| It has been proved that photosynthesis is the physiological basis in crop yield formation, and to some extent, the intensity of photosynthesis determines the level of crop production. At present, China is facing the dilemma to further increase yield in super hybrid rice breeding. In order to promote rice yield potentiality, the genetic engineering by which C4 photosynthetic enzyme genes are introduced and expressed to increase the photosynthetic efficiency has become an important strategy and the main content in super high-yield rice breeding.To explore the effects on improving photosynthetic characteristics by introducing C4 photosynthetic enzyme genes in different interactive forms,3 expression modules were set up, i.e., by Agrobacterium-mediated method, the maize cDNA of PEPC/PPDK and that of NADP-MDH/NADP-ME were transformed into Xianghui299 (R299), a super hybrid rice parental line, respectively, and through hybridization of two transgenic lines harbored with cDNA of PEPC/PPDK and that of NADP-MDH/NADP-ME, a new transgenic line combined with all four genes above was acquired. On this basis, the integration, transcription and expression of foreign C4 photosynthetic enzyme genes were systematically studied, and effects of the expression of different target genes were analyzed on the biological characteristics such as photosynthesis, stress tolerance, grain quality and yield. The major results were shown as below:(1) It was found that PEPC, PPDK, NADP-MDH and NADP-ME genes could be integrated, transcribed and expressed in rice leaves. The enzyme activity of PEPC and ME in transgenic rice plants were 1.3 to 5.9 and 1.4 to 6.8 times of those in wild type plants, respectively. The foreign C4 photosynthetic enzyme genes could be stably inherited and effectively expressed in progenies.(2) The average segregated percentage of the transgenic plants with target genes but selectable marker gene free (SMF) was 8.2%. Several selectable marker-free transgenic homozygous lines of T3 generation were obtained. The correlation coefficients of PEPC and NADP-MDH to Pn (photosynthetic rate) were 0.817 and 0.610, both of which are significant at the 5% level.(3) In the booting, heading, full heading stages, the average Pn of PEPC/PPDK transgenic restorer lines was higher than that of the control by 12.05%,8.14%,13.83%, respectively, while the average Pn of PEPC/PPDK transgenic rice hybrids was higher than that of the control by 3.59%,7.17%,9.51%, respectively.(4) From heading stage to mature stage, the SPAD value of chlorophyll in transgenic leaves declined gradually along with the reproductive process. The occurrence of turning point of chlorophyll SPAD (10DAF) was roughly consistent to the full heading stage, after which Pn declined dramatically.(5) Compared with the control, the transgenic restorer lines with PEPC/PPDK, NADP-MDH/NADP-ME or all four genes had a little bit lower CO2 compensation point and light compensation point but a higher light saturation point by 167μmol photos.m 2.m-1 and a higher light saturated photosynthetic rate by 10.97%. Under normal conditions, the transgenic plants displayed enhanced transpiration rate (VPD), stomatal conductance (Gs) and intercellular CO2 concentration (Ci) to some extent. Gs was positively and significantly correlated to Pn, and positively but not significantly correlated to VPD and Ci. It could be inferred that the enhanced stomatal conductance contributed a lot to its higher Pn.(6) Under drought stress, in heading stage the transgenic rice showed higher transpiration rate, intercellular CO2 concentration and stomatal conductance than the control; and compared with the control, the Pn and WUE (water use efficiency) of the transgenic rice were increased by 75%-85% and 15%-85%, respectively. Under high temperature stress, in seedling stage the transgenic rice showed higher ratio of 1st grade leaves. Under both drought and high temperature stress conditions, the transgenic rice had higher maximal photochemical efficiency of PSII (Fv/Fm) and higher PSII potential activity (Fv/F0). Therefore, it could be inferred that expression of the foreign C4 photosynthetic genes served a useful function for resistance to drought and high temperature.(7) The transgenic rice carrying C4 photosynthetic enzyme genes decreased in chalky rice percentage, chalky area and chalkiness to a different extent, and in some cases, the differences reached a statistically significant level between the transgenic rice and the control.(8) The transgenic restorer lines and their hybrids showed an increase in plant height, the number of spikelets per panicle and grain weight but a decrease in seed setting rate. Assessment of yield capacity test of the transgenic hybrids showed that three had a higher yield than the control hybrids by 7.45%,2.54% and 1.13%.On the whole, for PEPC/PPDK transgenic rice, it was superior to the others in enzyme expression, Pn, Gs, high temperature resistance, the number of spikelets per panicle and grain yield per plant. The NADP-MDH/NADP-ME transgenic rice had an advantage on drought resistance over the others. When it comes to the transgenic rice carrying all four genes, most traits had values between the PEPC/PPDK transgenic rice and the NADP-MDH/NADP-ME transgenic rice.The possible mechanism leading to this result and the problems such as faster degradation of chlorophyll and sharper decline of photosynthetic capacity in later growth stages, and lower spikelet fertility and yield advantage should be further studied.
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