Functional Analysis Of TaSSP1from Wheat And Its Homolog Gene At3G14067in Arabidopsis

Subtilisin-like proteases (subtilases) belong to the serine protease family(S8) which are widely distributed in all three kingdoms of life. This family of serine proteases is involved in multiple processes in plants, such as plants growth and development, response to environment changes and metabolic regulation. But their specific functions and regulation mechanism et al. are not entirely understanded. In the previous study of our group, a senescence-associated subtilisin-like serine protease was separated and purified from the dark-induced senescence wheat leaves, which was named TaSSPl (Triticum aestivum Subtilisin-like Serine Proteasel). The mass spectrometry data were used to alignment at NCBI protein database and only part of the protease amino acid sequence information was obtained for the database. However, the identified functions of this protease in wheat growth and development were not entirely clear. In this study, according to the known partial amino acid sequence of the protease, the RACE method was used to amplify the full sequence of TaSSP1protease gene. After the full-length sequence of TaSSPl gene was obtained, the CDS region of TaSSPl was used to compare in the MEROPS database and a protease of arabidopsis AT3G14067was found which with a homology was77.8percent compared to TaSSP1. The special function of AT3G14067protease was also not unclear in arabidopsis. Therefore, the methods of RT-PCR, real time quantitative PCR, western blotting, gradient gelatin-PAGE in gel proteolysis assay and transgenic et al. were used to furthermore understand the TaSSPl and AT3G14067function during plants development and response to environmental changes based on the results of the previous study. The main results obtained in this study were showed as follows:1) According to the known partial amino acid sequence of the TaSSP1, The RACE method was used to obtain the full-length sequence of wheat TaSSP1protease gene. The GenBank database accession No. was JX962746. This cDNA had a2361bp coding region, and was predicted to encode a protein of786amino acid residues, and that it contained a signal peptide, a19Inhibitor domain, a peptidase S8family domain, a protease-associated domain and a C-terminus domain as analyzed by NCBI’s Conserved Domain Database (CDD).In order to gain a better understanding of the functions of TaSSPl, wheat were cultured and treated with100μM ABA,6-BA, ethephon, and200mM NaCl, D-Mannitol, sucrose or16%PEG6000respectively, whereafter the mRNA, protein and activity levels of TaSSP1in wheat at stated different hours after treatment were analyzed by RT-PCR, fluorescent quantitative RT-PCR, western blotting and gradient gelatin-PAGE in gel proteolysis assay. The results showed that the transcriptional and translational expression activities of TaSSP1in ABA treated wheat were gradually up-regulated with the extension of whole treatment time, but were obviously down-regulated in6-BA treated wheat from the middle to late stages of treatment. The increasing expression levels of TaSSPl were also identified in dark-induced senescence and older wheat leaves. In addition, the TaSSPl expression activities could be obviously up-regulated in wheats treated with200mM NaCl, D-Mannitol and sucrose or16%PEG6000. Furthermore, TaSSP1was showed to be a metal-dependent protease because its activity could be activated by some divalent metal cations, such as Cu2+, Ca2+, Zn2+and Mn2+. The results above indicated the function of TaSSPl was not only related with leaf senescence, but also related with the development of wheat under environment stress, especially to osmosis stress. And suggested that TaSSPl might be important on the response of wheat to environment stresses through its regulation of both gene expression and protein turnover.2) As AT3G14067protein sequence structure was analyzed through bioinformatics method, we found that AT3G14067has very similar structure compared to TaSSPl, and that it also contained a signal peptide, a19Inhibitor domain, a peptidase S8family domain, a protease-associated domain and a C-terminus domain as analyzed by NCBI’s Conserved Domain Database (CDD).Combined with semi-quantitative RT-PCR for analysis tissue-specific expression of AT3G14067, The result showed that the highest expression sites were in the stem, rosette leaves and cauline leaves which was consistent with the results of the plant membrane protein database. The expression of AT3G14067in response to various plant hormones,200mM NaCl,200mM D-Mannitol,200mM sucrose and16%PEG6000was further analyzed by Real-time quantitative PCR, the results show that it is apparently induced by ABA,6-BA, ETH,200mM NaCl,200mM D-Mannitol,200mM sucrose and16%PEG6000. These results indicated that AT3G14067might also be important in arabidopsis on the response to hormones and abiotic stress.In wild type arabidopsis, we also found that after the rosette leaves were treated with dark, the expression of AT3G14067was up-regulated after12hours treatment and came to the highest level at48hours treatment. In addition, The increasing expression levels of AT3G14067was also identified in older rosette leaves. This may indicated that AT3G14067gene was also a senescence-associated gene.In germination experiments of arabidopsis seeds, the results were showed that the expression level of AT3G14067was significantly up-regulated when seeds were cultured in dark, blue light and far-red light situations. Among them, the phenomena above in dark and far-red light treatments were most obviously. This indicated that the responses of AT3G14067gene were different in the seed germination treated with different light quality.The results preliminary showed that the AT3G14067protease was not only associated with arabidopsis leaf senescence, but may also closely related to the growth and development of Arabidopsis.3) Over-expression transgenic arabidopsis AT3G14067OX-lines3,4,6were obtained and identified as homozygous by real time quantitative PCR method. Compared with wild type, mutant and over-expression lines phenotype were found that when the seeds germinated in the dark condition, the hypocotyl of AT3G14067gene over-expression lines elongation significantly while the mutant and wild type plants had no significant difference between them. Moreover, the bolting time was5days in advance compared to the over-expression lines and the wild-type. However, the seeds of plants will mature early between the over-expression and the mutant plants. Under natural growth conditions, the plants height of the over-expression lines were lower. In contrast, AT3G14067mutants plants show the opposite phenotype. During the same periods, the rosette leaves of over-expressing plants had deeper leaf senescence degree compared to wild-type and mutant plants.The physiological and biochemical analysis showed that when the Arabidopsis thaliana grow to the50d stage, the content of chlorophyll in rosette leaves of mutant was significantly higher than that in wild-type and over-expression lines. At the60d and70d stage, the content of soluble protein in rosette leaves of over-expression lines was obvious higher than that in wild-type, while it in mutant is similar to wild-type plants. In over-expression lines, the TBARS content was beginning to increase after40d stage, and clearly higher than that in wild-type and mutant in the60d stage. In the50d stage, the enzyme activity of POD, SOD and CAT was significantly decrease, and the content of proline was rising significantly compared with the wild-type and mutant.The expression quantity of senescence-related genes in Arabidopsis thaliana leaves were analyzed by qRT-PCR. The expression levels of SAG12in over-expressing lines were significantly increased and in mutant significantly reduced, while SAG13in over-expressing lines were slightly increased and in mutant slightly reduced. NAP and NAC2in over-expressing lines were highly expressed, but in mutant and wild-type ware not significantly difference. The expression profiles for AtMYB2in over-expressing lines was5times higher than in wild-type. The expression patterns of WRKY6and WRKY53in mutant have a slight increase and in over-expressing lines have raised two times. Only SAG29appeared upward trend in both over-expressing lines and mutant. These results further indicate that AT3G14067gene not only directly related to aging, but also possibly promoting aging through regulation of other aging-related genes in Arabidopsis.Using the qRT-PCR method to analysis the genes associated with the hypocotyls development. The expression profiles for BCB19, SOB7and DFL1in the mutant were all significantly increased and in over-expressing lines and wild-type have slightly different. FHY3only in mutant has a slight upward trend. The expression level of RLP21in wild-type and mutant were similar while in over-expressing lines was up-regulated. The expression patterns of DWARF6in the mutant was significantly up-regulated but in over-expressing lines had an obvious downward trend. FE11, RAFL23, CUL1, RPT1, NPL1, HY3, LCL5, MAX2were up-regulated in both over-expressing lines and mutant, but in the mutant have a higher degree.The genes related to flowering were analyzed by the qRT-PCR method. Both AP2and AT1G01460in mutant had increased tendency, but had no change in over-expressing lines and in wild-type. The expression level of AP1, AG, AGL2and LFY3in mutant and over-expressing lines were significantly reduced. The expression profiles for AGL9in the mutant and over-expressing lines were all significantly up-regulated. The expression pattern of FLC in mutant and wild-type had no changed but in over-expressing lines had a downward trend.These results above suggest that Arabidopsis thaliana AT3G14067gene may also have close relationship to the plant growth and development, such as the development of hypocotyl and flowering in Arabidopsis thaliana..