Isolation, Cloning, And Functional Study Of The Ripening-regulated Protein Gene From Camellia Oleifera Seeds

Tea-oil tree (Camellia oleifera), Tea-oil, belongs to Camellia, and is perennial small evergreen tree or large shrub, the original Chinese native tree. Tea-oil (Camellia oleifera), olive (Olea europaea), oil palm (Elaeis quineensis), and Coconut (Cocos nucifera) are known as the four woody oleiferous plants. Tea-oil tree is one of the most important ligneous edible oil trees in China. It is the only large cultivated area in the world.90% of its seed-oil is unsaturated fatty acid, such as oleic acid, linolaic acid, linolenic acid and so on. So it is a kind of excellent vegetable oil. It needs about a year from flower to mature in its life as well as the same stage of flower and fruit, so it has the long-life development period. The trade methods and techniques are hardly satisfied requirement of excellent varieties in tea-oil industrialization project. Therefore, together with gene engineering approaches and technologies, it is necessary for breeding the excellent varieties of early-fruit, and early matures. This paper make use of the Polymerase chain reaction (PCR), semi-quantitative RT-PCR, and the full-legnth DNA sequence of Camellia oleifera ripening regulated protein gene (CoRrp) and its expression patterns were studied, and the overexpression and small RNAi vectors were also constructed and transformed into the wild Arabidopsis thaliana, the major research conclusions are listed as follows.1. Isolation and cloning of the CoRrp gene Based on the constructed cDNA library and EST library by the Key Lab of Non-wood Forest Product of State Forestry Administration, Central South University of Forestry and Technology, and the C. oleifera ripening-regulated protein gene was firstly and successfully isolated and cloned. And its size of full-length cDNA is 1105 bp containing an open reading frame (ORF) of 966 bp with 5'and 3'untranslated regions (UTRs) and a long Poly (A) tail, designated as CoRrp (GenBank access no. FJ713027). It encodes a 25.27-kDa protein of 233 amino acid residues with the isoelectric point (PI) of 4.43. It shares the same sequence characteristics of the D-fragment DDDDDDDVD of E-fragment with that of tomato and potato, and amini acid comes to 53.16%, in which the content of Acid amino acids, i.g., Asparagine, Glutamine, Serine and Threonine, are high, and it is a half-membrane structure and non-secretory protein. Alpha helices are the main elements of the protein, and irregular coils disperse in the whole protein.2. The cloning of intron for the CoRrp gene The intron was obtained by PCR, and its length was 139 bp. The analysis result showed that the content of A+T is 69.78%, and of G+C is 30.22% in the intron, and belong to the intron of GT-AG, in which'there exists a similar promoter structure. The obtaining of the intron for the CoRrp gene can lay a basis for the further study of the gene overexpression.3. The study of the expression pattern of the CoRrp gene The expression differences among roots, stems, leaves, buds, and seeds of the different developing stages were analyzed by the semi-quantitative RT-PCR, and the results showed that there were plenty of expression in seeds, trace in buds, and none in roots, stems, leaves. The expression of the CoRrp gene varied with the stages of the seed development. The expression of the CoRrp gene has the spatial characteristics, and mainly expresses in seeds, and none expression in roots, stems, and leaves. On the other hand, the expression of the CoRrp gene also has temporal characteristics from the developing stages of seeds. There is tiny amount in the initial differentiate embryo, and when the seeds gradually bulging, the expression of CoRrp gene was gradually increasing from the sixth to seventh month of a year; in the eighth to nineth month, the ripping stage of C. oleifera, the expression of the CoRrp gene dramatic increased, and reached the top in the 10th month. And the results show that the CoRrp protein play important roles in the course of the seed mature.4. The constructed of overexpression vector and small RNA interference vector, and acquisition of transformed seedlings On the basis of the CDS sequence of AT2G18110 gene of Arabidopsis thaliana and the conserves sequence of the CoRrp gene, according to the principle of Tuschl, small RNA interference (sRNAi) was desgined, and constrcuted into the voctor pCAMBIA1304-35S, and the sRNAi expression vector was obtained; this study the genomic DNA sequence with intron CoRrp also was employed, and cloned into MCS of the down stream of CaMV 35S promoter of the vector pCAMBIA1304-35S. and the overexpression vector pCAMBIA1304-CoRrp was conducted by agrobacterium with the floral dip method, and transformed into Arabidopsis thaliana. And 43 of transformed pCAMBIA1304-CoRrp and 31 of transformed pCAMBIA1304-siRNA Arabidopsis thaliana seedlings were obtained.5. Overexpression of transformed Arabidopis thaliana and interference expression of the target gene in transformed Arabidopsis thalianaThe expression of target gene in the transformed Arabidopsis thaliana seedlings was detected with the semi-quantitative RT-PCR, and the results showed that there were significant differences between the transformed and the wild Arabidopsis thaliana seedlings. The expression of the target gene in the 12-selected-overexpression transformed-seedlings was much higher than that in the wild Arabidopsis thaliana seedlings. On the other hand, the expression of the target gene in the 12-selected-RNAi transformed-seedlings was much lower than that in the wild Arabidopsis thaliana seedlings. Compare with the expression of CoRrp and internal control gene Actin in the wild Arabidopsis thaliana seedlings, the expression of the 10-transformed pCAMBIA1304-siRNA is only half of the control seedlings.6. The regulation the CoRrp gene of in the seed mature of overexpression and RNA interference of transformed Arabidopsis thalianaThere was no significant difference in the overexpression and RNA interference of transformed Arabidopsis thaliana in the stage of nutrition growth, however, most of overexpression seeds ripe ahead of the normal, and of RNA interference of transformed Arabidopsis thaliana delay mature. According to the statistical results,43 seedlings (T2) of pCAMBIA1304-CoRrp Arabidopsis thaliana were obtained, of which 55.8% were matured three to four days ahead of the normal. And 31 seedlings of the pCAMBIA1304-siRNA transformed Arabidopsis thaliana were obtained, which of 64.5% were matured four to five days behind of the normal. Overall these results present that the ripening-regulated protein gene might play significant regulated roles in the C. oleifera seeds.Overall, the research of the expression pattern and function for the CoRrp gene will lay material and technique basis for the molecular breeding and cultivating new early fruit and early mature varieties, and thus possess significant meaning and application value.