Analysis Of Glycolysis Way And Study On The Function Of Aldolase Family Genes In Camellia Oleifera Seed

Camellia oleifera are specialty oil trees in China, which are planted in acidic red soil areas in the South Downs. Camellia oleifera are strongly supported by the state because of high quality tea oil. But the low yield per unit area has been the main factors restricting the rapid development of oil-tea industry. Conventional breeding of Camellia oleifera need long time and may be accompanied by genetic cumbersome. Using a new method of molecular design breeding to modify oil-tea, it not only could be directionally improve the single trait or multiple traits but also could greatly shorten the breeding cycle with out genetic cumbersome. Glycolytic pathway is the upstream metabolic pathway of oil synthesis in Camellia oleifera seeds. The glycolytic metablic rate directly affects the efficiency of fatty acid synthsis in Camellia oleifera seeds. Fructose-1,6-bisphosphate aldolase is the key regulation enzyme in the glycolytic pathway, which not only can regulate plant reproductive growth but can affect fat synthesis. In this paper, state trial oil-tea variety’Huashuo’as material, on the basis of the analysis of the digitized transcriptome and expression profiling database, the full-length cDNA of four key members of Camellia oleifera fructose bisphosphate aldolases(CoFBA) were cloned. The studies of CoFBA subcellular localization were carried out. The correlation between the four CoFBA gene expression and oil content in Camellia oleifera seeds was analyzed. After constructed over-expression and RNA interference vector which were transformed into wild-type Arabidopsis thaliana and Brassica napus, T1transgenic plants were obtained. It laid the scientific foundation for the development of Camellia oleifera molecular design breeding techniques. The main results are as follow:1. Molecular biology analysis of glycolytic pathway in Camellia oleifera seeds.Camellia oleifera seeds in fruit enlargement period (June) and oil peak period (October) were chosen as materials. The combined transcriptome of two periods and two different periods digitized expression profiling database were constructed. The data analysis showed that the Camellia oleifera glycolytic pathway contained306unigenes which were attributable to the23genes just as Phosphoglucomutase, Glucose-6-phosphatase, Glucose-6-phosphate isomerase, Fructose-bisphosphatase, N-acylglucosamine-6-phosphate2-epimerase, Phosphofructokinase, Aldolas, Triosephosphate isomerase, Glyceraldehyde-3-phosphate dehydrogenase, Bisphosphoglycerate mutase, Aldehyde ferredoxin oxidoreductase, Phosphoglyceromutase, Enolase, Phosphoenolpyruvate carboxykinase, Pyruvic kinase, Pyruvate dehydrogenase (acetyl-transferring), Dihydrolipoyllysine-residue acetyltransferase, Dihydrolipoic acid dehydrogenase, Pyruvate decarboxylase, L-lactate dehydrogenase, Aldehyde dehydrogenase, Alcohol dehydrogenase, Methanol dehydrogenase (cytochrome c) and alcohol dehydrogenase (cytochrome c). The majority of these genes were gene families, in which unigenes differentially expressed in different developmental period of Camellia oleifera seeds. The glycolytic pathway significant enrichment analysis of camellia oleifera seed expression profiling showed that, the expression abundance of regulating genes in this metabolic pathway increased overall. The initial expression26samples (June) were increased to the peak expression336samples (October). According to the glycolysis/gluconeogenesis metabolic pathway unigene data of Camellia oleifera seeds transcriptome and differential expression of functional genes of Camellia oleifera seeds’ glycolytic pathway in different developmental periods, the glycolytic pathway of Camellia oleifera seeds was concluded and drawn. Transcriptome database had11CoFBA unigene sequences, in which the two expression levels in oil synthesis peak period were3-4times than in fruit enlargement period, and significantly higher than the other regulation genes in glycolytic pathway. CoFBA were the key enzymes for oil synthesis of Camellia oleifera to impact from glycerol metabolic pathway to fat metabolic pathway directly.2. Gene cloning and bioinformatics analysis of CoFBAAccording to the data analysis of transcriptome, combined with cloning one by one and RACE, four CoFBA full-length cDNA were cloned and registered in NCBI, GenBank accession number for JN017093.1, JX914588, JX914589and JX914590. The four CoFBA genes were named as CoFBA1、CoFBA2、CoFBA3and CoFBA4. The four CoFBA proteins had no signal peptide and all hydrophilic proteins.They all contained typical FBA activity structure, which CoFBA1, CoFBA2, CoFBA4as ’VMFEGILLKPS’ and CoFBA2as ’VLLEGTLLKPN’. The CoFBA family divided into A and B subfamily, in which CoFBA2normalized B subfamily and CoFBA1, CoFBA3, CoFBA4classified as A subfamily. The results suggested what time and how much differentiation were related with active site, structure as well as function differences.The spatial structure predicted that CoFBA1and CoFBA4were similar with Babesia Mycobacterium1,6-bisphosphate aldolase3kx6A structure, while CoFBA2and CoFBA3were closed to rabbit muscle aldehyde reduction the enzyme3lgeB and P. falciparum diphosphate aldolase enzymes2pc4C structure respectively. 3. CoFB A protein subcellular localizationUsing gene gun bombardment of onion epidermal cells, CoFBA protein subcellular localization was studied. The results showed that CoFBA were not solely localized in the cytoplasm. CoFBA1may be localized in nucleus, CoFBA2may be localized in cytoplasm, CoFBA3may be localized in periplasm and CoFBA4may be localized in extracellular. But with bioinformatics prediction, CoFBA were located in outer membrane, cytoplasm, periplasm and outer membrane. But whether it was forecast information or was experimental results, it both reflected diversity of CoFBA expression site. Projections indicate that four aldolases were non-secreted protein. How CoFBA to be transported to the extracellular might be related to contain myristylated and glycosylation sites.4. Influence of CoFBA gene expression on oil content of Camellia oleifera seedsThe differences in oil content could be reflected from Camellia oleifera fruits and seeds phenotype. While the seed coat was milky white and seed kernel white before fruit expansion, oil content was only about10%. Whilee pisperm was dark brown and seed kernel oil yellow, the oil percentage rose to50%. Using real-time quantity PCR technology, the relative expression level in eight different developmental stages of CoFBA1、CoFBA2、CoFBA3and CoFBA4had been analyzed, which contrast with the relative expression level of CoACP, CoSAD and CoFAD2combined with oil content changes, the comprehensive correlation degree had been revealed by gray relational analysis. The comprehensive correlation were CoFBAl (.762817), CoFBA2(0.649344), CoFBA3(0.672756) and CoFBA4(.646704) respectively, which compared insignificant with enzyme of fatty acid metabolism such as CoACP (0.673442), CoSAD (0.699459) and CoFAD2(0.653404). The result showed that Camellia oleifera seeds oil synthesis was not only associated wit the key enzymes in fatty acid metabolism pathway but also the key enzymes in the glycolytic pathway. Because expression and regulation of CoFBA gene family had a certain impact on Camellia oleifera oil content, Camellia oleifera oil content could be improved through genetic improvement means.5. Constructing and transformation of overexpression vector and RNA interference vectorOverexpression vectors of CoFBA1, CoFBA2, CoFBA3, CoFBA4and RNA interference Gateway vector of CoFBA family were constructed as well as RNA interference Gateway vector of AtFBA family for contrast. The vectors had been transformed to wild Arabidopsis thaliana and the T1transgenic Arabidopsis thaliana had been obtained. Using Gateway technology, the expression vector was constructed and had been transformed to wild Brassica napus. T1transgenic Brassica napus ’inbred15’ had been screened. Through the comparison of phenotype, the biological function about promote reproductive growth, improving seeds yield then improving oil production of CoFBA1have been confirmed.