Analysis Of Tubers Development Related Differential Expression Genes By CDNA-AFLP In Yam(Dioscorea Opposita)

Dioscorea opposita tuber is one of the important factors limiting the yield of yam.Yam as a kind of important nutritional value of vegetables,medicinal and economic value,the quality and yield by domestic and foreign researchers.In this study,Bikeqi yam(butchery)tubers and Dechangyu yam(short crude)tubers were selected as experimental materials.cDNA-AFLP technology was used to screen the differentially expressed gene fragments associated with the development of two yam tubers.The secondary amplification,hybridization validation,cloning,sequencing and functional annotation analysis were carried out to search for different genes related to the growth and development of Chinese yam tubers.The exploration of these differentially expressed genes can provide scientific basis for revealing the molecular mechanisms of the growth and development of different yam tubers and the causes of differences in tuber shape during development.The main conclusions of this study are as follows:(1)Successfully constructed and further optimized the cDNA-AFLP silver-staining technology system that can be used to isolate differentially expressed genes related to tubers at different growth stages of different yam cultivars.According to the optimized system,the differentially expressed genes of yam tubers were analyzed.It was found that the same or different combinations of primers showed very rich polymorphisms in the amplification of three yam tubers.(2)Selecting 25 pairs of primer combinations with clear and polymorphic bands from the 256 pairs of selective amplification primer combinations,and amplify a total of689 polymorphic bands,the band lengths were mainly distributed in 100~1000 bp,of which 304 were up-regulated,which accounted for 44.1 % of the total number of amplified bands,and the size of the differential bands was mainly between 100~750 bp.There were 50 differential bands that were down-regulated.It occupied 7.3% of the total amplified bands,and the differential fragments were mainly concentrated between100~750 bp;the polymorphism ratio of the total differential fragments was 51.4%.(3)The 304 differential segments that were up-regulated were amplified twice and136 bands with clear and single bands were obtained,accounting for 44.7% of the total number of up-regulated differential fragments.The fragment size was mainlyconcentrated between 100~500 bp.(4)136 secondary amplification products were confirmed by reverse Southern blot,and 15 positive differentially amplified fragments were obtained,and 12 nucleotide sequences were obtained by sequencing.Among them,4 nucleotide sequences were specifically expressed in Mid-term expansion of slender type Bikeqi yam tuber.8nucleotide sequences were specifically expressed in short crude type Dahechangyu yam tuber,2 of them were specifically expressed in the middle stage of tuber,the other four were specifically expressed at the later stage of tuber.These 12 positive differentially expressed fragments may be specific expression genes related to the growth and development of Chinese yam tubers.(5)After the 12 nucleotide sequences were annotated and functionally annotated by NCBI BLAST,9 nucleotide sequences were highly similar to the known sequences.and the functions involved mainly differentiation and development,chaperone proteins,and ribonucleoproteins,energy metabolism,protein synthesis,transcription regulation,signal transduction,etc..2 nucleotide sequences no moer than 50%.And 1 nucleotide sequence has no homologous protein in the database.They may be genes of unknown sequence and related to the growth and development of yam tubers.