本实验室在研究小麦叶片衰老过程中蛋白水解酶同工酶的变化时，发现一种了性质较为稳定的蛋白酶。进而纯化了该蛋白酶，并通过质谱鉴定以及对所得数据在NCBI数据库中比对，结果显示其属于枯草杆菌类（subtilisin-like）丝氨酸型蛋白酶，因此命名为TaSSP1 (Triticum aestivum Subtilisin-like Serine Protease1 )；随后，通过对该蛋白酶抗原性分析，以其mRNA序列设计兼并引物，克隆到了与该蛋白酶抗原性相关的718bp基因片段（Triticum aestivum Subtilisin-like Serine Protease 1-fragment，TaSSP1-f）. 接着，在此基础上，成功地在大肠杆菌中表达了该基因片段的蛋白产物，并制得了该蛋白酶片段的抗血清. 近期，通过RACE（rapid amplification of cDNA ends）方法，成功获得了该蛋白酶基因的全序列，该序列全长为2361bp。 本论文工作在上述研究基础上，首先，对去除信号肽的TaSSP1全基因序列以及其不同结构域的基因序列进行了原核表达，获得该蛋白酶的不同蛋白片段，然后通过活性分析等研究，以期阐明该蛋白酶激活的结构学机制。其次，分别从核酸和蛋白水平对小麦体内该蛋白酶的表达情况进行分析鉴定，以期进一步明确该蛋白酶的生理功能。 通过Signal4.0软件分析, 知道上述蛋白酶序列的前27个氨基酸序列是该蛋白的信号肽区域。然后，将去除信号肽的TaSSP1基因重组到表达载体pET24b上，成功构建了pET24b-TaSSP1-31-786 重组表达质粒，并将重组质粒转入大肠杆菌Rosetta中诱导表达，诱导条件：温度30℃，时间3h， IPTG浓度为0.5 mM。在菌液OD600值为0.8左右时，超声破碎菌体后通过SDS-PAGE电泳检测发现菌液中诱导表达的蛋白分子量（约56KD）比理论蛋白的分子量（78KD）要小。而western-blot检测发现，诱导表达的蛋白可以和本实验室制备的该蛋白酶片段抗血清进行免疫结合。同时根据该蛋白酶不同结构域的基因序列结构，分别构建了pET24b-TaSSP1-133-450 ，pET24b- TaSSP1-133-786两个重组质粒，并按照上述相同方法转入大肠杆菌Rosetta中诱导表达，相同条件下超声破碎菌体后通过SDS-PAGE电泳检测，但结果均未发现表达的预期相应蛋白。 在TaSSP1基因生理功能的研究中，实验材料为扬麦158，小麦生长采用定期灌浇Hoagland营养液的水培方式，在小麦长至15天时，营养液中分别加入10μM脱落酸ABA（Abscisic Acid）与22μM 6-苄氨基嘌呤6-BA（6-Benzyl Aminopurine）对小麦进行处理。然后取不同处理时间的小麦叶片（仅取小麦第三片叶），提取RNA，采用半定量和荧光定量RT-PCR两种方法分析在不同的激素和不同时间处理下，小麦TaSSP1在转录水平上表达量的变化。结果显示，在ABA处理条件下，随着处理时间的增加从0h、1h、3h、到7h，TaSSP1基因在RNA水平的表达有逐渐增加的趋势，然后恢复到天然水平；而在6-BA处理下，在处理的72h内，除1h外，随着处理时间的增加，TaSSP1基因的RNA表达水平呈现逐渐下降趋势。 同样条件下取不同激素和不同时间处理的小麦叶片，提取小麦叶片的粗酶液为样液，分别采用SDS-PAGE 和Western-blot方法，检测了小麦枯草杆菌类丝氨酸型蛋白酶TaSSP1 蛋白水平表达的变化情况。结果显示，在ABA处理后的0到12h时，小麦TaSSP1 基因在蛋白水平上的表达量呈现增加趋势，而后到72h内一直保持比对照高的水平； 而6-BA处理后的0h到7h内，小麦TaSSP1 基因在蛋白水平上的表达量呈现逐渐降低的趋势，而后的72h内一直保持在比对照低的水平上。 在小麦长至15天时，取去除其两端的小麦第三片叶，暗处理1、2、3和4天后，进行Western-blot和含明胶的5％-15％浓度梯度的聚丙烯酰胺凝胶电泳分析鉴定，结果显示，随着暗处理时间的增加，小麦叶片TaSSP1 的活性逐渐增强,并伴随着Rubisco的逐渐减少；分子量为78KD的TaSSP1会逐渐降解为分子量约为71KD、66KD、57KD的片段，其中，71KD片段的量逐渐减少，而57KD的片段的量逐渐增加；71KD、66KD的片段是TaSSP1的活化中间体，而57KD的片段为降解片段，不表现酶活性。

In our laboratory, found a quite stable endopeptidase isoenzyme related leaf senescence in wheat leaves. Then purified the protease and identified it by MS (mass spectrometry). A comparative data analysis of protein sequence in NCBI databases showed that the protease is a Subtilisin (subtilase-like) protease. We named it as TaSSP1 (Triticum aestivum Subtilisin-like Serine Protease1). Furthermore, designed degenerate primers based on antigenic analysis and mRNA sequence of TaSSP1, which is related to the antigenicity of the protease and 718bp in length. On this basis, We has been successfully expressed the protein product of TaSSP1-f in Escherichia Coli and prepared the antiserum of TaSSP1-f. Recently, We obtained the protease gene sequence by RACE (rapid amplification of amplification of cDNA ends). The full length of TaSSP1 sequence is 2361bp. This experiment is based on the results above. Firstly, complete genome sequence of TaSSP1 without the fragment encoding signal peptide and the genome sequence of different domains are expressed in prokaryotic cells to obtain different protein fragments of the protease. By analyzing the activity of different protein fragments, we hope to elucidate the mechanism of TaSSP1 being activated in vivo. Secondly, in order to farther understand the physiological functions of the protease, the expression of TaSSP1 in the wheat was analyzed in the level of protein and nucleic acid respectively. Software Signal4.0 analysis showed that the first 27 amino acid sequence of TaSSP1 is the signal peptide region of the protein.The TaSSP1 without the fragment encoding signal peptide was cloned into expression plasmid pET24b, and then constructed the recombinant expression plasmid pET24b-TaSSP1-31-786. On that basis, we the recombinant plasmid was transferred into E. coli Rosetta and expressed by IPTG-inducing. The best inducing expression conditions for protein products: 30℃, 3 h, 0.5 mM IPTG, and OD600 =0.8. Analyzed by SDS-PAGE after breaking the cell by using ultrasonic waves，we found that the molecular weight of expressed protein was about 56KD, which is smaller than targeted value (about 78.8KD) of TaSSP1-31-786 protein. However, the expressed protein could be hybridized with the antiserum prepared in our laboratory by Western-blot. We believe that the recombinant protein is the degradation bands of TaSSP1.Meanwhile, according to the gene sequence of TaSSP1’s different domains, the two recombinant plasmids pET24b-TaSSP1-133-786 and pET24b-TaSSP1-133-450 were also constructed and transferred into E. coli Rosetta respectively, but analyzed by SDS-PAGE，there is no expressed proteins found in Rosetta cells after IPTG-inducing at the same condition. In the study of the physiological function of TaSSP1, we chose the wheat (Triticum aestivum L.cv. Yangmai158) as experiment materials grown in Hoagland culture solution. After 15 days, abscisic acid (ABA) and 6-benzylaminopurine (6-BA) were added into the culture solution at the final concentration of 10μM and 22μM respectively. Than,we only took the third wheat leaves at different processing time, extracted RNA from the leaves and studied the expesssion of the TaSSP1 in transcriptional level by semi-quantitative RT-PCR and fluorescence quantitative RT-PCR. The results showed that the transcriptional expesssion level of TaSSP1 gradually increased in wheat leaves treated with ABA from 0h to 7 or 12h, and then gradually restored to the natural leave; while the transcriptional expesssion level of TaSSP1 in wheat leaves treated with 6-BA decreased gradually with treatment processing time. At the same time, the third wheat leaves treated at different processing time were also chosen and extracted as the crude enzyme solution. The protein level changes of TaSSP1 in the crude enzyme solution were detected by using SDS-PAGE and Western-blot. The results were showed that the protein expression level of TaSSP1 also slightly increased after the wheat was treated by ABA from 0h to 12h; While the the protein expression level of TaSSP1 in the wheat treated by 6-BA from 0h to 7h gradually also decreased，and then kept the lower level than that untreated. After the wheats grew for 15 days, the third wheat leaves removed both ends were dark-induced for 1d, 2d, 3d and 4d. The results detected by using gelatin-GPAGE (5%-15%) and Western-blot showed that with the increasing of the time of dark treatment, the activity of TaSSP1 in wheat leaves gradually increased and accompanied by a gradual reduction of Rubisco; TaSSP1 will gradually degraded and form the fragment of 71 KD, 66KD, 57kD；The fragment of 71 KD gradually decreased and The fragment of 57 KD gradually increased; The fragments of 71 KD and 66KD were the actived form, but the fragments of 57 was the degraded form with no activity.