【作者】 刘茜；

【导师】 李明军；

【作者基本信息】 西北农林科技大学 ， 果树学， 2023， 硕士

【摘要】 植物碳代谢是生长发育最基础的生理途径之一,在维持其正常生长、保持源库平衡、响应胁迫等方面发挥重要作用。果糖作为碳代谢过程中的关键代谢物,影响植物碳信号及分配调控。果糖激酶(FRK)是调控果糖含量的关键酶,MdFRK2是苹果中调控果糖含量的关键基因。为了系统阐述MdFRK2对植物碳分配的影响,本研究中利用不同启动子驱动的MdFRK2过表达转基因苹果及杨树等材料,对不同组织糖含量、酶活性及相关基因表达进行分析,探索MdFRK2过表达转基因株系不同组织碳分配的差异及其机制。获得的主要结果如下:1.35S启动子驱动的MdFRK2过表达影响了苹果不同组织中的糖含量、糖代谢相关基因表达和糖代谢相关酶活性。对MdFRK2转基因苹果不同组织中糖含量测定发现,成熟果实中果糖、葡萄糖、山梨醇等较野生型均显著增加;茎、幼叶中的果糖含量较野生型降低,但成熟叶、幼果中的果糖含量较野生型增加;除成熟叶外其余组织蔗糖含量较野生型均有一定程度的增加。对不同组织糖代谢相关酶活性分析表明,成熟叶、成熟果实、幼果磷酸蔗糖合成酶(SPS)活性增强,蔗糖合成能力增加;幼果、幼叶蔗糖合酶(SUSY)活性增强,蔗糖卸载能力增加。对不同组织糖代谢相关基因相对表达量分析发现,成熟叶中与蔗糖合成相关的MdSPS1/6分别增长了3.9倍和1.7倍;成熟果实中MdSPS6表达量较野生型增加了34.5%;幼果中MdSUSY3相对表达量增加了1.43倍,幼叶中MdSUSY2相对表达量增加了9.2%,茎尖中MdSUSY1增加了1.2倍。这些结果表明,35S启动子驱动的MdFRK2过表达影响不同组织糖含量与源库碳分配有关。2.为了证明苹果MdFRK2过表达对杨树不同组织糖含量及源库碳分配的影响,对35S启动子驱动的MdFRK2过表达转基因杨树的相关指标进行分析。结果表明,过表达MdFRK2后,成熟叶、幼叶、茎、茎尖果糖含量较野生型显著降低,成熟叶蔗糖含量增加;幼叶中蔗糖、葡萄糖、半乳糖含量均显著增加。茎中蔗糖、半乳糖、葡萄糖含量均降低,但纤维素含量增加。对不同组织糖代谢相关酶活性分析表明,成熟叶、幼叶、茎、茎尖中SPS活性较野生型显著增加,幼叶、茎中SUSY活性较野生型显著增加。这些结果表明,35S启动子驱动的MdFRK2过表达在杨树中也能够影响不同组织间的碳分配。3.为了进一步探明MdFRK2表达的组织特异性与不同组织碳水化合物代谢的关系,对MdFRK2自身启动子驱动的MdFRK2转基因杨树与35S启动子驱动的转基因杨树的相关指标进行分析。研究结果表明,两类转基因株系茎中的果糖及蔗糖含量降低,纤维素含量增加,而35S启动子驱动的转基因杨树茎中纤维素含量高于MdFRK2自身启动子驱动的MdFRK2转基因杨树株系。更多还原

【Abstract】 Carbon metabolism is one of the most basic physiological pathways for plant growth and development.It plays an important role in maintaining normal growth,maintaining source-sink balance,and responding to stress.Fructose,as a key metabolite in carbon metabolism,affects plant carbon signal and distribution regulation.Fructose kinase(FRK)is a key enzyme regulating fructose content,and MdFRK2 is a key gene regulating fructose content in apple.In order to systematically elaborate the effect of MdFRK2 on plant carbon allocation,in this study,MdFRK2 overexpression transgenic apple and poplar materials driven by different promoters were used to analyze the sugar content,enzyme activity and related gene expression in different tissues,and to explore the difference and mechanism of carbon allocation in different tissues of MdFRK2 overexpression transgenic lines.The main results are as follows :1.The overexpression of MdFRK2 driven by 35 S promoter affected the expression of sugar metabolism related genes and the activity of sugar metabolism related enzymes in different tissues of apple,and affected the sugar content in different tissues.The determination of sugar content in different tissues of MdFRK2 transgenic apple showed that fructose,glucose and sorbitol in mature fruit were significantly increased compared with wild type.The fructose content in stems and young leaves was lower than that in wild type,but the fructose content in mature leaves and young fruits was higher than that in wild type.Except for mature leaves,the sucrose content of other tissues increased to a certain extent compared with the wild type.The analysis of enzyme activities related to sugar metabolism in different tissues showed that the SPS activity and sucrose synthesis ability of mature leaves,mature fruits and young fruits increased.The SUSY activity of young fruits and leaves increased,and the sucrose unloading ability increased.The relative expression of genes related to sugar metabolism in different tissues showed that MdSPS1/6 related to sucrose synthesis in mature leaves increased by 3.9 times and 1.7 times,respectively.The expression of MdSPS6 in mature fruit increased by 34.5% compared with wild type.The relative expression of MdSUSY3 in young fruit increased by 1.43 times,the relative expression of MdSUSY2 in young leaf increased by 9.2%,and the relative expression of MdSUSY1 in shoot tip increased by 1.2 times.These results suggest that overexpression of MdFRK2 driven by the 35 S promoter affects glucose content in different tissues and is associated with source-sink carbon allocation.2.In order to confirm the effect of MdFRK2 overexpression on sugar content and carbon distribution in different tissues of poplar,the related indexes of MdFRK2 overexpression transgenic poplar driven by 35 S promoter were analyzed.The results showed that after overexpression of MdFRK2,the fructose content of mature leaves,young leaves,stems and shoot tips was significantly lower than that of wild type,and the sucrose content of mature leaves was increased.The contents of sucrose,glucose and galactose in young leaves increased significantly.The contents of sucrose,galactose and glucose in stems decreased,but the content of cellulose increased.The analysis of enzyme activities related to glucose metabolism in different tissues showed that the SPS activity in mature leaves,young leaves,stems and stem tips was significantly higher than that in wild type,and the SUSY activity in young leaves and stems was significantly higher than that in wild type.These results indicate that overexpression of MdFRK2 driven by the 35 S promoter can also affect carbon allocation between different tissues in poplar.3.In order to further explore the relationship between the tissue specificity of MdFRK2 expression and carbohydrate metabolism in different tissues,the related indexes of MdFRK2 transgenic poplars driven by MdFRK2 promoter and 35 S promoter were analyzed.The results showed that the two types of overexpression lines reduced the fructose content in the stem,reduced the sucrose content in the stem,but increased the cellulose content in the stem.The cellulose content in the stem of the 35 S promoter-driven transgenic poplar was higher than that of the MdFRK2 self-promoter-driven MdFRK2 transgenic poplar lines.更多还原