Establishment Of CDNA-AFLP Technic With Two Low-frequency Cut Enzyme In Maize And The Differential Expression Analysis Of Genes Correlated To Maize Ear Number

cDNA—AFLP combines the strongpoint of AFLP and RT-PCR into a useful technique for the study of gene expression and transcript profiling.Because of its high reproducibility,accuracy and reliability,it is widely applied to comprehensive and systematical analyses of organismal transcriptome.But it will cost a lot of money to accomplish cDNA—AFLP.In our study,we established a cDNA-AFLP technique that used two average cutting frequency restriction enzymes combination.It is less costly but effective.Therefore,the cDNA-AFLP technique established was used to analyze differential expression of genes correlated to ear number of maize.The results were as the follows:1.Establishment of cDNA-AFLP technic with two low-frequency cut enzyme in maizeThe RNAiso Regent,GenerayGK3092 kit,Guanidine Thiocyanate were used to extract total RNA from seedling leaves of QCL1094,a maize inbred line.With the PrimeScript^TM 1st Strand cDNA Synthesis Kit,the first single strand cDNA was gained through the reverse transcription from the total RNA.The second strand cDNA was synthesized by using RNase H,E.coil DNA polymerase I and E.coil DNA ligase, and filled in the ends by T4 DNA polymerase.The double-strand cDNA was cut with two low-frequency cut enzyme,EcoR I and Pst I,and ligated to two adapters by T4 DNA ligase.The cDNA fragments were pre-amplified with a pair of primers EA00/P00 and detected by 1%agarose gel electrophoresis.Then the pre-amplified fragments were select-amplified with another pair of primers EA01/P01 and detected by 6%denaturing PAGE.The results indicated that the RNA extraction,cDNA synthesis,cDNA-AFLP pre-amplification and select- amplification by above procedures were effective.The technical system established is economical and effective,and a good reference to the application of cDNA-AFLP technology.2.Differential expression analysis of genes correlated to ear number of maizeUsing QCL6004 with multiple ears,QCL1010 with double ears,and QCL1007, QCL1168,QCl1178,AL29-2 with single ear as parents,their near-isogenic materials were constructed through continuing back cross.The seedling leaves,leaves and growing tips during ear differentiation period,silks,and kernels before maturation of the parents and near-isogenic materials.were analyzed by using the cDNA-AFLP technic established with 36 pairs of primers composed of 6 strands of EA serial primers combined with 6 strands of P serial primers.At present,6 fragments of differential expression have been found,found 2 fragments of differential expression in silks period of BC3,found 4 fragments of differential expression in seedling leaves period of BC4.Their sequenceing is underway.