Geno Me-wide Identification Of ANT Gene Family, And The Functional Studies Of SLAIL1-2in Tomato

ANT (AINTEGUMENTA) transcription factors family is a kind of DNA bindingprotein, which is unique in the plant kingdom and is able to regulate gene expression atmRNA transcription level. ANT gene family is the member of the AP2subfamily witchis in the AP2/ERF family of transcription factors in plant. AP2family membersincluded2AP2/ERF domains involves extensively in regulation of plant organsdevelopment. Tomato (Solanum lycopersicum) is one of the important worldwideplanting, processing materials, the most important representatives of solanaceae crops,and also the most important model plant for the studies on fleshy fruit development andmaturation. However, there is little research on ANT gene family in tomato.We also constructed the SlAIL1-2expression vector, antisense inhibition vector andGFP fusion expression vector to study the subcellular localization of SlAIL1-2and itsresponds to hormones. For further search, we analyzed the expression patterns ofdifferent parts in floral organ and in different period of pistil by Quantitative PCR. Wegot several SlAIL1-2over expression and antisense inhibition transgenic plants,observed the T0generation plants’ phenotype and detected the expression of relatedgenes. Then, we can preliminary supposed that SlAIL1-2plays an important role in theprocess of tomato development. The main experimental results are as follows:①In order to identify tomato ANT family members, we used Arabidopsis aminoacid sequences as reference sequences to did the homologous retrieval through Tomatogenome database. We identified5members which located in the tomato chromosome4,5,7,2and11. The amino acid length they encoded is between464aa (SlAIL7)~654aa(SlAIL1-2), their molecular weight size is between50.70KD~72.00KD, theoreticalisoelectric point is between6.03(SlAIL7)~8.54(SlAIL5), and they all have7or8introns.②In the structure analysis of tomato ANT family, we found that these fivemembers all included2AP2conservative structure domain(R1domain and R2domain).Each domain contained about70highly conservative amino acids sequence and had4specific motifs (Motif2,3,4were in front of the R1domain sequence, while Motif1included10aa insertion in R1domain). Five tomato ANT genes all had completelyconservative domain structure. ③In the phylogenetic relationship analysis of tomato ANT family, Arabidopsisthaliana AP2/ERF gene family members were used to construct phylogenetic tree.Based on Boostrap data, different AP2/ERF genes are divided into3groups (euANT,basalANT and ERF-like). The results showed that, like Arabidopsis thaliana ANTfamily, tomato ANT members also had2highly conservative AP2/ERF domain,belonged to the euANT branch.④Real-time PCR analysis indicated that all of the ANT genes showedexpression in multiple plant tissues, with higher levels of expression in young tissues ascompared with older tissues. ANT genes were low in stem and leaf tissues; mainlyexpressed in flower organ (Bud and flower); detected in immature fruit while very lowin mature green, break, yellow fruit and red fruit stage.⑤The SlAIL1-2gene has been cloned and bioinformatics analysis showed thatSlAIL1-2encoded654amino acids. Its promoter prediction showed that SlAIL1-2maybe related to thermal stress response, the response of salicylic acid, meristem and lightrelated process. Subcellular localization analysis indicated that SlAIL1-2existed in thenucleus, which could predict SlAIL1-2interact with transcription factors and regulateplant growth. In responses of hormones treatment, expressions of SlAIL1-2under KTand ABA were obviously improved.⑥Real-time PCR results of SlAIL1-2in different organizations of flower anddifferent periods of pistil showed that SlAIL1-2mainly expressed in the pistil. Itshighest expression level was at the day of pollination, then gradually weakened.⑦In order to further research on SlAIL1-2, we observed the morphologicalcharacteristics of SlAIL1-2over expression and antisense inhibition transgenic plantscarefully. It showed that SlAIL1-2inhibition expression lead to shape changes of leavesand color changes of both leaves and fruit peels. The expression of lycopenemetabolism related enzyme genes were tested, it supposed that SlAIL1-2can regulatethe color of tomato leaves and fruit peels.