| Sedoheptulose-1,7-bisphosphatase is pivotal to the Calvin cycle, where it catalyses thedephosphorylation of sedoheptulose-1,7-bisphosphate （SBP）. cyFBPase is also very important in thesucrose-biosynthesis pathway, catalysing the first irreversible reaction in the conversion of triosephosphates to sucrose.As we know sucrose is the carrier of source-sink transportation These two arelimiting velocity enzymes in the two pathway.In this study we choose two genes, thesedoheptulose-1,7-biphosphatase（SBPase） gene and cytoplasmic fructose1,6-biphosphatase（cyFBPase） gene, This study is to combine them to construct transgenic plant expression vectors totransformate of tobacco.In an effort to improve plant photosynthetic efficiency at the same time,improve plant sucrose synthesis and transport efficiency.In the larger extent, to promote the growth ofplants. The main studies are as follows：（1）SBPase role in RuBP regeneration pathway in the Calvin cycle. It has been proposed tobe one of the key regulators of flow of carbon through the Calvin cycle. In this study, we clonedSBPase gene from the Brassica napus and analyzed its cDNA and genome sequences. The genomesequences of SBPase gene are1820bp,including six introns, and its sheared transcript are1185bp,encoding375amino acid residues. This gene encodes a protein with a chloroplast targeting peptideand a disulfide bond area-CGGTAC.Show that the regulating mode is REDOX regulation of enzymeactivity. Cytoplasmic fructose1,6-biphosphatase （cyFBPase） activity has a great influence on sucrosesynthesis efficiency. The Brassica napus L genome sequences of cy-FBPase gene are1890bp,including six introns, and its sheared transcript are1017bp, encoding338amino acidresidues.The two genes are both conserved in plant, This shows that they are very important for plantgrowth and development.（2）We connected the the two genes to plant expression vector pBI121respectively, andsuccessfully constructed two plant expression vectors pGBI-sbpase and pGBI-fbpase，and the binaryplant vector pGBI-sbp-fbp which contains both SBPase and cyFBPase genes.These plant expressionvectors are used for transformation of tobacco.Detection of gene expression and enzyme activity indifferent transgenic plants, The plants with two genes overexpression have manifested significantadvantages in gene express and enzymatic activity. Real-Time PCR analysis showed that mRNAexpression of SBPase gene and cy-FBPase in bivalent plants are3.93times and4.36times asnon-transgenic controls respectively;and the enzymatic activity are1.45times and1.77times asnon-transgenic controls.While the SBPase gene mRNA expression and enzymatic activity of inSBPase single overexpression plants are3.43times and1.35times as non-transgenic controls,andcy-FBPase gene mRNA expression and enzymatic activity of in it’s single overexpression plants are3.76times and1.55times as non-transgenic controls.The overexpression of one gene of SBPase orcy-FBPase will stimulate the other.（3）Determinated of the photosynthesis rate of transgenic plants，and indicates that these bivalent plants and these SBPase single overexpression plants showed higher photosynthetic rate thanthat of wild type，they are9.88μmol·m-2·s-1and9.52μmol·m-2·s-1，increased by25%and20%respectively. While the photosynthesis rate of cyFBPase single overexpression plants is8.22μmol·m-2·s-1，increased by4%than wild type control.These results suggested that the SBPaseactivity plays an important role for the improvement of photosynthetic rate.We also found thatphotosynthetic rate of the old leaves of bivalent plants and cyFBPase single overexpression plantsdecreased at a slower pace，this suggested that increased the cyFBPase activity have somethinghelpful to delay senescence.（4）Detection of the starch and sucrose content in leaves and stems of transgenic plants andnon-transgenic controls. That in bivalent plants is higher than any other plants, are1.53times and2.0times as non-transgenic controls respectively, Compared with non-transgenic controls these contentincreased by27%and10%in SBPase single overexpression plants.But in cyFBPase singleoverexpression plants,the starch content reduced by14%and sucrose content increased by59%compared to wild type plants. Moreover the ratio of sucrose and starch in cyFBPase singleoverexpression plants is highest.The cyFBPase activity influence the sucrose content in stems moreobviously than that in leaves.The higher enzyme activity in stem tissue the sucrose content increasedsignificantly.（5）The statistical data of Plant high, leaf area, stem diameter, ratio of dry weight and freshweight,the onset of flowering and number of flowering are all analyzed in plant growth process.Comprehensive analysis of the above data,the bivalent plants showed obvious growth advantage.（6）cy-FBPase enzyme activity has important effect to enhance drought tolerance of plants, thesucrose content in root increased significantly in bivalent plant and single cyFBPase overexpressplant after drought treatment20days. Can be10times as much as control, with the result that increasedrought tolerance of plants.The overexpression research of SBPase gene and cyFBPase gene in plant shows that they bothhave positive effects on plant growth,and the bivalent plants showed obvious growth advantage andsignificant effect of drought resistance than other plants. Therefore, this research for geneticengineering of high yield and plant stress resistance research is very important. |
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