Construction Of Mixed Full-length CDNA Library And Cloning Of RGA Fragments In Peanut

So far as I know, the majority of plant disease resistance genes are constitutive expression; even though also reported for induced gene, such as xal, which has been the only induced gene reported in the paper. Though someone argued that even if genes of constitutive expression would still enhance low-level in bacteria-induced condition, that also indicated whether the scalar on the expression of genes increased does not affect detection.Otherwise, the expression will not be able to determine whether the volume was increased or decreased. In this study, so a mixed full-length cDNA Library with whole tissues was directly constructed and a study related to a preliminary analysis of biological information was carried in progress; aiming to lay a solid foundation for functional genomics study of peanut for our laboratory. And some fragments for resistance gene analogues (RGA) have also been cloned through homology cloning in this study, on purpose that pre-ready work should be done to do a good job for cloning full-length resistance-related genes. Specific results are as follows:RNA was extracted from the various tissues or organs of the peanut, and mRNA was isolated from an equivalent RNA mixtures. Double-stranded cDNA was synthesized by the improved SMART technology. It was then digested by the restriction endonuclease Sfi I. After fractionation the cDNA fragments were connected to plasmid vector and a mixed full-length cDNA library was constructed successfully in the peanut. The obtained primary library contained 1.98×106 clones which have been enough for a rare gene screening. The insert fragments and its size were confirmed by both PCR and restriction enzyme digestion. It showed that the library had a high rate of recombinant with the inserts varied from 1000 to 2000bp and the average size about 1300bp. Sixteen clones were randomly selected for sequencing analysis and nine of them were full-length cDNA. It indicated that this library could be used for full-length genes screening and low abundance genes cloning. The full-length cDNA library was stocked after amplification, the titer of which was estimated as 8.5×109cfu/ml, meeting the requirements of the following experiments.Clones were randomly selected and then sequenced from 5'-end, obtaining a total of 400 high quality EST sequences. A preliminary study was conducted on the biological information analysis. Bases composition analysis and BLAST comparison results show that between the known genomes of dicotyledon Arabidopsis thaliana and monocot rice, it is more similar to Arabidopsis thaliana for peanut, and it is in accord with traditional plant classification results. However BLASTX results also show that the obtained sequences of peanut seem to be more similar to those of vine grapes than to those of peanut on web; which ranks only in the fourteenth from the point of sequence similarity. At the basis of BLASTX, GO annotation was carried out using BLAST2GO, which showed that they contained various functional gene categories basically. The theoretical evidence has been provided for the feasibility of large-scale subsequent sequencing, as well as the necessary clues for related research.A total of five group degenerate primers were designed according to the result of multiple sequence alignment for entile RGA sequences in peanut. RGA fragments were amplified using homology cloning on the peanut variety Minhua number 6. By BLASTN sequence analysis confirmed that there is 37 RGA sequences sequence, 18 sequences of which have been submitted; GenBank numbers are EU639668-EU639685. In the five pairs of primers, in terms of the sequencing results of the amplified fragments, there is one pair of primer whose amplified products contained 90 percent of sequences similar to RGA sequences, and a pair of primer set with the lowest proportion of RGA still amplified over 20 percent of RGA sequences. It was showed that there was no sequence obtained in the study similar to known sequences of RGA according to the sequencing results. So this study shows that it is more efficiently to amplify corresponding RGA fragments by means of the RGA sequence known to this species, which provides a shortcut for following RGA study and use.In comparison with other species, peanut genomics study is still very weak on the whole. This study can extend the scope of peanut functional genomics research and lay a foundation for the eventual settlement on the issues related to peanut improvement.