Analysis Of Differentially Expressed Genes In Rice Leaves Attacked By Rice Leaffolder And Exogenous MeJA Treatment

In this study, the gene expression profile and differentially expressed genes of rice attacked by rice leaffolder (RLF) was analysized by cDNA-AFLP technique and SSH, which was further verified by Real time PCR of 4 TDFs. Aexpression vector, for the further expression analysis of membrane associated salt-inducible protein(TDF518) vector was constructed with by gateway cloning technology. The main results were as followings:1,cDNA-AFLP technique were employed to elucidate the transcription profile of rice attacked by RLF feeding and exogenous MeJA treatment. Sixteen pairs of primers were used for selective PCR amplification and total 12000 TDFs were acquired. 240 differential TDFs were isolated and sequenced, of which 164 shared similarities with known or putative functional proteins in GenBank. Among the changed 154TDFs against RFL feeding in rice leaves, 94 TDFs were up-regulated, mainly including Auxin Efflux Carrier family protein, Chaperone protein, early-responsive to dehydration protein, PHD-finger family protein, and 60 TDFs were down-regulated, including RNA polymerase beta' subunit-2, subtilisin-like protease, Heat shock protein and so on. Among the changed 119 TDFs in rice leaves under the MeJA treatment, 70 TDFs were up-regulated, mainly including RNA polymerase beta' subunit-2 , PHD-finger family protein , ferredoxinand, early-responsive to dehydration protein, and 49 TDFs were down-regulated, including Auxin Efflux Carrier family protein, Heat shock protein and so on. Under the RLF-feeding and MeJA treatment in rice leaf, 42 up-regulated and 12 down-regulated TDFs were found and respectively including PHD-finger protein, auxin-repressed protein, early-responsive to dehydration protein and prolyl carboxypeptidase like protein, Ribulose bisphosphate carboxylase large chain precursor, Heat shock protein and so on.2,Genes of Rice to RLF feeding were studied by using SSH technique. SSH was an simple and effective technique for the differentially expressed gene screening and was well known for the superiority on the application for cloning the differentially expressed gene. The differentially expressed genes were effectively enriched and cloned by the subtractive hybridization and PCR of two cycles. Two subtractive libraries of rice including 135 cDNA clones were constructed in the study. Some inserted cDNA clones were sequenced. and 33 cDNA sequences of Rice to RLF feeding SSH library were obtained. Among the 33 cDNA clones, there are 13 clones that were thought to be in line with the cDNA-AFLP result and including NAC-domain containing protein 90, auxin-repressed protein, light-induced protein 1 -like, alanine aminotransferase and so on.3,The expression of 4 candidate genes in rice to RLF feeding and exogenous MeJA treatment was analyzed by Real-Time PCR. The results showed that under the RLF feeding stress the expression of protein phosphatase 2c (501TDF), membrane associated salt-inducible protein (518TDF) and PHD-fmger protein (974TDF) was started after 2 hours, attained the higher level after 6 hours and was slightly lower as the following time. And the three genes were expressed slightly under the MeJA treatment. The gene which encoded the ferredoxin-1 (764TDF) was slightly expressed under the RLF feeding stress, however, strongly expressed under the MeJA treatment. The cDNA-AFLP technique was verified the reliability for the transcript profile due to the accord with the Real-Time PCR.4,A gene about the membrane associated salt-inducible protein(518TDF) was obtained and the related entry vector and destination vector were constructed by Gateway TM cloning technology.