【作者】 黄诚梅；

【导师】 杨丽涛；

【作者基本信息】 广西大学 ， 作物栽培学与耕作学， 2007， 博士

【摘要】 甘蔗是我国重要的糖料和能源作物，主要分布在广西、广东、云南、海南等南方黄、红壤地区，而且90％以上的种植面积为缺乏灌溉的旱坡地，季节性干旱是限制我国甘蔗生产的首要环境因素。在广西各大植蔗区，大部分植蔗区基本上都没有灌溉条件，而且由于坡地保水能力差，不能充分利用自然降水，每年都出现不同程度的旱害，特别是春、秋旱，这是制约甘蔗获得高产高糖的最主要因素。脯氨酸是目前所知生物界分布最广的渗透保护物质之一，干旱、高盐、高低温及重金属等非生物胁迫都会导致细菌、动物、植物、藻类中脯氨酸大量累积。弄清甘蔗植株体内脯氨酸在胁迫诱导之下的生物合成与代谢变化，以及介入脯氨酸代谢的基因调控，了解其对提高甘蔗耐逆性的调控分子机制，将有助于利用脯氨酸基因工程改良甘蔗抗逆性。本研究以甘蔗（Saccharum officinarum L．）品种：桂糖21号（GT21），新台糖16号（ROC16）和新台糖22号（ROC22）等3个品种为材料，用聚乙二醇（polyethylene glycol，PEG）6000处理模拟水分胁迫条件，探讨其对甘蔗生长前期与伸长期间叶中脯氨酸积累及其代谢相关酶活性的影响，以阐明脯氨酸代谢与甘蔗耐水分胁迫能力之间的关系。同时，通过PEG6000进行模拟水分胁迫诱导甘蔗△¹-吡咯啉-5-羧酸合成酶（Saccharum officinarum L．△^-1-pyrroline-5-carboxylate synthetase，ScP5CS）基因的表达，采用同源克隆方法从甘蔗中克隆出ScP5CS基因，并对该基因核苷酸与推断的氨基酸序列进行了结构分析。为了进一步验证甘蔗ScP5CS基因的功能，通过构建ScP5CS基因植物反义表达载体遗传转化烟草，分析该基因在转基因烟草植株中的表达及其抗渗透胁迫功能，以初步探讨该基因的功能。主要研究结果如下：1．在甘蔗生长前期，经胁迫处理，对于游离脯氨酸的变化，ROC16在处理后第1 d其含量就明显上升，GT21在处理2 d后也开始上升，而ROC22在处理后12 d内其含量仍增加不明显。P5CS酶活性经胁迫处理都较之未处理的表现活跃，GT21与ROC16在胁迫处理后其酶活性表现上升趋势，而ROC22变化不明显。鸟氨酸转氨酶（ornithine-δ-aminotransferase，δ-OAT）酶活性变化不明显。对于脯氨酸脱氢酶（proline dehydrogenase，ProDH）酶活性，GT21与ROC16在处理1 d后稍有上升但在7 d后都有下降，而ROC22在第7 d其酶活性就开始提高。2．在甘蔗伸长期，PEG处理后第4 d，GT21与ROC16叶中游离脯氨酸含量明显上升，但ROC22在处理后6 d内其含量增加仍不明显。P5CS酶活性变化因品种而异，ROC22在处理后第6 d才明显上升，其他2个品种则在胁迫处理1 d后即明显提高。δ-OAT酶活性变化不明显。ROC22在处理1 d后其ProDH酶活性稍有提高，而其他2个品种则下降。3．通过探讨PEG胁迫处理下甘蔗生长前期与伸长期间叶中脯氨酸积累及其代谢相关酶活性的变化，表明了在渗透胁迫下，甘蔗叶中游离脯氨酸大量积累，其生物合成中谷氨酸→脯氨酸途径比精氨酸→鸟氨酸→脯氨酸途径更占优势地位。脯氨酸降解受抑制和其合成对脯氨酸积累有同等意义。4．P5CS是脯氨酸生物合成中谷氨酸途径的限速酶，本研究以甘蔗品种ROC22为材料，采用同源克隆方法从基因组DNA中获得两条特异扩增带，初步确定为甘蔗ScP5CS基因组片段，其中ScPS₁片段长度为2016 bp，包含有7个内含子，8个外显子，其中仅有977 bp的外显子编码序列，GenBank注册号为EF620362；将外显子序列与ScP5CS mRNA（EU005373）相比，同源率达到99．6％。而ScPS₂片段大小与ScPS₁外显子序列一样，长度为977 bp，同源率达到99．6％，但缺失了内含子，与ScP5CS mRNA（EU005373）相比，同源率也达到99．6％，这表明了ScPS₂具有大多数返座假基因鲜明的特征：完全缺失存在于功能基因中的间隔序列，即内含子序列。因此，初步推测基因片段ScPS₂为返座假基因序列。5．采用同源克隆与RT-PCR法从甘蔗品种ROC22中得到甘蔗ScP5CS基因的编码区cDNA序列，其长度为2151 bp，为一个完整的ORF，编码716个氨基酸，GenBank注册号为EU005373。核苷酸序列与前人已注册的甘蔗P5CS（EF155655）相比，同源率达到98％，但推断编码的氨基酸同源率仅为92％。该基因具有P5CS共有的结构域与结合位点：Putative ATP-bindingsite；Putative leucine domains；Glu-5-kinase domain；Putative NADPH-bindingdomain；Conserved GSA-DH domain保守区，脯氨酸反馈抑制作用位点。与前人已注册的甘蔗P5CS基因氨基酸序列（ABM30223）比较，在γ-GK保守域（Glu-5-kinase domain）内氨基酸变化较大，而与水稻、小麦的相比，其变化较小。因此，推断为甘蔗P5CS基因家族中的一个新成员。6．用长度分别为384 bp（位置为1318～1701 bp）和954 bp（位置为1163～2166 bp），包含在甘蔗ScP5CS基因的γ-谷氨酰磷酸还原酶（Gamma-glutamyl phosphate reductase，γ-GPR）区域内的两个甘蔗ScP5CS基因片段反向插入植物表达载体pBI121的克隆位点上，由CaMV35S启动子控制两个基因片段的表达，构建了植物表达载体pBI121／Sc-PCS₁与pBI121／Sc-PCS₂。用甘蔗ScP5CS基因反义植物表达载体遗传转化烟草。甘蔗ScP5CS基因在烟草中的反义表达，部分抑制了烟草P5CS基因的活力。对转基因烟草植株进行NaCl与PEG6000胁迫培养，发现转入ScP5CS反义基因片段的烟草植株矮小，叶片容易发黄，根系发育迟缓，根长缩短，而且转入短片段Sc-PCS₁（384 bp）与长片段Sc-PCS₂（954bp）的转基因植株生长受到抑制效果一样；而转入空载体pBI121的烟草和对照烟草植株一样，叶色和叶片的主脉均较绿，新叶长出正常，发根相对早，根长稍长。更多还原

【Abstract】 Sugarcane （Saccharum officinarum L.） is an important sugar and energy crop in China. The major sugarcane and sugar production in China is dominently located in southern region includes such as Guangxi, Guangdong, Yunnan and Hainan provinces. Drought is the most important abiotic stress limiting sugar productivity in China because about 90% of sugarcane is grown in the upland areas where irrigation is not available. In Guangxi, drought occurs very often in the major sugarcane growing areas, especially in the spring and autumn, which affects cane yield and sugar productivity seriously.Proline accumulation in response to various environmental stresses such as drought, excessive salinity, high or low temperature and heavy metal, has been described in many phylogenetically diverse organisms such as bacterial, mammalian, plants and algae. Efforts toward understanding the proline biosynthetic pathway in sugarcane and to unravel the role of proline in response to osmotic stress may help us to use gene engineering related proline to enhance the resistance of sugarcane under osmotic stress.Three sugarcane varieties, GT21, ROC16 and ROC22, were used as experimental materials. The plants were treated with 25% polyethylene glycol （PEG） 6000 at early and elongation stages, respectively. Effects of PEG stress on proline accumulation and the activities of the key enzymes in leaves of sugarcane were investigated in the present study. At the same time, the isolation and the structure of the sequences of nucleotide acid and deduced amino acid encoding the ScP5CS {Saccharum officinarum L. Δ¹-pyrroline-5-carboxylate synthetase) gene were reported. And the functions of this gene were analyzed by transferring the anti-sense mRNA of ScP5CS gene into tobacco. Expression and resistance under osmotic stress of the sugarcane ScP5CS were analyzed in the transgenic tobacco. The main results as follows:1. At the early growth stage, free proline content in ROC16 increased 1 d after starting of the osmotic stress treatment, that in GT21 also increased 2 d after the starting of the treatment, while there was no difference between the treatment and the control for the variety ROC22 during 12 d. The same results for the activity ofΔ¹-pyrroline-5-carboxylate synthetase （P5CS） were obtained after the stress treatment, the activity in GT21 and ROC16 increased after treatment, but it was contrary in ROC22. For the activities of ornithine-δ-aminotransferase （δ-OAT）, there was no remarkable difference between the treatment and control. The proline dehydrogenase （ProDH） activity in GT21 and ROC16 increased at firstly then decreased after 7 d while that in ROC22 increased 7 d after the starting of the treatment.2. At the elongation stage, free proline content in GT21 and ROC16 increased 4 d after the starting of the treatment, while there was no difference between the treatment and the control for the variety ROC22 during 6 d. The same results for the P5CS activity were obtained after the stress treatment, the P5CS activity in ROC22 did not increase until 6 d after the starting of the treatment. For the activities of 5-OAT, there was no remarkable difference between the treatment and the control. The ProDH activity in ROC22 increased after treatment, but it was contrary in the other varieties.3. Effects of water stress on proline content, activities of P5CS,δ-OAT and ProDH in leaves were studied. The results showed that proline is largely accumulated in sugarcane under the stress treatment. Whereas proline can be synthesized from either glutamate or ornithine in plants, the experiments indicate that glutamate, rather than ornithine, is primary precursor for proline biosynthesis in osmotically stressed sugarcane. And the accumulation of proline occurred as the result of both the activation of proline biosynthesis and the inactivation of proline degradation.4. P5CS is the rate-limiting enzyme in proline biosynthesis of glutamate pathway. In the present study, the sugarcane variety ROC22 was used as the experimental material. By homologous based cloning, two specific fragments isolated from sugarcane genome DNA with specific primers, which were confirmed as the sugarcane ScP5CS genes, naming ScPS₁ and ScPS₂. The length of sequence ScPS₁ was 2016 bp, containing 7 introns and 8 extrons, and the GenBank accession number is EF620362. Comparing the extron sequence 977 bp of ScPSi with that of ScP5CS mRNA （EU005373）, it showed high identity （99.6%）. And the length of ScPS₂ was the same as the transcribed sequence of ScPS₁, showing high identity with the transcribed sequence of ScPS₁ （99.6%） and with ScP5CS mRNA （EU005373） sequence （99.6%）. But the introns were absent in the ScPS₂, which is the feature of the pseudogene. So the ScPS₂ was confirmed as the pseudogene.5. With the sugarcane variety ROC22 as experimental material, the cDNA sequence of ScP5CS gene in sugarcane with 2151 bp in length was isolated by homologous cloning, which contained an open reading frame encoding a protein of 716 amino acids, with GenBank accession number EF620362. Comparing the sequence of ScP5CS with that of ScP5CS （EF155655） reported, the nucleotide acid showed high identity （98%）, but the deduced amino acid was only 92%. The deduced protein contains putative ATP-binding site, putative leucine domains, Glu-5-kinase domain, putative NADPH-binding domain, conserved GSA-DH domain and feedback inhibition site. Besides, there were more differences in Glu-5-kinase domain from the deduced amino acid of ScP5CS （EF155655）, but less for the P5CSs from rice （Oryza sativa） and wheat （Triticum aestivum）. So it is confirmed this gene is a new gene of sugarcane P5CS.6. The antisenced expression vectors pBI121/Sc-PCS₁ and pBI121/Sc-PCS₂ were constructed by respectively inserting two different fragments, which were 384 bp （position 1318-1701 bp） and 954 bp （position 1163-2166 bp）, of ScP5CS gene, into the plant expression vector pBI121 downstream controlled by CaMV35S promoter. These fragments were contained in gamma-glutamyl phosphate reductase domain. The antisenced expression vectors of ScP5CS gene were used to transformed tobacco plants. The transformed antisence ScP5CS in tobacco inhibited the expression of tobacco P5CS. The transgenic plants grew slowlier, the leaves were easy to get etiolational and the root system development was stunted and the roots were shorter under the NaCl and PEG stress as compared with the control. And the same results were obtained for both the shorter sequence Sc-PCS₁ （384 bp） and the longer sequence SC-PCS2 （954 bp） after the stress treatment, but it was contrary for the plants with the transformed vector pBI121 and the non-transgenic （negative） control.更多还原