Cloning And Functional Analysis Of A T-DNF Activation-tagged Altered Development Program Mutant Gene In Nicotiana Tabacum

We screened an tobacco activation tagging mutant in the T-DNA activation-tagged mutation group,a dominant mutant mainly for dwarf and compact leaves compared to the wild type Hong Da. Flanking sequence amplification and two-primer test determined the insertion location of the T-DNA tag,quantitative RT-PCR test identified the over-expressed gene which causes the mutation type. In this research, we got 4 conclusions as follows:(1) We screened an tobacco activation tagging mutant in the T-DNA activation-tagged mutation group. We identified that this mutation type was caused by single dominant gene according to the phenotype segregations of the T1, T2 and T3 lines. Using FPNI-PCR, we got the flanking sequences in the tobacco genome, sequence alignment results with the tobacco genome database showed that the mutant has two insertion sites, which located in chromosome1 and chromosome10 respectively.Two-primer test showed that the mutation phenotype is caused by the insertion in chromosome10,flanking sequence amplification of the transformed plants without mutation phenotype also confirmed this conclusion.(2) Using tobacco genome database, we found 5 genes within 120 kb upstream and downstream of the insertion site, with the closest gene Ntab0245760 71 kb from the insertion site. To determine which gene was altered in its expression level, we examined the expression levels of the 5 genes by RT-PCR.Only one gene, Ntab0245760, was over-expressed, while the expression of all the other genes remained mostly unchanged, suggesting that over-expression of Ntab0245760 could be responsible for the mutation phenotypes.(3) To confirm the conclusions we got above, we over-expressed Ntab0245760 in wild-type tobacco by a over-expression vector. The transgenic plants recapitulated most of the phenotypes in mutation type. Some of the transgenic plants showed more severe phenotypes of smaller plant size and highly compact leaves. The expression level of Ntab0245760 correlated with the severity of the phenotypes. These data indicate that the mutation type were indeed caused by over-expression of Ntab0245760, a member of MATE gene family.(4) We identified 131 MATE genes in common tobacco by bioinformatics, and they were classified into 4 subfamilies. We also found that subfamily 3 was distinct from other subfamilies,including the conserved domain prediction, TM prediction and subcellular location prediction, there was the same situation in subfamily 3 in Arabidopsis. Ntab0245760 belongs to subfamily 4, which has the Arabidopsis At ZRZ and At ADP1 in the same subfamily. Previous research has shown that over-expression of At ZRZ and At ADP1 caused similar mutation phenotype with Ntab0245760, and At ADP1 was also proved to associate with auxin biosynthesis. The lower auxin content in the mutant probably showed that Ntab0245760 has similar function with At ADP1...