【作者】 黄旭新；

【导师】 周晓阳； 陈少良；

【作者基本信息】 北京林业大学 ， 植物学， 2011， 硕士

【摘要】 秋茄(Kandelia candel(L.)Druce)是红树的一种,属于非泌盐型红树,长期生活在海边,具有很强的耐盐性。前期研究显示,秋茄苗木KcBURP基因与KcC2H2型锌指蛋白基因在盐胁迫下表达明显上调,提示这两个基因可能对秋茄耐盐性有所贡献。本文以秋茄为材料,利用前期Microarry分析所获得的KcBURP及KcC2H2型锌指蛋白基因的cDNA序列设计引物,通过RT-PCR,获得这两个基因cDNA的开放读码框(ORF)。序列分析结果显示KcBURP基因的ORF长1131bp,编码一个等电点为9.07、分子量为39.8kD、由375个氨基酸组成的多肽蛋白。KcC2H2型锌指蛋白基因ORF长738bp,由245氨基酸组成,蛋白分子量约26.1kD,理论等电点为8.65。将秋茄KcBURP基因构建到植物表达载pCAMBIA-2300中,转化烟草。选取10个卡那霉素抗性株系进行基因组PCR鉴定,数据表明其中的8个株系为转基因株系。PT-PCR的结果显示在2个转基因株系中KcBURP得到过表达。150 mM NaCl处理后,野生型明显萎蔫,过表达转基因植株长势良好。进一步对野生型和转基因烟草进行光合作用测定,结果显示100mM盐处理显著降低了野生型烟草的净光合速率,而对转基因植株叶片的光合作用影响较小。盐浓度达到200mM时,转基因植株及野生型烟草净光合速率都明显降低,盐胁迫解除后,转基因烟草光合作用的恢复情况明显好于野生型烟草。说明KcBURP的过表达提高了烟草的抗盐性。另外,将KcC2H2型锌指蛋白基因全长cDNA构建到pK7WG2D上,转化烟草,选取8个株系进行基因组PCR鉴定,初步鉴定获得转基因植株。更多还原

【Abstract】 Kandelia candel is one of salt-tolerant mangrove species and habitats in seaside area. Preliminary studies have shown that KcBURP and KcC2H2 zinc finger gene of Kandelia candel seedlings were significantly up-regulated under salt stress, suggesting that they contributed to salt tolerance of Kandelia candel.In the presented study, the cDNA coding for the open reading frames (ORF) of KcBURP and KcC2H2 zinc finger genes of Kandelia candel were obtained by a PCR approach using the primers designed from the sequence information of Microarry cDNA. Sequencing analysis indicate that the ORF of KcBURP has a size of 1131bp, encoding for a protein of 375 amino acid with an isoelectric point of 9.07 and a molecular weight of 39886 Da.The ORF of KcC2H2 zinc finger gene has a size of 738bp, encoding for a protein of 245 amino acid with an isoelectric point of 8.65 and a molecular weight of 26174Da.The gene construct of the KcBURP under the control of constitutive promoter pCAMBIA-2300 was transformed into tobacco. Ten individual KcBRP-transformed tobacco plants were identified by PCR. The PCR result show that eight of ten plants were KcBURP-positive. While there were only two plants showed over expression when the KcBURP-transformed tobacco plants were tested by RT-PCR. Salt tolerance examinations show that the KcBURP-transformed tobacco plants grew better than the wildtype under 150 mM NaCl. Furthermore, net photosynthetic rate of KcB URP-transformed tobacco plants were higher than the wildtype under 100 and 200 mM of NaCl saline, although the leaf photosynthesis was reduced by salt treatment. It is noting that the photosynthetic rate of KcBURP-transformed tobacco plants recovered rapidly than the’wildtype after the salt relief. Our result suggest that KcBURP gene is beneficial for plant salt tolerance. The gene construct of KcC2H2 the under the control of constitutive promoter pK7WG2D was transformed into tobacco. We identified seven KcC2H2-transformed tobacco plants by means of PCR test.更多还原