The Isolation Of Banana Receptor-like Protein Kinase(RLPKs) Gene, Construction Of RNAi Plant Expression Vector And Optimization Of Banana Regeneration

The aim of the research was to produce the regeneration plant of the banana via secondary somatic embryogenesis and to isolate a banana receptor-like protein kinase (RLPKs)gene of ripening-associated,then translated it to banana embryogenic callus to identify the gene's function using RNAi plant expression vector.Optimizing the regeneration of banana.Male flower buds were exised from 1-month old male flower clusters,the inner layers of bracts were peeled off with forceps until the flower bud was cut into four segments longitudinally through the shoot tip and then transversely into 1-mm segments.Via secondary somatic embryogenesis,namely:immature male flower embryoid somatic embryos mature embryos bourgeoning plantlets,we have gained 9 regeneration banana plantlets.The plantlets gained via this approach was unicellular and current protocols for somatic embryogenesis using embrogenic cells cultures have the potential to produce non-chimeric plants.This protocol can also make the culture initiation in a short period of time,only about 6 months, shortening the time of banana regeneration.The research set up a way to produce banana regeneration plantlets via secondary somatic embryogenesis.Isolating the banana like-receptor protein kinase(RPKs) gene of ripening-associated.On the basis of cDNA segment gained via SSH and cDNA microarray, we isolated a 1689bp gene segment using plaque hybridization and RACE.The result of BLAST showed 40% homology between the segment and the banana RPKs.The result of RACE showed that we would gain an about 1800bp DNA segment of 5'-end,and an about 1000bp DNA segment of 3'-end.Creating RNAi plant expression vector.The RNAi vector in this research is a ihpRNA constuct, containing sense/anti-sense arms and an intron,which had a consistently enhancing effect. S.Varsha Wesley (2001) used hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species,intron-cintaining constructs(ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing.The degree of silencing with these constructs was much greater than that obtains using either co-suppression or anti-sense constructs.This system may facilitate the large-scale determination and discovery of plant gene functions.