| As model plant, Arabidopsis thaliana is widely used in plant molecular biology, genetics and developmental biology. Unknown function of Arabidopsis thaliana gene needs to explore and research generally through the transgenic plant. a variety of plant expression vectors for unknown function of gene AtBNU-X in Arabidopsis were cloned and constructed. And then, those expression vectors were transformed into inflorescence of plants by agrobacterium-mediated method and transgenic plants were obtained. The study laid a foundation for further study of this gene. The main results are as follows:(1)Bioinformatics analysis of gene AtBNU-X:Sequencing results show that full-length cDNA are 1219 bp, including 5' UTR of 76bp,3' UTR of 351bp and 792bp open reading fragment (ORF) which encoding 263aa protein about 29.03KD. The protein secondary structure of AtBNU-X mainly consists ofα-helix andβ-sheet, and without any transmembrane region. It was predicted that the possible phosphorylation site of protein may totally have 24 (including Ser:20; Thr:2; Tyr:1), and the protein located in the nucleus.(2)We found that expression of AtBNU-X gene significantly increased in green tissues and roots after exposed to 4℃,Nacl and Mannitol for 12 hours;but significantly decreased after treatment with 38℃for 3 hours.These results indicate that AtBNU-X gene is involved in response to stress.(3) Construction of plant over-expression vector by Gateway clone technique and transgenic plants were obtained;at the base of pEarlygate 101 with 35S promoter, we successfully constructed gene AtBNU-X and the homologous gene AtBNX of over-expression vector pEarlygate101-AtBNU-X and pEarlygate101-AtBNX in Arabidopsis thaliana. The two recombinant plasmids were transformed into agrobacterium tumefaciens GV3101,then transformed into Arabidopsis thaliana according to the method of floral dip, and screened of transgenic Arabidopsis of T1 generation in 1/2MS medium containing Basta resistant. we found that the plants were larger, the color of cotyledons were darker in positive transgenic plants, plants were gradually dying in non-transgene, then 3 T1 transgenic plants were obtained, respectively. And screened transgenic plant of T2 generation, we found that most of T2 transgenic lines with segregation rate of 3:1, but there were not marked differences in phenotypes compared with the wild plants.(4) Construction of RNAi vector transgenic plants were obtained;Using the artificial microRNA and pGEM-T easy, we successfully constructed RNA interference expression vector: pSN1301-Ami-AtBNU-X, pSN1301-Ami-AtBNX and pSN1301-Ami-AtBNU-X/AtBNX. Three recombinant plasmids were transformed into Arabidopsis by Agrobacterium tumefaciens-mediated method, then 12 T1 transgenic plants were obtained transformed pSN1301-Ami-AtBNU-X interference vector, and most of T2 transgenic lines with significant segregation rate of 3:1. However, it was not significantly different phenotypes compared with wild plants.For pSN1301-Ami-AtBNU-X/AtBNX interference vector, we have been obtained 3 T1 transgenic plants, and which were cultivated now.(5) Construction of promoter and GUS fusion expression vector of plant transgenic plants were obtained;2kb promoter sequences were amplified from the 5' upstream fragments of AtBNU-X by PCR, and which was cloned into expression vector of pMDC 163containing GUS gene as the reporter gene. Then we successfully constructed fusion expression vector of pMDC163-AtBNU-X. With Hygromycin resistance for screened, we found that cotyledons were dark green, roots and true leaves were normal. However, non-transgenic plants were gradually dying. We have obtained 3 T1 transgenic plants.
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