摘要
高产育种始终是水稻最重要的育种目标之一。为了提高水稻的产量水平,一些研究者提出了这样一种思路：改进作物光合作用效率和光合产物的分配进而提高水稻产量。淀粉和蔗糖是植物光合作用形成的主要终产物。在C3植物中,卡尔文循环中形成的磷酸丙糖(Triosephosphate, TP)决定了淀粉和蔗糖的合成与分配。总体而言,大部分TP通过位于叶绿体被膜上的磷酸丙糖转运器(triose phosphate translocator, TPT)与无机磷酸基团(pi)的对等交换被转移至细胞质,并在细胞质内的一系列酶促反应下合成蔗糖,而滞留在叶绿体内的TP可在一系列酶促反应下合成淀粉。胞质型无机焦磷酸化酶(Inorganic pyrophosphatase, PPase)可在细胞质内催化焦磷酸基团(ppi)分解为磷酸基团(pi)。通过调节ppi和pi的比例,可以有机协调蔗糖合成反应、淀粉合成以及质体中的糖酵解反应。因此,从理论上推测过表达外源无机焦磷酸化酶基因时,可不断使细胞质中的ppi转化为pi,刺激细胞质中的酶促反应向有利于蔗糖合成方向进行,同时也为TP通过TPT进入细胞质提供充足的交换体pi[1]。由于蔗糖合成量的增加必将消耗更多的光合原初产物,从而促进源叶片的光合作用,增加植物光合产物的合成。
蔗糖是植物碳转运的主要形式。蔗糖在维管束中的长距离运输需要ppi的存在。若人为操作PPase基因在植物体内恒定而持续的表达,可能会导致维管束中的ppi也被分解为pi,造成蔗糖在韧皮部的装载受阻,不能及时被输送到植物需要的各个部位。前人研究表明特异启动PPase基因的表达可避免这一问题。本研究尝试利用叶肉细胞特异表达启动子驱动PPase基因的表达,同时为了降低转基因安全性风险,利用水稻自身的胞质型无机焦磷酸化酶基因OsIPl作为靶基因,研究其在水稻产量形成中的潜在效应。
另外,不同源库类型的品种对植物光合产物的需求也不同,光合产物的分配常受到库需要量的支配,改变光合产物的分配后,对产量的影响可能也不相同。因此,本研究还将分析OsIPl基因在不同源库类型品种中是否存在效应差异的问题,或曰其更适用于在那类品种上加以应用。研究主要结论如下：
1、以6个不同穗型的水稻品种为试验材料,通过剪叶和疏花处理,明确了各水稻品种的源、库特征。结果表明：4个粳稻品种中,中超123穗型最大,籽粒充实度较差,有明显的两次灌浆现象,为典型“源限制型”品种；穗粒并重型品种恢236表现库大源足,粒／叶和粒重／叶均最小,但籽粒充实度也较差,为“源大库大流不畅”；多穗型品种武陵粳1号源库较协调,同化物质运转能力较强。同为多穗型品种农垦57,结实率、籽粒充实度均最高,单株总库容量最小,属于“库限制型”品种。2个籼稻品种中,扬稻6号表现与武陵粳1号相似,为“源库协调型”；R6547籽粒充实度较好,即使在剪叶处理下灌浆后期物质也有向茎秆再积累的现象,属于“库限制型”品种。
2、利用生物信息学对水稻中约30个无机焦磷酸化酶编码基因进行了氨基酸序列比较、结构域特征、亚细胞定位预测以及上游顺式元件释义等分析,进而克隆了1个预测为编码胞质型可溶性无机焦磷酸化酶的基因OsIPl (Os04g0687100)利用水稻叶肉细胞特异启动子cyFBPase驱动OsIPl基因的表达,构建了嵌合基因cyFBPase:OsIPl的双T-DNA植物表达载体。
3、通过农杆菌转化法将嵌合基因cyFBPase:OsIPl分别导入“源限制型”水稻品种中超123和“库限制型”品种农垦57中,分别获得了20、28个阳性转基因植株。根据QPCR分析和农艺性状比较,筛选高表达量且农艺性状没有明显改变的T2转基因纯合株系进行产量比较试验,分析在水稻叶肉细胞中特异性过表达OsIPl基因的效应。结果显示：
1)在营养生长期,转基因株系的最高茎蘖数和穗数相比各受体亲本均有不同程度的提高,且在分蘖性较强的品种上的提高效应更为明显；
2)在籽粒灌浆结实期,叶片、叶鞘中可溶性总糖、蔗糖和淀粉含量在整个灌浆期的变化趋势与对照相似,但是在灌浆高峰期时,多数转基因植株的叶片和叶鞘中的蔗糖含量显著高于对照。
3)转基因株系的单株产量较野生型对照呈现不同程度的增加,其中“源限制型”品种中超123转基因株系单株产量较对照增幅达显著水平,而“库限制型”品种农垦57的相关转基因株系增产未达显著水平。此外,在单株干物总重上,转基因株系相对野生型对照均有显著提高。
以上结果表明：cyFBPase:OsIPl基因的过表达均能使两种类型的品种生产更多的光合产物,产量较对照也不同程度增加,表明利用叶肉细胞过表达OsIPl基因这一策略改进水稻的产量具有一定的可行性,但在不同源库类型的品种中的增产效应有所差异。
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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