Cloning,Prokaryotic Expression And Primary Study On Physiology Function Of TASSP1from Wheat

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 and718bp 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 TaSSPl sequence is2361bp.This experiment is based on the results above. Firstly, complete genome sequence of TaSSP1without 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 TaSSPl being activated in vivo. Secondly, in order to farther understand the physiological functions of the protease, the expression of TaSSP1in the wheat was analyzed in the level of protein and nucleic acid respectively.Software Signal4.0analysis showed that the first27amino acid sequence of TaSSP1is the signal peptide region of the protein.The TaSSP1without 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℃,3h,0.5mM 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 about56KD, which is smaller than targeted value (about78.8KD) of TaSSP1-31-786protein. 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-TaSSP1-133-786and pET24b-TaSSP1-133-450were 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. After15days, abscisic acid (ABA) and6-benzylaminopurine (6-BA) were added into the culture solution at the final concentration of10μM and22μM respectively. Than,we only took the third wheat leaves at different processing time, extracted RNA from the leaves and studied the expesssion of the TaSSP1in transcriptional level by semi-quantitative RT-PCR and fluorescence quantitative RT-PCR. The results showed that the transcriptional expesssion level of TaSSPl gradually increased in wheat leaves treated with ABA from Oh to7or12h, and then gradually restored to the natural leave; while the transcriptional expesssion level of TaSSPl in wheat leaves treated with6-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 TaSSP1in the crude enzyme solution were detected by using SDS-PAGE and Western-blot. The results were showed that the protein expression level of TaSSP1also slightly increased after the wheat was treated by ABA from Oh to12h; While the the protein expression level of TaSSPl in the wheat treated by6-BA from Oh to7h gradually also decreased, and then kept the lower level than that untreated.After the wheats grew for15days, the third wheat leaves removed both ends were dark-induced for Id,2d,3d and4d. 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 TaSSP1in wheat leaves gradually increased and accompanied by a gradual reduction of Rubisco; TaSSP1will gradually degraded and form the fragment of71KD,66KD,57kD; The fragment of71KD gradually decreased and The fragment of57KD gradually increased; The fragments of71KD and66KD were the actived form, but the fragments of57was the degraded form with no activity.