Studies On Rubisco Stability In Flag Leaves Of Different Rice Varieties (Oryza Sativa L.) After Flowering

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC4.1.1.39) is a bisfunctional enzyme which catalyzes the fixation of CO2and involves in photorespiration of C3plants. Rubisco content and activity in higher plants are important to the function of photosynthesis, closely related to genotypes and growth conditions, and influence the formation of yield and quality. Rubisco in plants is composed of large subunit and small subunit, prone to structure disintegrated when the plants grow in the adverse conditions. Flag leaves of rice are the main functional blades to photosynthesis product supply in the grains formation. This research attempts to analyze dynamically the difference of stability in different genotypes of Rubisco in the aspects of Rubisco content, activity, gene expression, the subunit protein expression, and in vitro degradation characteristics, in order to provide genetic physiological basis for functional stability evaluation of rice Rubisco, and breeding and cultivation of rice.This study selected four rice varieties (Nippobare, N22, R49, ITA312) as the experimental materials, of which the photosynthetic physiological indexes, Rubisco activity, gene transcriptional expression of Rubisco large subunit and small subunit (rbcL and rbcS), protein expression of Rubisco large subunit at different times (5,10,5and20days after flowering) in the filling stage were determined dynamically, and the condition of Rubisco enzyme degradation in vitro was explored preliminarily.The main results are as follows:1. SPAD value, chlorophyll content, net photosynthetic rate (Pn), Rubisco activity of the four varieties after flowering were all in a downward trend from the fifth to twentyth day after flowering, but there were some differences between the extent of variation in species. Pairwise correlation between Rubisco content, initial activity and Pn both reached over0.9, and there were differences between varieties. Of these varieties, pairwise correlation of Nippobare was the best.2. Gene expression of Rubisco large and small subunit were of the same tendency. The expression quantity firstly increased from the fifth to fifteenth day after flowering, reached the highest on the fifteenth day, and then decreased. Rubisco content in the varieties Nippobare and R49was on the decline, which in N22and ITA312first increased and lowered later after reaching the maximum on the tenth day. It was speculated that Rubisco gene transcription and translation were not synchronized that caused the different tendency between Rubisco gene expression and content, which might also be associated with the differences of varieties and demand for nitrogen level. Gene expression of rca first increased and decreased later after reaching the maximum on the tenth day, but the highest enzyme activity mostly appeared on the fifth day after flowering. It was possibly caused by Rubisco content, which reached the maximum on on the fifth day after flowering, and the content was the key determinant of activity at the moment.3. By using online software to predict the antigenic determinants of rbcL amino acid sequence (GenBank Accession: P0C510) to predict and combined with the analysis of characteristics, a length of valid antigenic peptide sequence was selected, and Rubisco large subunit specific antibody (anti-rbcL) of high antibody titer was successful prepared. The result of western blot experiments showed that large subunit content of Nippobare and R49Rubisco was on the decline, while N22and ITA312’s first increased and then decreased later, consistent with the trend of Rubisco content determination result. According to the result of multiple sequences alignment, the antibody can be used to detected changes of Rubisco large subunit in wheat, rapeseed and maize, which was verified.4. In vitro degradation experiment of Rubisco:when Rubisco rude enzyme of the20th day’s flag leaves after flowering in Nippobare, R49and ITA312was treated under the condition of33℃, pH5.0or5.5for0to4hours, Rubisco was degraded, with the extension of time, which was more obvious;20to50℃, pH5.0or5.5treated for2hours, the degradation was of different degree, most obvious when30-35℃, and most of Rubisco large subunit was degraded completely or not degraded actually when over45℃. The results of SDS-PAGE and western blot showed Rubisco larege subunit of Nippobare and ITA312were more stable than that of R49. Research rice Rubisco degradation conditions laid the foundation to further study on the sensitivity of rice Rubisco to proteolytic enzymes.Gene and protein expression level of Rubisco large subunit in the results of this study can be used as an important reference index for rice photosynthetic function evaluation, and the prepared rbcL antibody can quickly detect large subunit protein level in rice, wheat, rape and maize, with practical value.