| Introducing C4 high photosynthetic efficiency system to C3 crops as wheat can improve the genetic basis of photosynthesis of C3 crops and which can greatly improved photosynthetic efficiency and then increase biological and grain yields of wheat has become a hot research topic. To study in detail about C4 photosynthetic gene’s photosynthetic characteristics and photosynthetic property in C3 crops has very important scientific significance to carry out the C3 crop breeding for high photosynthetic efficiency. In order to further improve the wheat photosynthetic characteristics and yield level, In this study, we had transferred maize C4 high photosynthetic efficiency NADP-ME gene into wheat and analyzed molecular genetics characteristics and physiological property of the transgenic wheat, The result is to study the C4 NADP-ME effect of photosynthetic function of wheat, and explore a new way of improving wheat yield potential.1. Molecular characteristics identification of a maize C4- high photosynthesis gene NADP-ME in wheat background.In this study, T3 transgenic lines and wild controls were identified by PCR and Southern blotting, The results showed that the NADP-ME was integrated in different chromosome loci with 2 copies in both two transgenic lines 10T(9)-1-1 and 10T(9)-225-4.We also used Western blot to analysis the expression of the T3 transgenic lines and receptor controls. The transgenic lines Protein expression were increased 1.90-2.77 and 2.50-2.95 times compared with controls, respectively.So, the author inferred that the foreign NADP-ME had a corrected expression in wheat.2. Analysis the transcription and expression characteristics of a maize C4-NADP-ME in wheat.The qRT-PCR results indicated that all the transgenic lines showed a higher transcription of photosynthetic relative genes than controls:the NADP-ME gene transcription level regulation:Anthesis> Heading>Jointing> 7th day after anthesis>15th day after anthesis;The activities of NADP-ME in different organs of transgenic lines were higher than WT at various growing stage, the activities of NADP-ME in different organs regulation:Leaf> Peduncle> Stem> Ear, the activities of NADP-ME in growing stage regulation:Anthesis> Heading>Jointing> 7th day after anthesis>15th day after anthesis. The activities of NADP-ME in leaf of the transgenic lines 10T(9)-1-1 at Jointing、Heading and the 7th day after anthesis,compared with controls, significantly increased 1.69、1.46 and 2.33 times respectively; The transgenic lines 10T(9)-225-4 of the activities of NADP-ME in leaf at growing stage of the Heading,7th day after anthesis% 15th day after anthesis, compared with controls, significantly increased 1.58、2.12andl.75 times respectively; Jointing,the most significantly increased 2.52 times.3. Analysis the Photosynthetic and Agronomic Traits of a maize C4-NADP-ME in wheat.The net photosynthetic rate of various transgenic wheat lines showed lower than WT at different growing stage. different growing stage of net photosynthetic rate regulation: Heading> Anthesis >Jointing> 7th day after anthesis>15th day after anthesis. including heading, the net photosynthetic rate of various transgenic wheat lines 10T(9)-1-1 and 10T(9)-225-4 were:24.27mol M-2 S1 and 26.23mol M2 s-1, compared with controls, significantly declined 14.93% and 8.06% times respectively. Diurnal variation of gas exchange parameters of transgenic wheat and Zhoumai 23 was determined at different growth stages (heading, anthesis,7th day after anthesis and 15th day after anthesis). At four determined times Pn variation tendencys were same, which show two Peak curve. The results show net photosynthetic rate of transgenic wheat was obviously declined at the four stages, the intercellular CO2 concentration (Ci) was increased significantly, Stomatal conductance (Gs) and transpiration rate (Tr) were reduced significantly, compared with the control.Response curves of net photosynthetic rate respectively to light intensity、CO2 concentration and temperature were measured based on T3 generation of transgenic nadp-me gene wheat and the control at the anthesis stage. comparing with the control, Saturation intensity of transgenic wheat was 1400.33 μmol·mg-1 min-1, significantly declined 12.44%; C02 compensation point was 106.40 umol.mol-1, improved by 21.16% and the carboxylase efficiency was 0.0585, drops by 30.19%.There was no difference for net photosynthetic rate between transgenic wheat and control when temperture during 20-25℃, their optimum temperature were all at 30℃.but, when temperature blow 20℃ or high 25℃, the net photosynthetic rate of transgenic nadp-me gene wheat was declined. the results showed that the ability of CO2 assimilation of transgenic wheat was declined.Compared with controls, T3 transgenic lines 10T(9)-1-1 and 10T(9)-225-4 at sing plant weight、grain yield per plant、1000-grain weight were respectively declined by 3.10%、 0.58%、4.06% and 1.60%、3.84%、3.92%, including thousand grain weight were significantly lower than those of the control in transgenic wheat. There was no difference for the plant height spike number per and number per spike of the transgenic lines compared with controls.4. Analysis the decline in photosynthetic rate mechanism of a maize C4-NADP-ME in wheat.The analysis of the transgenic lines and control malate and pyruvate content in the flag leaves showed that transgenic wheat lines 10T(9)-1-1 and 10T(9)-225-4 malate content were declined 2.2% and 8.8%, pyruvate content were declined 8.3% and 25% respectively. After spraying of malate, transgenic wheat and controls were increased by 5.80% and 1.16% respectively, there are no different between them. The stomatal conductance for transgenic wheat and controls were 253.30 mmol·m-2·s-1 and 288.50mmol·m-2·s-1 respectively, The transgenic wheat of stomatal opening rate and stomatal conductance were reduced 15.12% and 12.20% respectively, compared with controls. So the content of malic acid in flag leaves of regulation of flag leaf stomatal opening rate and stomatal conductance, the declined of the transgenic wheat’s flag leaf stomatal opening rate and stomatal conductance were caused by flag leaf malate content, then lead to a decline in net photosynthetic rate.5. Analysis the net photosynthetic rate determination of a maize C4- PEPC+NADP-ME in wheat.The net photosynthetic rate of maize C4-NADP-ME wheat were 25.53μmol·m-2·s-1, compared with controls, declined by 2.98%; The net photosynthetic rate of maize C4-PEPC and C4-PEPC+NADP-ME wheat were 26.93μmol·m-2·s-1 and 27.90μmol·m-2·s-1, compared with controls, significantly declined 5.43% and 9.25% times respectively.The results described above indicated that the transgenic wheat expressing maize NADP-ME gene showed lower photosynthetic characteristics and yield than untransformed plant, only integrate nadp-me gene can’t improve it’s photosynthetic and yield in the wheat. |
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