Construction Of A SSH Library Of The Young Banana Fruit And Bioinformatics Analysis On ESTs Sequence

Banana (Musa spp.) identified as the fourth major grain crops after rice, wheat, corn by the UN Food and Agriculture Organization, and which is the most popular fruits in some industrialized countries. As one of the most important crops in tropical, subtropical regions of the world, Banana has an important role in China’s tropical agriculture and society. The fruit of crop is rich in nutrients, so it is an important food source for humans. The fruit is an important part of higher plant reproductive organ, and the formation and development of fruit are a complex process controlled by different genes and regulations. So it is one of the important fields of plant developmental biology, and the research about it has become an important frontier of Plant Science in plant fruit development. Banana flower can format fruit without pollination, directly fromed the real cell division and cell expansion. And the banana fruit is a typical respiration climacteric fruit. The molecular biology research about banana fruit is mainly focused on fruit ripening stage, which aims to improve the value of the goods, to clarify the mechanism of mature banana fruit development, but on the other stage reported less.The development of flower, bracts, young fruit is carried out in the pseudostem,and when the fruit is extracted from the pseudostem, it basically completed the split of the cells during fruit development. In my research, according to the characteristics of the fruit development, the young fruits of Musa AA were treated as tester and young leaves as driver. Suppression subtractive hybridization (SSH) was utilized for the isolation of cDNA fragments for differentially expressed genes, and the sequences were analyzed by Bioinformatics, to lay the foundation for the next step of related fruit development genes isolation and screening, to provide the basis for genetic improvement of banana fruit characteristics and banana growth regulation.This experiment optimized fruit and leaf RNA extraction methods, and the extraction of CTAB method by our group of modified of the RNA quality can be very good to meet the subtractive library construction demand. At last, the banana fruit SSH library was constructed successfully. From 982 colonies of quality inspection,749 positive clones were obtained,563 effective single sequence obtained by sequencing, and the gain rate was 75.2%.And GenBank dbEST base sequence comparison,505 EST sequences had homology banana sources of EST, accounting for 89.70%, which proved the reliability of the sequences obtained from banana. And Nucleotide collection (nr/nt) database for homology comparison, it involved 89 kinds, and 342 EST with known genes have high similarity, accounted for 60.75%, there were 101 unknown function genes. Compared with the nonredundant protein database, it involved 87 kinds,544 homologous had known genes, accounted for 96.63%, and 19 are no matching protein, accounted for 3.37%.563 EST sequences were spliced into 262 unigenes, with Gene Ontology (GO) functional annotation, which were divided into 28 categories.138 unigenes belongs to the cellular component,166 unigenes belongs to the biological process, and 168 unigenes belongs to the molecular function. The results showed that the difference of banana fruit development is mainly caused by the functional differences inside the cell and organelle, and the results reflected the difference in fruit development was mainly affected by some factors, which should be integrated into its mechanism of action. The process of metabolism and cell were accounted for the majority of the proportion, indicating that cell during the fruit development of strong metabolic activity and those related to metabolism and gene to have provided the material basis and the energy to support the development of fruit. In KEGG pathway,50 unigenes of 262 were positioned to 62 significant gene interaction pathways, and were annotated 136 times, different signal pathways involved different gene numbers.In the electronic localization,7 unigenes had no sequence similarity in the banana genome sequences, so those were unable to locate. The remaining 255 can be positioned at the 11 chromosomes, localization rate reached 97.33%. Coding sequences of 255 unigenes were mapped in 682 regions of genome, which has 133 positioning in negative chain, accounted for 52.16%,122 positioning in positive-strand genome, accounted for 47.84%.The number of unigenes positioning in the positive and negative chain roughly the same.In functional analysis, there are 6 unigenes relates to the development process, and they all play a role in the developing embryo. Metabolic pathway mainly involved in carbon metabolism and nitrogen metabolism, which play an important role in energy and material metabolism during fruit development.