| High-yielding breeding is one of the most important rice breeding aims. For increasing rice grain production, some researchers have proposed a hypothese of increasing yield by improving photosynthetic efficiency and altering partitioning of photosynthetic products.. Photosynthesis results in the synthesis of two primary carbohydrates in plants, sucrose and starch. The triose phosphate (TP) synthesized from the calvin cycle determines the synthesis and distribution of starch and sucrose in C3plants. In general, most of TP can be transported out of chloroplasts into the cytosol through the exchange of inorganic phosphate ion (pi) and the inner chloroplast membrance localized triose-phosphate translocator. In cytosol, the TP will be used for sucrose synthesis by a series of enzymatic reaction, while in chloroplast it was synthesized starch under a series of enzymatic reaction. Soluble cytoplasmic inorganic pyrophosphatases (ppase) hydrolyze inorganic pyrophosphate (ppi) to two ions of inorganic phosphate (pi) in cytoplasm. By adjusting the proportion of ppi and pi, sucrose/starch synthesis, glycolysis in plasmid can be properly coordinate with each other. Therefore, over-expression exogenous PPase could continually catalyze ppi converting to pi in cytosol, which will stimulate enzymatic reaction towards the sucrose synthesis, and will provide sufficient pi for the TP exported from the stroma to the cytosol. The increased amount of sucrose synthesis will consume more photosynthetic products, which will in turn increase photosynthesis and photoassimilate of source leaves.Sucrose is the main form of carbohydrates transported from source to sink tissues in higher plants. Sucrose biosynthesis in the mesophyll cytosol requires pi, while sucrose transport in vascular bundle requires certain concentration of ppi. As a result, constitutive expression ppase gene in plant would lead to the ppi in vascular bundle decomposed into pi. This will cause sucrose that can not be loaded into the phloem, which will affect it transported to plant tissues in time. Previous studies have shown that specific expression of an exogenous ppase gene in mesophyll tissue can avoid this problem. For reducing the transgenic risk, in this thesis, we cloned an inorganic pyrophosphatase gene OsIPl from rice, and put it under the rice mesophyll-specific promoter "cyFBPase" to produce a construct cyFBPase:OsIP1. Through Agrobacteria mediated transformation, we transformed it into rice for studying its potential effects on rice yield increase. Moreover, different source-sink types of rice varieties are different on the requirement of more photosynthetic products, therefore, if their distribution of photosynthesis products were changed in a same way, their yield formation would be affected differently. Consequently, in this thesis, we also evaluated the effects of expression OsIPl gene between different source-sink types of rice varieties, and the result could provide the clue that which kind of varieties are more suitable to be improved by OsIPl.The results are as follows:1、Through the treatments of leaf-cutting (cutting off half of the flag leave when panicle emergence) and spikelet thinning (cutting off odd primary branches when panicle emergence), six rice cultivars with various panicle characteristics were investigated for identifying source-limiting and sink-limiting varieties, respectively. The results indicated that among the four japonica rice varieties, Zhongchao123had the largest panicle than those of the others, and showed poorly grain filling characteristics with two times of grain filling. This indicated that Zhongchao123was source-limiting variety. Hui236showed large sink and ample source, poor grain filling capacity, and least ratio of grain number/leaf and grain weight/leaf. This indicated that the transport capacity of photosynthesis products (source) to grain (sink) was not smooth in Hui236. Wuling jing1had more panicles as well as the stronger capacity of the translocating assimilation product, which showed the source coordinated with sink was very well. Nongken57, on the contrary, although it was like Wuling jing1with more panicles, it had the highest seed-setting percentage and grain filling degree. This indicated that the sink capacity of Nongken57was the smallest compared to the other three varieties, therefore it was classified into sink-limiting variety. For the two indica rice varieties, Yangdao6belonged to the type with good source-sink coordination like Wulingjing1, while R6547was considered a typical sink-limiting variety, which showed well grain filling degree, and even after the treatment of leaf-cut there still had photosynthesis products transported into stems at the later filling stage.2、Total of30inorganic pyrophosphatase genes predicted in rice were firstly analyzed by a series of bioinformatics methods, such as protein clusters, amino acids sequence alignment, protein subcellular localization prediction and cis-elements analysis. Among them, one gene (Os04g0687100) that was considered encoding soluble cytoplasmic inorganic pyrophosphatase, named as OsIPl, was cloned. The rice mesophyll-specific promoter "cyFBPase", from a cytosolic fructose-1,6-bisphosphatase gene, was used to drive OsIPl to make a chimeric gene cyFBPase:OsIP1in a double T-DNA plant expression vector.3、We transfered this chimeric gene, cyFBPase:OsIPl, into different rice varieties, including source-limiting variety Zhongchao123and sink-limiting variety Nongken57by using Agrobacterium-mediated method, and respectively obtained20,28independent positive transformants. We selected some To transgenic plants with high expression level by real-time PCR and generated their T2homozygous lines with similar morphological and agronomic traits to their wild-type parents. The effects of over-expressed OsIPl specifically in the mesophyll cell were estimated by investigating tiller dynamics, soluble and stored carbohydrate contents in leaf blade and in leaf sheath, dry weight, yield and so on. The preliminary results showed as follows:1) During the vegetative stage, the tiller capability and panicle number of transgenic lines increased to a different extent compared to the corresponding wild type. The increased effect seems to be more obvious in the high tillering variety, Nongken57than that in R6547.2) At different time-testing point, during the grain-filling stage, changing trends of the soluble sugar, sucrose and starch content in leaf blade and leaf sheath of transgenic lines were consistent with those of the wild types. However, at the peak grain-filling stage, the sucrose content of leaf blade and leaf sheath in the most transgenic lines was significantly increased comparing to their wild parents.3) Compared with the wild types, the dry weights and grain yields of transgenic lines increased in different extents. The grain yield of transgenic lines in Zhongchao123background were all significantly higher than that of the wild type, while the yield increasement of Nongken57transgenic lines did not reach significant level.In summary, we tried a new strategy to improve the photosynthesis production of two source-sink type rice varieties by specific over-expression of OsIPl gene in mesophyll cells. We found that compared with the wild type, the grain yield of transgenic lines were increased to a different extent. The results suggested that this strategy was feasible to improve rice grain yield although the various effects of OsIP1gene on different types of source-sink rice varieties were found. |
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