【作者】 孙金月；

【导师】 陈珈；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2003， 博士

【摘要】 实现源、库的协调是小麦获得高产的生理基础。TPT是光合碳代谢与胞质其它代谢沟通的桥梁，是源、库间同化物运输的关键调控部位。研究TPT的性质、TPT在同化物分配中的凋节功能及其TPT基因的表达调控，对于提高光合作用同化物利用效率有着重要意义。但是至今对重要粮食作物小麦TPT的运输性质、生理功能及其基因表达凋控的研究尚无报导。本文研究了小麦TPT蛋白的定位、运输动力学及其对同化物分配的调节功能，TPT基因表达的影响因子，为协调源、库间的同化物分配，提高光合同化物的利用效率，增加作物产量提供依据。 根据我们已克隆的小麦TPT cDNA序列（GenBank登录号AF314182）推测的蛋白氨基酸序列，经软件分析表明羧基端的13个氨基酸序列可能是TPT的抗原决定簇。以人工合成的13肽（N’-KAK IEE EKR AKA A-C’）为半抗原，与BSA偶联后，免疫兔子获得抗血清，效价为1:500。利用TPT抗体进行Western blotting分析表明，小麦TPT蛋白仅存在于叶绿体被膜上，而在液泡膜和线粒体膜上不存在。通过TPT不可逆抑制剂[H³]₂-DIDS标记和SDS-PAGE分析表明，小麦TPT蛋白是叶绿体被膜中最主要的蛋白，约占被膜总蛋白的15％，其分子量为35kD。 用小麦完整叶绿体，采用硅油离心法进行磷酸二羟丙酮（TP）、磷酸烯醇式丙酮酸（PEP）、葡萄糖-6-磷酸（G6P）与Pi反向交换的TPT运输动力学测定。结果表明，DHAP／Pi的最大运输活性最高，PEP／Pi次之，G6P／Pi最低。这些运输底物的Km值（由小至大）表示TPT与底物的亲和性，亲和性由高到低依次为DHAP、Pi、PEP和G6P，证明TPT的最适运输底物和运输方式为DHAP／Pi的反向交换。TPT转运活性受光的调节，光可提高V_max，但光照并不能明显改变TPT对底物的亲和性（即TPT对其运输底物的Km值仅受光的微弱影响）。黑暗条件下，DHAP的V_max为21.4μmol／mg chl·h，K_m为0.019mmol／L。光照处理使DHAP的V_max，增加54.67％，K_m值下降10.5％，表明光在调节TP合成的同时也促进了TPT的运输活性。用TPT特异性抑制剂DIDS处理的叶绿体，无论黑暗还是光照，所有底物的运输均被强烈抑制。光照条件下抑制达90-95％。 叶绿体内光合同化物磷酸丙糖（TP）有3个去向，即卡尔文循环中RuBP的再生、外运到胞质中合成蔗糖送往库器官和叶绿体内合成临时淀粉。利用TPT的专一性抑制剂比较光照条件下离体叶绿体中的终端产物淀粉合成量的差异，估算出TPT外运TP的量。当TPT被DIDS抑制，失去子运输能力时，可使叶绿体内淀粉合成增加14倍，与淀粉合成途径相关的上游代谢物PGA、TP、FBP、F6P、G6P的含量均有明显增加，增幅在25％以上。证明在保证卡尔文循环正常运转的前提下，通过TPT外运到胞质中参与蔗糖合成和其它代谢活动的TP约占93.6％，而用于叶绿体内合成淀粉的TP仅占6.4％。因此，小麦是一种典型的糖叶型植物。对光合效率不同的小麦品种进行TPT的Western blotting分析，结果表明中、低等光合效率品种的TPT表达量均较低，而高光合效率小麦品种的TPT表达量较高。因此，在对更多的小麦品种进行验证后，有可能把TPT表达的丰度作为筛选高产小麦品种的参考指标之一。 Northern blotting分析表明，渗透胁迫处理使小麦幼苗TPT在转录水平上的表达增强，但具有相同渗透势的葡萄糖等溶液处理使TPT在转录水平上的表达明显降低。在葡萄糖对小麦TPT表达调节中，低浓度范围内，即0.1-100μmol／L的葡萄糖有激活效应，当浓度大于1mmol／L时为抑制作用。用 5 mmol／L葡萄糖溶液处理小麦幼苗 id后，TPT基因的表达开始下降，第 4 d时受到强烈的抑制。 为了验证己糖激酶（HXK）是否参与葡萄糖对小麦TPT表达的调节，用r干CR方法克隆到一长度为 581 hp的 HXK CDNA片段，部分构建了 dSANAi真核表达载体pBI－HXK－RNAi。电激法转化小麦原生质体，检测 HXK活性为对照的 9．62％，表明获得了 pBIHXK－RNAi瞬时表达的原生质体。采用’H．DIDS特异标记TPT，比较野生和转化原生质体中葡萄糖、多种葡萄糖类似物以及 HXK抑制剂（葡萄糖胺）对 TPT表达量的影响。结果表明只有葡萄糖仰 mmol／L)或者能被 HXK磷酸化的葡萄糖类似物2－deoxyglucose与活化的HXK共存时，TPT的表达才被抑制。初步分析认为，葡萄糖可能是TPT对糖应答反应的信号分子，HXK对葡萄糖的磷酸化为糖应答反应所必需。更多还原

【Abstract】 The partitioning of photosynthate between source and sink is the physiological basis for acquiring a high yield in wheat, and triose phosphate/ phosphate translocator (TPT) may be the first regulation point for this partitioning. Understanding the characteristics of TPT, its regulation on the distribution of assimilates and the expression of tpt gene may be critical for improving the utilization rate of photosynthetic assimilates. But up to now it is unknown about the transport characteristics, the physiological function of TPT, and the expression of tpt gene in wheat. In this paper, we described the localization and transport kinetics of TPT, analyzed its physiological role in the distribution of assimilates, explored the factors that effect the expression of tpt, and studied the role of hexokinase (HXK) in the expression of tpt gene regulated by glucose in wheat, which will provide clues for coordinating the partitioning of assimilates between source and sink, improving the utilization rate of photosynthate, and increasing crop yield.A peptide with a length of 13 amino acids (N’-KAK. IEE EKR AKA A-C’) was synthesized according to the putative amino acid sequence by 3’ end of wheat tpt cDNA (39 bp) cloned by us. The peptide coupled with bovine serum albumin (BSA) was used to inject rabbit to prepare antibody. The ratio of the prepared antibody against TPT was 1:500. Western blotting analysis showed that TPT only exists in the envelope membrane of wheat chloroplasts, but not in the membrane of vacuoles and mitochondrials. SDS-PAGE and labeling of TPT with [H3]2-DIDS (4, 4’-diisothiocyano-2, 2’-stilbenedisulfonate, DIDS), an irreversible specific inhibitor, indicated that TPT is a 35 kD chloroplast membrane protein and makes up about 15 % of the total membrane proteins of chloroplasts.Silicone-oil-layer centrifugation system was employed to study the kinetic properties of TPT, which catalyzes the counter exchange of dihydroxyacetone phosphate (DHAP)/phosphoenol -pyruvate (PEP)/glucose-6-phosphate (G6P) and inorganic phosphate (Pi). The maximal transport activity of TPT was the highest for DHAP/Pi, followed by PEP/Pi and G6P/Pi. The Km of TPT was the lowest for DHAP, followed by Pi, PEP and G6P, so the most preferred substrate of TPT is DHAP. The Vmas and Km of DHAP were 21.4 umol/mg chl-h and 0.019 mmol/L respectively in the dark. In the light, Vmax of DHAP increased by 54.67 %, and Km decreased by 10.5 %. Thus, the transport of TPT appears to be regulated by light, and it can increase Vmax, but illumination does not change the affinity of TPT to substrates, that is to say that light has few effects on the Km of substrates transported by TPT. The transport of the various substrates is strongly inhibited by DIDS whether in dark or in light.The transport activity of TPT being inhibited leads to an obvious increase of the content of many metabolites and an accumulation of starch in chloroplasts. A change of the enzyme acitvity in wheat chloroplasts also provide a support for the flux of photosynthate to starch. Wheat is a kind of sugar-leaf plants. TPT plays an important role in the regulation on the distribution of assimilates in wheat chloroplasts and in the transport of photosynthetic products from source to sink. Its function is to exportphotosynthetic assimilates out of chloroplasts efficiently and participate in all kinds of metabolism in cytosol.Northern blotting analysis demonstrated that the expression of tpt gene increased under osmotic stress treatments in seedlings of Jing 411, but decreased obviously under the treatments with glucose, sucrose and frucrose. The expression of tpt decreased clearly in wheat seedlings when the concentration of glucose was above 5 mmol/L for 4 days. Western blotting analysis showed that the expression of tpt of the cultivar Jing 411, with a middle photosynthetic rate, Zhongpin 97-37 and Zhong 924, with a low photosynthetic rate, is lower than No 6 Wenmai and No 16 Jinan, with a higher photosynthetic rate, so the abundance of expression of tpt may act as更多还原