臺灣博碩士論文加值系統

Relative to C3 plants (e.g. rice, wheat, and potato), C4 plants (e.g. maize, sorghum, and sugarcane) have several agronomic advantages, such as high CO2 assimilation rate, low photorespiration rate, and high nitrogen and water use efficiency. In order to improve the growth and yield of rice, attempt has been made to install a functional C4 pathway into rice by overexpressing several key C4 photosynthesis enzymes via molecular genetic engineering. Three genes encoding C4 photosynthesis enzymes have been previously introduced into rice independently: maize phosphoenolpyruvate (PEPC), maize pyruvate, orthophosphate dikinase (PPDK) and rice NADP dependent malic enzyme (NADP-ME). To achieve a functional C4 pathway, it is necessary to simultaneously express these enzymes in the same rice plant. The major goal of my research was to integrate these transgenes into the same rice plant for further study. The homozygous PEPC/PPDK or CK transgenic rice plant overexpressing these two maize C4 photosynthesis enzymes was cross-hybridized with homozygous ME transgenic rice plant to obtain a three-way hybrid (PEPC/PPDK/NADP-ME or CKM). Homozygous CKM lines that overexpress these three enzymes were screened for further molecular and physiological characterization. Integration of these genes into rice genome was confirmed by Southern blot analysis. Expression levels of these three genes in various transgenic rice plants were determined by RT-PCR, northern blot analysis, western immunoblot analysis and enzyme activity assay. Photosynthetic physiological measurements were performed to assess the effects of overexpressing one, two, or three of these C4 photosynthesis enzymes in transgenic rice plants. The results show that these transgenic rice plants have comparable or high photosynthetic rates under different environmental conditions compared to the non-transgenic wild type plants. The higher photosynthetic capacity may be related to higher stomatal conductance to CO2 and enhanced tolerance to photoinhibition. The higher photosynthetic capacity of these transgenic rice plants may be related to a higher stomatal conductance to CO2 and an enhanced tolerance to photoinhibition.

Acknowledgements...........................................................................................Ⅰ
Abstract.............................................................................................................Ⅱ
List of figures....................................................................................................Ⅵ
List of tables......................................................................................................Ⅷ

Ⅰ. Introduction..……………………………………………………………............ 1
1. Overexpression of PEPC......................................................................... 6
2. Overexpression of PPDK..........................................................................9
3. Overexpression of NADP-MDH..............................................................10
4. Overexpression of NADP-ME.................................................................11
5. Overexpression of PCK...........................................................................12
6. Overexpression of multiple C4 genes......................................................13

Ⅱ Materials and methods
1. Plant material and growth condition.....................................................17
2. Cross-hybridization.................................................................................17
3. Pollen viability assay...............................................................................18
4. Enzyme activity assay.............................................................................18
5. Isolation of genomic DNA for PCR.........................................................19
6. PCR conditions........................................................................................20
7. Isolation of genomic DNA for Southern blot analysis............................21
8. Southern blot analysis............................................................................22
9. Total RNA extraction...............................................................................23
10. Synthesis of first-strand cDNA and RT-PCR.......................................23
11. Northern blot analysis..........................................................................24
12. Western immunoblot analysis..............................................................25
13. Gas exchange measurements...............................................................25
14. Photo-oxidative stress response...........................................................26

Ⅲ. Results
1. Cross-hybridization.................................................................................28
2. Initial screening of F1 hybrids by enzyme activity assay......................29
3. Screening of homozygous CKM transgenic lines...................................30
4. Confirmation of homozygousity in F3 generation by PCR....................32
5. Southern blot analysis............................................................................36
6. RNA expression analysis.........................................................................40
7. Protein expression analysis....................................................................43
8. Gas exchange experiment.......................................................................47
9. Photo-oxidative stress response..............................................................57
10. δ13C analysis........................................................................................60

Ⅳ. Discussion
1. Integration and expression of C4 genes in transgenic rice plants.......63
2. Photosynthetic performance..................................................................65
3. Tolerance to photoinhibition..................................................................67
4. Photosynthetic discrimination against 13C...........................................69

Ⅴ. Conclusions..................................................................................................71

Ⅵ. References...................................................................................................72




















List of figures

Figure 1. Initial screening of homozygous CKM lines by enzyme activity assay.......................31

Figure 2. Screening of homozygous NADP-ME lines from the F3 progeny of
CKM line 18 by PCR...........................................................................................33

Figure 3. Screening of homozygous NADP-ME lines from the F3 progeny of
CKM line 34 by PCR...........................................................................................34

Figure 4. Screening of homozygous PPDK lines from the F3 progeny of
CKM line 18 by PCR...........................................................................................35

Figure 5. Screening of homozygous PPDK lines from the F3 progeny of
CKM line 34 by PCR...........................................................................................37

Figure 6. Screening of homozygous PPDK lines from the F4 progeny of
CKM line 34-10 by PCR......................................................................................38

Figure 7. Southern blot analysis of expression of three C4 transgenes
in various transgenic and wild type rice plants................................................39

Figure 8. RT-PCR analysis of expression of three C4 transgenes
in various transgenic and wild type rice plants................................................41

Figure 9. Northern blot analysis of expression of three C4 transgenes
in various transgenic and wild type rice plants................................................42

Figure 10. Western blot analysis of expression of three C4 transgenes
in various transgenic rice, wild type rice and maize plants............................44

Figure 11. Enzyme activities of PEP carboxylase, Pyruvate, Pi dikinase,
and NADP-malic enzyme in various transgenic rice, wild type rice
and maize plants...............................................................................................46


Figure 12. Photosynthetic responses to temperature of CKM transgenic rice
line 18-8, wild type rice and maize plants.......................................................48

Figure 13. Photosynthetic responses to CO2 of various transgenic rice,
wild type rice and maize plants.......................................................................49

Figure 14. Photosynthetic responses to light of various transgenic rice,
wild type rice and maize plants.......................................................................52

Figure 15. Stomatal conductance and intercellular CO2 concentration
as a function of external CO2 concentration in various
transgenic rice, wild type rice and maize plants............................................55

Figure 16. Stomatal conductance and intercellular CO2 concentration
as a function of light in various transgenic rice, wild type rice
and maize plants...............................................................................................56

Figure 17. Fv/Fm before and after and % inhibition in Fv/Fm after
methyl viologen and high light treatments in leaves of
various transgenic and wild type rice plants...................................................58

Figure 18. NPQ before and after and % inhibition of NPQ after
methyl viologen and high light treatments in leaves of
various transgenic and wild type rice plants...................................................59











List of tables

Table 1. Carboxylation efficiency, CO2 compensation point and apparent
quantum yield of various transgenic rice, wild type rice
and maize plants..................................................................................................54

Table 2. Photosynthesis rates of various transgenic and wild type rice measured at 30℃, 375 μl/l CO2 and 1000 μmol/m2/s....................................................................................61

Table 3. Leaf δ13C contents for various transgenic rice, wild type rice and
maize plants..........................................................................................................62

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