【作者】 孙海伟；

【导师】 张丽霞； 冯殿齐；

【作者基本信息】 山东农业大学 ， 茶学， 2012， 博士

【摘要】 自然环境下，低温胁迫是植物主要的环境灾害因素，也是许多农作物区域性和季节性的限定因素，往往会影响植物的生长发育和作物生产率，从而导致严重的经济损失。传统的植物育种方法在改善植物耐冻性方面具有一定的局限性，利用现代生物学技术对植物（尤其是木本植物）进行抗冻性改良，具有高效性和针对性，可以加速高抗冻性植物新品种的选育进程。沙冬青肌醇半乳糖苷合成酶基因（galactinol synthase gene of Ammopiptanthusmongolicus）AmGS是从抗寒性强、能忍耐冬季-30℃以下低温的沙冬青中筛选得到。经过RACE扩增得到了AmGS基因的全长cDNA序列。然后分别克隆到原核表达载体pET-22b和真核表达载体pCAMBIA2300-35S-OCS中，建成了AmGS基因的细菌遗传转化表达载体pET-22b-AmGS和红叶石楠遗传转化表达载体pCAMBIA2300-AmGS。然后分别转化到大肠杆菌和和红叶石楠中，获得了转AmGS基因的大肠杆菌和转AmGS基因的红叶石楠。初步筛选出抗寒性提高的转基因红叶石楠株系，为用基因工程技术对茶树等常绿树种进行抗寒性改良提供了重要参考和技术储备。主要研究结果如下：1、通过AmGS基因的原核表达研究，AmGS基因成功转化大肠杆菌菌株，在低温下转基因大肠杆菌比转化空载体的大肠杆菌存活率高，说明我们克隆到了预期的AmGS基因。2、建立了红叶石楠高效再生体系，外植体应选择水培嫩芽，最佳启动培养基：MS+6-BA1.0mg/L+NAA0.1mg/L；不定芽诱导培养基：MS+6-BA2.0mg/L+NAA0.5mg/L；茎段再生分化诱导：茎段平铺在MS+6-BA2.0mg/L+NAA0.1mg/L培养基上培养；生根培养：IBA浓度为生根限制因子，培养基为1/2MS+NAA0.05mg/L+IBA0.1mg/L+蔗糖10g/L。3、构建了植物转基因表达载体pCAMBIA2300-AmGS，研究了农杆菌介导转化后植株的死亡率、分化率、分化芽数、芽苗长度、芽苗颜色与遗传转化有关的指标，发现浸染部位是最重要的影响因子，茎尖是最佳的浸染部位，确定了AmGS基因转化红叶石楠的转化方案为：选取红叶石楠组培苗茎尖，用携带pCAMBIA2300-AmGS表达载体的农杆菌原液（OD600≈0.4）的1/2浓度，浸染10min，然后水平放置于附加25mg/L乙酰丁香酮（AS）的MS分化培养基上，25℃下黑暗条件下共培养2d，转入50mg/L卡那霉素浓度的MS培养基进行筛选培养。4、本研究中筛选得到32个抗卡那霉素的转基因植株，取其中的R6、R7、R8、R9、R10、R11、R12进行了茎段再生分化培养，对培养的第一代进行PCR分析表明转基因阳性株系有R6、R7、R8和R10。对R6、R7、R8和R10进行Southern杂交分析表明，AmGS基因已经整合到R6、R7和R8三个转基因株系的基因组DNA中。对单拷贝的R6和R7进行RT-PCR分析结果表明，导入的AmGS基因在转基因植株中的转录水平上表达。5、转基因植株的遗传传递和遗传稳定性检测发现，在C1、C2和C3继代植株中，个别植株出现过PCR阴性的结果，但从C4至C6继代植株中都没出现PCR阴性的植株，这表明C4以后的继代植株基本趋向稳定；也表明导入的AmGS基因在R6、R7两个转基因株系中能稳定地传递到后代转基因植株中。6、抗冻表型分析表明，在不同低温条件下，转基因植株都比野生型植株表现出较高的存活率，抗寒能力明显优于野生型植株，说明导入的抗冻基因提高了红叶石楠的抗寒性。7、不同低温处理下转基因植株的生理生化指标研究表明，低温处理后两个转基因株系相对电导率升高的程度明显低于野生型植株。R6株系的LT50是-12.93℃，R7株系的LT50是-12.63℃，野生型植株的LT50是-8.25℃，两转基因株系的LT50分别比对照下降了4.68℃和4.38℃，说明转基因株系的抗寒能力有了明显提高。转基因红叶石楠的可溶性蛋白、可溶性糖、游离脯氨酸、MDA等的测定结果也支持了转基因植株在低温处理后受到的危害减轻，从另一个方面反映了转基因植株的抗寒性得到了改良。8、转AmGS基因红叶石楠对土壤微生物影响的初步检测表明，转基因株系和非转基因株系的微生物类群主要有细菌、真菌和放线菌，以细菌为主要微生物群落。转基因红叶石楠根际的土壤微生物数量有变化，其中细菌菌群数量变化最大，但对总体组成影响不大，仍以细菌数量最多，真菌和放线菌次之；在细菌菌群中以芽孢杆菌属的细菌为主。本研究为用基因工程技术对茶树等树木进行抗寒性改良提供了重要理论参考和技术储备，本研究中筛选出的R6、R7两个转基因新株系为进一步培育类茶植物红叶石楠的抗寒品种提供了选择材料。进一步的研究还在进行中。更多还原

【Abstract】 Cold stress is a major environmental factor that limits the agricultural productivity of plants.Low temperature has strong impact on the survival and geographical distribution of plants.Cold stress often affects plant growth and productivity, which causes significant losses.Traditional plant breeding approaches have had limited success in improving freezingtolerance.Using the modern biological technology to improve the cold-tolerance of plants(especially woody plants) with higher efficency and specility, can accelerate the breedingprocess.An antifreeze gene AmGS (galactinol synthase gene of Ammopiptanthus mongolicus) wasisolated from Ammopiptanthus mongolicus, which has strong cold-resistance and couldendure low temperature below-30℃. In this study we obtained whole cDNA sequence thencloned into pET-22b (an prokaryotic expression vector) and pCAMBIA2300-35S-OCS (aneukaryoticexpression vector) respectively. A bacteria transformation vector pET-22b-AmGSand a plant transformation vector pCAMBIA2300-AmGS were constructed respectively. Andthen were transformed into E.coli and Photinia×fraseri, obtained the transgenic E.coli andtransgenic Photinia×fraseri of AmGS gene.The major results are as follows:1. The AmGS gene was successfully transformed into E. coli strain. The survival rate oftransgenic E. coli was higher than that of the nontransformed of E. coli at low temperatures.This fact support that the cloned AmGS gene is correct.2. Established efficient regeneration system of Photinia×fraseri. The best explant was waterculture shoots. Best initial medium was MS+6-BA1.0mg/L+NAA0.1mg/L, Formulacombination of adventitious bud induction was MS+6-BA2.0mg/L+NAA0.5mg/L, thisformula had a large number of axillary buds, sturdy seedlings, the color dark green.The bestmedium for regeneration differentiation of stem segments was MS+6-BA2.0mg/L+NAA0.1mg/L.IBA concentration as the main rooting culture limiting factor, the best rootingmedium was MS+NAA0.05mg/L+IBA0.1mg/L+sucrose10g/L.3. The plant expression vectors pCAMBIA2033-AmGS was constructed.We investigated themortality rate，differentiation rate，differentiation number of buds，sprouts length，Shoot color of transgenic plants.Found that impact site was the the major factor which effect oftransformation.The best Agrobacterium-mediated AmGS gene transformation program wasthat，Select shoot tip, with Agrobacterium stock solution (OD600≈0.4)1/2concentration, dip10min, put on the MS differentiation medium additional25mg/L acetosyringone (AS), at25℃under dark conditions,2d, then transferred to the concentration of50mg/L kanamycinMS medium filter culture.4. In total,32kanamycin resistant plants were obtained. From them, seven lines (R6, R7, R8,R9, R10, R11, R12) were identified by PCR, four (R6, R7, R8, R10) showed PCR positive,which were futher identified by Southern hybridization. Results indicated that3lines (R6, R7and8) showed positive hybridization results. These results indicated that the AmGS gene hasintegrated into the genomic DNA of transgenic plants of R6, R7and R8lines. RT-PCRanalysis on R6and R7showed that, the imported AmGS gene expressed at transcriptionallevel in transgenic plants.5. Transgenic plant heredity and genetic stability examination found that, in C1, C2and C3subculture plants, few plants appeared in PCR negative results, from C4to C6subcultureplant could not appear PCR negative plants. These results indicate that the transgene AmGScan be transferred to the offspring in the transgenic lines R6, R76. Cold resistance phenotype analysis showed that, under different temperature conditions,transgenic plants showed higher survival rate and much better cold resistance ability thancontrol wild-type plants.It proved AmGS gene enhanced the cold tolerance of transgenic plant.7. REC（relative electric conductivity）test results showed that, after low temperaturetreatment, the REC increasing level of transgenic plants was obviously lower than that ofuntransformed control plants. The LT50of R6strain was-12.93℃, R7strain was-12.63℃,untransformed control plant was-8.25℃. The LT50value of transgenic strains was obviouslylower than that of the untransformed control plants. These results proved that transgenicstrains cold resistant ability has been improved.The soluble protein, soluble sugars, free proline and MDA determination results of transgenicPhotinia×fraseri also support the harm suffered reduce after cold treatment, reflected that thecold resistance of transgenic plants improved.8. Initial test of rhizosphere soil microbial effects of transgenic plant showed that transgeniclines and genetically modified strains of microorganism are bacteria, fungi and actinomycetes,bacteria as the main microbial community. Transgenic plant rhizosphere soil microbialquantity change, in which the bacterial flora changes in maximum, but had little effect on the overall composition.Still bacteria has the largest number, fungi and Actinomyces in thesecond, Bacillus bacteria mainly in the bacterial flora.This study provide important theoretical reference and technical reserves to using geneticengineering techniques improved cold resistance of trees.This study screened R6, R7twotransgenic cold resistant lines and provides a selection of materials. Further research is still inprogress.更多还原