The Construction Of Full-Length CDNA Library During The Oil-Formation Phase Of The Cotton Seed & The Cloning Of Pepc Gene

Cotton is an important economic crop and oil crop as well as an important strategic material.Its cultivation area is large and it can be widely distributed and at the same time it is fairly adaptable.In China,the cotton acreage has stabilized at more than 8000 mu,total output has reached more than seven million tons.The cottonseed as the main by-product of cotton production has reached 13 million tons. The seed contents rich oil which is about 30%-45%is an important source of oil-bearing crops.So far, the report on the formation of cottonoil related genetic research is rarely found both at home and abroad. In this study,we took the Gossypium barbadense L.development seeds as materials,and constructed the full-length cDNA library of cotton seeds during the oil -formation phase of the Gossypium barbadense L.according to the technique in the instruction of CLONTECH company Creator^TM SMARTM PCR cDNA Library Construction Kit.We also made EST sequencing and analysis based on this library.The capacity of the primarily built library was 5×10⁶ and the library titer was 1.5×10⁸ cfu·mL^-1.We randomly selected 180 clones in the library to make PCR testing and the results showed that the length of the inserted fragments were mainly between 1.0-2.0 kb.And then we randomly picked 2170 clones to make the 3' end of one-way sequencing and got a total of 1831 effective sequence with the average sequence length up to 423bp which contained 1459 pieces of Unigenes.We made a BLAST comparison between all the acquired EST sequence with the NCBI database of nt and nr and found that 67.51%of the EST sequence showed a higher degree of homology with the existing nucleic acid sequence;It also showed 75.12%of the EST with a higher protein sequence homology;And there were a total of 38 EST sequences having something to do with the oil formation which accounted for 2.60 percent of all Unigenes.Phosphoenolpyruvate shuttle enzyme(PEPC) is a fixation enzyme of CO₂ which catalyzed from phosphoenolpyruvate(PEP) to generate acetic acid grassland(OAA) reaction and mainly played multiple roles in energy and metabolism in the biosynthesis.This study cloned a new pepc gene from 7124 Gossypium barbadense L.by using RACE and chromosome walking(GenomeWalking) technology according to the published the PEPC gene EST sequence and named it Gb.pepc3.The full-length of the sequence was 3259bp which contained a 2910 bp open reading frame encoded 969 amino acids.Its estimated molecular weight was 110.7 Kd and the isoelectric point was 6.08 I.It had shown high similarity between Gb.pepc3 and the C3 type pepc of the other reported plants through the comparison of Gb.pepc3 protein homology and the analysis phylogenetic trees which was above 75%. FQ-PCR results showed that Gb.pepc3 was wide spreaded in various organizations of the cotton among which the expression in the embryo was the highest and the expression in the fiber was relatively lower. The expression Gb.pepc3 changed in various stages of seed development:the Gb.pepc3 expression reached its peak 15 days after the flowering of Gossypium barbadense L.whereas the expression of Gb.pepc3 reached the peak 20 days after the flowering of the upland cotton.In addition,there were significant differences between the expression of upland cotton and Gossypium barbadense L..The cloning of PEPC gene cotton had great significance on research and application of cotton PEPC geneis. I would contribute to the understanding the mechanism of photosynthesis of cotton and provide theoretical and technical support in increasing the oil content of cotton seed.