【作者】 陈晓；

【导师】 徐朗莱；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 本实验室研究发现,小麦叶片中存在着能够降解1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco)大亚基(LSU)为51KDa的相关蛋白酶,而且该蛋白酶在其分离纯化过程中与Rubisco紧密结合。本文以小麦3E158(Triticum aestivum L.cv.3E158)和扬麦158(Triticum aestivum L. cv. Yangmai158)叶片为实验材料,通过天然梯度聚丙烯酰胺凝胶电泳(GPAGE)后切胶回收得到的Rubisco,应用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)分离Rubisco蛋白亚基及蛋白酶,分别采用SDS-PAGE、加明胶的活性SDS-PAGE鉴定蛋白质和检测蛋白酶的方式,研究了该蛋白酶的生化特性和合成的细胞定位,并对与Rubisco紧密结合的蛋白酶的分离纯化进行了进一步尝试。其主要研究结果简述如下：1、与Rubisco大亚基降解相关的蛋白酶的生化特性小麦粗酶提取液经过非变性梯度(5%-10%)聚丙烯酰胺凝胶电泳后,通过切胶方法回收含有Rubisco的凝胶,我们得到了纯度很高的Rubisco全酶。将这种Rubisco全酶在不同的温度下进行处理2h,发现降解LSU的蛋白酶的最适温度为40～50℃。研究不同浓度的ATP对Rubisco大亚基的降解影响,结果表明,在添加1mmol·L-1ATP下能加速LSU降解,并降解成很多小的片段；添加的ATP浓度过高或过低,LSU降解程度相对较弱。选取小麦初生叶(平均叶长2cm)为材料,切胶回收得到纯度很高的Rubisco全酶。将这种Rubisco全酶在40℃下保温处理6h后,通过SDS-PAGE检测,发现能够清晰的得到Rubisco大亚基被降解为51KDa的条带；同时,对上述样品煮沸5min,再进行适宜温度下保温反应6h,发现此时LSU不再发生降解。因此,我们认为,降解Rubisco大亚基为51KDa的相关蛋白酶在小麦叶片的生长初期即已存在。2、与Rubisco大亚基降解相关的蛋白酶合成的细胞定位选用两种蛋白质合成抑制剂—放线菌酮(核基因编码蛋白质合成抑制剂)和林可霉素(叶绿体基因编码蛋白质合成抑制剂),分别浸泡处理暗处的小麦叶片。不同浓度(0.01、0.1和lmg/ml)的上述抑制剂作用叶片后,提取叶片粗酶液,通过SDS-PAGE检测LSU的降解情况,结果显示,林可霉素抑制LSU降解作用更明显。此外,1mg/ml林可霉素的抑制作用更好。1mg/ml的两种抑制剂,单独及其混合溶液暗处浸泡处理叶片1d,然后在pH7.5的Tis-HCl缓冲体系和pH5.5的柠檬酸缓冲体系下,通过聚丙烯酰胺电泳及切胶回收得到相应缓冲体系下的Rubisco全酶,SDS-PAGE检测Rubisco降解为51KDa的情况。实验结果显示,在两种pH条件下,林可霉素均能较强地抑制蛋白酶对Rubisco大亚基的降解。此外,1mg/ml抑制剂暗处浸泡处理叶片不同的时间,结果发现,随着处理时间增加,林可霉素处理的Rubisco大亚基发生降解的情况越少。由此,我们可以确定,叶片暗诱导衰老过程中合成产生的特异性降解LSU的该蛋白酶可能主要是由叶绿体基因编码的。3、分离纯化结合Rubisco并降解大亚基的蛋白酶切胶回收得到的Rubisco样品,过mono-Q强阴离子交换柱,收集Rubisco峰进行脱盐,浓缩后SDS-PAGE检测。结果表明,Rubisco仍会降解,即Rubisco样品通过强阴离子交换柱分离依然没有将该蛋白酶和Rubisco分开。切胶回收得到的浓度很高的Rubisco酶液进行非完全变性(样液不进行加热煮沸)的SDS-PAGE,电泳后用TritionX-100脱去SDS。然后,分别切胶回收整块凝胶中Rubisco大亚基、小亚基所在处及分离胶中除大、小亚基位置之外的成分,最后分别将大亚基之外的其他各成分加入大亚基成分中,SDS凝胶电泳检测发现,Rubisco全酶经过SDS凝胶电泳后,降解Rubisco大亚基的蛋白酶能够与Rubisco大、小亚基分开,而这种蛋白酶可能不止一种,其中占主导地位的蛋白酶分子量可能比大亚基大。通过对切胶回收的Rubisco全酶进行添加明胶的SDS-PAGE活性电泳检测,也发现Rubisco切胶回收酶液中存在着一种分子量可能高于大亚基的蛋白酶。更多还原

【Abstract】 In our previous research, we found a kind of protease closely binding to Rubisco (Ribulose1,5-bisphosphate carboxylase/oxygenase) could degrade its large subunit (LSU) to51KDa. On the basis of former studies, using the leaf of wheat (Triticum aestivum L.cv.3E158) and Yangmai158(Triticum aestivum L., Yangmai158) as material, Rubisco holoenzyme was first obtained by gel cut after non-denaturing gradient polyacrylamind gel electrophoresis (GPAGE), and then the degradation of LSU and characters of the protease were identified by conventional and improved SDS-PAGE. The biochemical characteristics, synthesis localization and separation of the protease were mainly studied, And a preliminary attempt about its purification was conducted. The main results are summarized as following:1. The biochemical characteristics of the proteaseAfter non-denaturing gradient (5%-10%) gel electrophoresis of crude enzyme solution from wheat leaves, the high purity Rubisco holoenzyme was got by gel cut acording the location of Rubisco on the gel. When the solutions of Rubisco holoenzyme were incubated at30～60℃for2h, we found that there was a optimal activity of the protease which could degradate LSU at40～50℃. The effects of different concentrations of ATP on the degradation of LSU showed that the LSU could be quickly degraded into many small fragments with appending1mmol·L-1ATP.Selected the firstborn leaf as material, the Rubisco holoenzyme got by gel cut was incubated at40℃for6h, Detected, the degradation fragments of LSU could be clearly observed, While this degradation phenomenon of LSU could be eliminated by the treatment of Rubisco holoenzyme boiled for5minutes. The results above showed that the protease binding closely to Rubisco, which could degrade LSU, had existed in the young leaf of wheat.2. The synthesis location of of the proteaseTwo kinds of protein synthesis inhibitor-cycloheximide (inhibitor of nuclear gene coding proteins) and lincomycin (inhibitor of chloroplast gene coding proteins) respectively, treated the wheat leaves under dark condition in vitro. The degradation of LSU in crude enzyme solution from the leaf treated by different concentrations (0.01,0.1and1mg/ml) of the inhibitors was detected by SDS-PAGE. The results showed that lincomycin possessed more pronounced inhibition of LSU degradation. In addition, the inhibition displayed better in the treatments with lmg/ml of inhibitors.The leaves treated with lmg/ml of the two inhibitors and the mixed solution under dark for1day, Rubisco holoenzyme obtained by gel cut after GPAGE was soaked in the pH7.5Tris-HCl buffer and pH5.5citrate buffer system, and the LSU degration in soaked solutions was detected by SDS-PAGE. The results showed that, under the two pH conditions, lincomycin could more restrain the degradation of LSU. In addition, the longer the treatment time carried through, the stronger the degree of LSU degradation inhibition of lincomycin at lmg/ml was. So, we could conclud that, the protease synthesized during the dark-induced, was encoded mainly by the chloroplast genes.3. Separation of the protease closely binding to the Rubisco.The Rubisco in the Rubisco extraction solution from gel-cut after GPAGE was separated by the mono-Q strong anion-exchange chromatography, desalting, concentration and SDS-PAGE. The results showed that, Rubisco degradation would continue to carry out after strong anion exchange chromatography. That is to say, the protease, which could degrade LSU, was still closely binding to the Rubisco.The Rubisco extraction from gel-cut was Electrophoresis by nonfull-denatured SDS-PAGE, in which the sample was not boiled, SDS was took off with TritionX-100. After that, the LSU, SSU, and other components in the Rubisco holoenzyme were first obtained by gel cut. All the other ingredients were added to the LSU, and detected respectively, we can found that the protease that degraded LSU could be separated from the Rubisco extraction after SDS-PAGE. In addition, When the Rubisco extraction from gel-cut was detected by Gel-SDS-PAGE, results showed that there was more than one protease to be able to degrade LSU in Rubisco extraction, and a protease, that molecular weight is higher than the LSU, had more important role in LSU degradation.更多还原