【作者】 潘欣；

【导师】 朱延明；

【作者基本信息】 东北农业大学 ， 植物学， 2010， 硕士

【摘要】 水稻是世界上重要的粮食作物,世界上3/4以上的人口以水稻为主食,但是低温、干旱、盐碱等因素严重影响水稻产量和品质,每年都会造成不同程度的损失。据统计,世界干旱、半干旱地区占地球陆地面积的33%,其危害相当于其它自然灾害之和。盐碱地占地球陆地面积的7.6%。另外,还有许多地区经常受低温冷害的影响。而我国约有670万公顷盐碱土耕地,还有2000万公顷盐碱荒地尚待开发。黑龙江省的盐碱地集中在松嫩平原地区,分布面积广,盐渍化程度高,其中尤以三肇、安达、大庆等地盐碱地分布最为集中,大面积的盐碱地严重影响农作物的产量,对黑龙江省的经济造成极大的损失。随着工业现代化的发展,灌溉地和塑料大棚面积不断扩大,土壤次生盐渍化日趋严重。随着人口的不断增加、耕地面积的日益减少和生态环境的急剧恶化,粮食供求矛盾将日益凸现,因此,如何提高作物抗干旱、盐渍和低温的能力是一个全世界都亟待解决的问题。传统育种学家已经在提高植物抗逆性方面进行了长期的探索与研究,但常规育种方法存在着工作量大、周期长、耗资多、效率低、可利用基因资源有限等弊端,而通过基因工程手段进行分子育种则避免了以上缺点。近年来,随着对植物逆境胁迫的分子反应和信号传递机理研究的逐渐深入,研究者已发现从改良或增强一个关键调控基因的调控能力着手,也许是提高作物抗逆性更为有效的方法和途径,因此,进行这些调控基因的克隆和功能鉴定是植物抗逆基因工程研究的重要前提条件,筛选并获获得具有抗逆形状的转基因植株植物,为改良不断恶化的生态环境提供了重要依据。本研究以本实验室获得的转钙依赖的蛋白激酶基因OsCDPK7和促分裂素原活化蛋白激酶基因OsMAPK4基因的T2代和T3代水稻作为试材,对T2代植株进行了耐盐碱的初步筛选,并研究了株高和死亡率；将收获的T3水稻,进行了纯合体的筛选及耐盐碱的抗逆性检测,同时研究了外源基因的表达情况和主要的耐盐碱性状。通过本研究可以获得具有耐盐碱的转基因水稻,用于黑龙江省盐碱地的改良,研究结果将为植物基因工程改良盐碱地提供了新思路,具有一定的理论和应用价值,为最终培育可在盐碱地正常生长的转基因水稻提供了重要资源。主要研究结果如下所示：1转OsMAPK4、OsCDPK7基因水稻植株的遗传稳定性分析1.1转OsMAPK4、OsCDPK7基因水稻植株PCR检测对转OsMAPK4、OsCDPK7基因水T2代植株和T3代植株进行了PCR检测,PCR检测结果表明,转OsMAPK4、OsCDPK7基因水稻T2代植株和T3代植株均能扩增出目的基因；初步证明转基因水稻T2代植株和T3代植株中均含有目的基因,目的基因已经稳定遗传到后代。1.2转基因抗性植株Southern杂交检测为了进一步验证转OsMAPK4、OsCDPK7基因水稻的遗传稳定性,Southern杂交此部分试验正在进行中。2转OsMAPK4、OsCDPK7基因水稻T：植株遗传特性的分析对转OsMAPK4、OsCDPK7基因水稻T2代植株进行了耐盐碱的初步抗性检测,调查和分析了株高和死亡率,结果表明,转基因水稻的株高变化量大于对照水稻株高的变化量,转基因水稻的死亡率也低于对照水稻；其中E12启动子调控的转OsMAPK4、OsCDPK7基因水稻T2代植株株高变化量大于rd29A启动子调控的转OsMAPK4、OsCDPK7基因水稻T2代植株株高变化量,E12启动子调控的转OsMAPK4、OsCDPK7基因水稻T2代植株死亡率低于rd29A启动子调控的转OsMAPK4、OsCDPK7基因水稻T2代死亡率,转OsMAPK4、OsCDPK7基因水稻T2代植株具有较高的抗盐碱性。3转OsMAPK4、OsCDPK7基因水稻T3植株遗传特性的分析对转OsMAPK4、OsCDPK7基因水稻T3代植株进行了耐盐碱的抗性检测,调查和分析了株高和盐碱伤害指数,结果表明,转基因水稻T3代植株的株高变化量大于对照水稻株高的变化量,转基因水稻的盐碱伤害指数也明显低于对照水稻；E12启动子调控的转OsMAPK4、OsCDPK7基因水稻T3代植株株高变化量大于rd29A启动子调控的转OsMAPK4、OsCDPK7基因水稻T3代植株株高变化量,E12启动子调控的转OsMAPK4、OsCDPK7基因水稻T3代植株盐碱危害指数低于rd29A启动子调控的转OsMAPK4、OsCDPK7基因水稻T3代盐碱危害指数,具有较高的耐盐碱性,转OsMAPK4、OsCDPK7基因水稻T3代植株具有较高的抗盐碱性。更多还原

【Abstract】 Rice is the world’s important food crops,the 3/4 people of the world eat rice.But salinity, drought and low temperature and other abiotic stresses is the most harmful rice production and quality every year. According to statistics, the arid area and Semi-Arid Area accounts for 33 percent of the world.The damage is equivalent to the total of other natural disasters. Saline accounted for 7.6 percent of the land area. In addition, there are many areas often affected by low temperature stress.In China a total area of saline-alkali land is about 6.7 million hectares, includingmillion hectares potentials only saline-alkali land in Heilongjiang Province reached more than 9,000 hectares, has seriously hampered agricultural production in Heilongjiang Province. Relative to the changing external environment, enhance their salt and alkali resistance of plants to be more effective mitigation of the impact of salinity on the protection of the natural environment and the maintenance of world food production has an important role. Therefore, how to improve the ecological environment of plants to survive, how to improve the crop salinity, to achieve rapid agriculture, sustainable development has become an urgent need to resolve key issues.Traditional breeders have carried out long-term exploration and study to improve plant resistance,but traditional breeding methods has the problem of huge workload, long growth periods and cost more,low efficiency, the genetic resources available, through such means of genetic engineering molecular breeding avoid above shortcomings.In recent years, with studying the stress on plant molecular reactions and adversity signal transmission mechanism,researchers have found that gradually improved or increase ability to control gene regulation, is perhaps the methods and ways to more effectively improve crops resistance, the regulation of gene cloning and function is the prerequisites of plant gene engineering research, screening and obtain the transgenic plants with resistibility,and provides an important basis for the worsening ecological environment.In our study, utilized rd29A and E12 respectively promoter regulation from rice transformed with OsCDPK7 gene and OsMAPK4 gene of T2 and T3 transgenic plant as the tested varieties.Studying the preliminary screening of T2 generation of plants on salt or alkali stress for plant growth and plant death..homozygote.。Harvesting T3 transgenic plant,to the screening of homozygote and salt and alkali resistance testing, at the same time, research gene expression and the main characters of the salt and the alkali.Through this research we can be obtained transgenic rice with the ability to resist the alkali and saline, used in in Heilongjiang province. The results showed that provided a new idea to improve for plant genetic engineering modified saline and alkali, has certain theoretical and practical value. Providing an important resource for the final cultivation of normal growth of transgenic rice.The major findings are as follows:1.Genetic Stability Analysis of Transgenic Rice1.1 Genetic stability analyses of target genes in T2 generation and T3 generationThe genome DNA of T2 generation and T3 generation were determined by PCR analysis.The target genes were amplified in transgenic rice with PCR; which validated that the transgenic plants T2 generation and T3 generation co-integrated with OsMAPK4 and OCCDPK7 genes.1.2.Southern blotSouthern blot, this part of its research is in progress.2. Genetic Character Analysis to Transgenic Rice of T2 generationStudying the preliminary screening of T2 generation of plants on salt or alkali stress for plant height and plant mortality homozygote.The results showed that the variation of height of transgenic rice more than the control and the mortality rate is below the control rice Which the variation of plant height of transgenic rice which is the regulation of E12 promoters OsMAPK4,OsCDPK7 gene more than the transgenic rice which is the regulation of rd29A promoters OsMAPK4, OsCDPK7 gene.the plant mortality of transgenic Rice which is the regulation of E12 promoters OsMAPK4, OsCDPK7 gene was lower the transgenic Rice which is the regulation of rd29A promoters OsMAPK4, OsCDPK7 gene.3. Genetic Character Analysis to Transgenic Rice of T3 generationStudyed the screening of homozygote of transgenic rice of T3 generation and the screening of transgenic rice on salt or alkali resistance testing for the plant height and the index of salt or alkaline tolerance. The results showed that the variation of height of transgenic rice more than the control and the index of salt or alkaline tolerance.was below the control rice Which the variation of plant height of transgenic rice which is the regulation of E12 promoters OsMAPK4, OsCDPK7 gene more than the transgenic rice which is the regulation of rd29A promoters OsMAPK4, OsCDPK7 gene, the index of salt or alkaline tolerance.of transgenic rice which is the regulation of E12 promoters OsMAPK4, OsCDPK7 gene was lower the transgenic rice which is the regulation of rd29A promoters OsMAPK4, OsCDPK7 gene.更多还原