The Functional Analysis Of An Unknown Gene In The EST Library Of Thellungiella Halophila

As days pass by, the salt-stress of soil has came to be a mainly stress to plantall over the world. The area of soil suit for agriculture shrunk every year. About20 percent farmland and about a half of the irrigated farmland had been turnedinto pickled land. We have to find ways to survive from the crisis of drought andpickled.We selected an unknown gene (BI698705) named Novel protein gene in theλZap-cDNA library(a EST Library) of the Thellungiella halophila treated with200mM NaCl. The reason why we interested in this Novel protein is that it's highperformed in this library in contrast with the control and 11 duplicates whoselevel also high are detected using about 1580 tags. The Novel's mRNA is veryabundant after the Thellungiella halophila is treated with NaCl , so we guess thenovel protein maybe have some special function in the regulation ofphysiology .After analysing this protein's sequence with the bioinformaticalmeans' help, we hypothesized it locates in the nucleolus. We got to know that thisgene is 474bp long and encodes 158 amino acid after analyzing its characteristicsof the sequence, structure and express character. Using Southern Blotting, wehave known the gene is single copy in the genome of the Thellungiella halophila .We were glad to find out that this protein is small (about 18KD),so we have reasonto hypothesized that the protein may be able to control transcribe when beinduced . So we did research with its functions and interactions with otherproteins as our stress.Northern blotting showed that the gene expressed very high after theThellungiella halophila be treated with various concentrations of NaCl: Whenthe Thellungiella halophila is treated with 200mM NaCl , the gene expressed levelgo higher and higher with the time passed by and after 48 hours it reached themaximal point.Treating with NaCl with different concentration for 24h, we find that thegene expressed in all groups, especially in the group of treated with 150 mMNaCl, We only to found that the gene's expressing slightly dropped in this NaClconcentration.Also, we grown Thellungiella halophila under low temperature of 4℃ fordifferent hours. Analyzed with Northern blotting, we found that in 3 hours thelevel of the novel gene expressing was remarkably enhanced. But the level of thegene expressing slightly drops in a long treat. While the plant was hot shot with40℃ there was not evidently induced the different between those groups.In the RNAi transgenic Thellungiella halophila, we treated the plant withlong period of 45 days, but there is no evident phynotype . We treated this kindof plant with a long time of NaCl(200mmol/L),there also no evident differentwith wild type.In the over-expressing transgenic Arabidopsis Thellungiella 45 days afterseeding, we treated the plant also with 200mM NaCl for 24 hours, and we got theresult that the transgenic plant grown better than the wild type.In the experiment of stress of oxygenation, the transgenic plants were fedwith 0.5μM Methyl Viologen (MV) from seeding. We found that the MV wasmore harmful to the wild type. We also do some same treat to the transgenicArabidopsis with 2μM abscisin acid (ABA), we found that the seeding rate ofthe transgenic type was low that the contral.We also expressed the gene in vivo. We established a synthesis system inprokaryote, and we also purified the protein with a GST kit from Promega.After purification we immunized rabbits with the unknown protein as anantigen.To find out the function of the unknown protein, we try to localized theprotein via a gfp-novel gene express vector in onion epidermis. And we get thepicture of the co-localization of GFP-NOVEL protein by a confocal. We foundthe novel protein localized in the cell nuclear.What is more interesting, we get same unexpected protein from thepurification. Those unexpected protein were purified as well as the GST-novelprotein. We analyzed these unexpected protein by LC-LC-MS, and quarried thesequence by the SEGUEST.To sum up, a lot of proofs such as cDNA sequence of the Novel gene fromthe λZap-cDNA library show that Novel protein may be in the nucleolus and itmay have key functions in regulatting of the genes related to NaCl.