Cloning And Functional Analysis Of ANL2 Transcription Factorin Chimonanthus Praecox

Wintersweet(Chimonanthus praecox l.), belongs to Calycanthaceae, is a kind of highly ornamental tree species and proud of blooming in severe winter with elegant color and fragrans. In the research of Wintersweet, the varieties of Wintersweet are divided into Concolor Group, Intermedius Group and Patens Group.The Concolor Group is famous for yellow color, and the Intermedius Group is famous for puple color in inside perianth and yellow color in outside perianth, with highly ornamental value.In Arabidopsis thaliana, ANL2 is a kind of transcription factor that can control the distribution of anthocyanin in leaves and stem, also related to the cellular organization of the primary root. Here, an ANL2 in Wintersweet has been cloned, and the gene was inserted into p CAMBIA2300 to construct the plant binary expression vector, and the construct vector was transformed into the model plant tabacco, wildtype Arabidopsis thaliana and mutant, mediated by Agrobacterium.1. The full-length of Cp ANL2 was cloned and assured by bioinformatic analysis.2. RT-PCR of Cp ANL2 was carried out, to find the difference experssion between different varieties and flowering phase. According to the ressult,we found that Cp ANL2 may regulate the upstream gene of genes related to the synthesis of quercetin, so Cp ANL2 can affect the synthesis of anthocyanin indircetly.3. Cp ANL2 was digested and inserted in to p CAMBIA2300 to construct the plant binary expression vector and was transferred into Agrobacterium tumefaciens GV3101 by heat shock method. The Cp ANL2 gene was transferred into wildtype Arabidopsis thaliana, 6 plants of T1 generation of wildtype Arabidopsis were transformed successfully, 24 plants of T2 generation of wildtype Arabidopsis were transformed successfully. The Cp ANL2 gene was transferred into Arabidopsis mutants, 21 plants of T1 generation of Arabidopsis mutants were transformed successfully. The Cp ANL2 gene was transferred into wild tobacco, and 20 plants were transformed successfully.4. Compared to the wildtype Arabidopsis, the positive plant got from over-expression has different phenotype, positive plants of T1 generation having deeper green leaves and deeper purple stems, 1 positive plant of T1 generation having purple stems and leaves, 4 positive plants’ of T2 generation having purple leaf stalks and stems. 2 positive plants from mutant Arabidopsis were found purple subepidemal of leaves, and the epidemal is also deeper than the wildtype Arabidopsis and mutant plants. Andthe stems of positive plants were purple, while mutant plants’ stems were green. All of the results of phenotype’s change revealed that Cp ANL2 can affect anthocyanin’s synthesis and distribution.