{反应中能产生1.1 kb大小的预期DNA片段，而且该片段可与刃叮D基因探针进行特异性杂交。进一步对这4株花生的后代 （T它代，共21株）进行PCR检测，其中有7株呈阳性。这表明经花粉管通道介导转化的二洲D基因已整合进花生基因组中，并己初步获得稳定遗传。转基因花生种子（}’:代）在l月0 mmol/IJ NaCI溶液处王‘}‘一卜}’}勺发芽率比对照提高21.9%。花粉管通道介导秋茄总ONA和海边月见草总I)触转化花生的研究。转基因花生种子门’:代)在1斗0 mm。1/L NaCI溶液处理卜‘的发芽率有明显的提高。与对照相比，转秋茄总洲A花生和转海边月见草总0入八花生的发芽率分别提高了31.8%、28.4%。更多还原

【Abstract】 Salt stress negatively impacts agricultural yield throughout the world affecting production whether it is for subsistence or economic gain. The United Nations Environment Program estimates that approximately 20 % of agricultural land and 50 % of cropland in the world is salt-stressed. Modern molecular genetics and plant transformation advances have made salt stress feasible to assess biotechnological strategies based on engineered biosynthetic pathways, targeted gene or protein expression or alteration of the natural stress responsiveness of genes for development of salt tolerant crops. Previous works with model transgenic plants have demonstrated that cellular accumulation of mannitol can alleviate salt stress. The rationale behind this strategy is that mannitol can stabilize proteins and cellular structures and can increase the osmotic pressure of the plant cell under salt stress.In order to test this hypothesis in peanut, transgenic peanut (Arachis hypogaea L. cv. Shanyou 523) to express the mtlD gene (encoding mannitol-1-phosphate dehydrogenase) for the biosynthesis of mannitol via genetic transformation mediated by Agrobacterium tumefaciens and the pollen-tube pathway were engineered in this reasearch. Furthermore, the exogenous total DNA ofKandelia candel L. Druce and Oenothera litlaralis Schlect were also introduced into the peanut by the pollen-tube pathway technique.The main results and achievements contained in this thesis are as follows: 1. Established a highly efficient genetic transformation system using the peanut leaf as starting material. In this system, leaf explants were preconditioned for 3 days on BM medium [Murashige and Skoog (MS) salts basal medium supplemented with 30 g/L sucrose, 2 mg/L AgNO3, 6 mg/L Glutamine and a hormone combination of 0.8 mg/L onaphthaleneacetic acid (NAA) and 3 mg/L 6-benzyladenine (BA) solidified with 0.7 % agar, pH 5.8], followed by co-cultivation with A. tumefaciens EHA105 carrying binary vector pBIMTL, the latter of which contains a mtlD gene and a neomycin phosphotransferase (ATT/7) gene, on BM medium for 2 days in the dark. Then the explants were washed three times with steriled water and placed onto BM mediumcontaining 80 mg/L neomycin and 500 mg/L carbenicillin. After 5-6 weeks, the explants with buds were transferred to EM medium [MS basal medium supplemented with 30 g/1 sucrose, 2 mg/L AgN03 and 3 mg/L BA solidified with 0.7 % agar, pH 5.8] with 80 mg/L neomycin and 500 mg/L carbenicillin for the elongation of buds. 3-4 weeks after cultured, 2- to 3-cm-high elongated shoots were excised and planted on RM medium [MS basal medium supplemented with 30 g/L sucrose, 2 mg/L AgNOa , and 0.8 mg/L NAA solidified with 0.7 % agar, pH 5.8] with 40 mg/L neomycin for rooting. When the roots occurred, about 3 weeks later, the plantlets were planted in sand for further growth.2. The transformed T0 generations obtained through A. tumefaciens-medialed transformation above, were tested positive for mtlD genes by polymerase chain reaction (PCR) analysis. Results of PCR-Southern blot also confirmed that the mtlD gene was integrated into the peanut genome. The transformation rate was 5.3 % in this research.3. Plasmid pBlMTL, which contained mtlD gene and NPTH gene, was also employed as donor and introduced into peanut after self-pollination by pollen-tube pathway. PCR analysis and PCR-Southern blot of the transformed TO, TI, T2 generations also indicated the mtlD gene v/as integrated into the peanut genome successfully. The transformation rate was 5.9 % through this method. The germination rates of transgenic mtlD peanut were increased 21.9 % compared with control plants.4. The exogenous total DNA of Kandelia candel L. Druce and Oenothera littaralis Schlect were also introduced into the peanut through the pollen-tube pathway technique. The salt tolerance experiment results showed that the seeds from transgenic plants have better germination than control plant in the 140 mmol/L NaCl medium. The germination rates of transgenic peanut were e更多还原