| Litchi(litchi chinensis Sonn.)is an evergreen tree which belongs to the family of Sapindaceae,and originated from southern China.Litchi has a long history of cultivation in China and is mainly distributed in Guangdong,Guangxi,Fujian,Hainan,Sichuan,Yunnan and Taiwan provinces,which is characterized as the distinguishing fruit in the south of China.Litchi is a typical arillate fruit with aril(fruit pulp)serves as the edible part.An ovule develops into a seed and ovary wall develops into a pericarp(skin).Litchi has diverse skin color ranging from bright red to purplish red.Some cultivars even display non-red or uneven red pericarp phenotype.The skin color of flesh fruit largely determines its exterior quality and affects its commercial value.Anthocyanins accumulation in vacuole is the main color pigment of litchi pericarp.The biosynthesis of anthocyanins,however,is a very complicated process,which is influenced by many factors and co-controlled by internal and external factors.Previous studies have demonstrated the key structural genes and regulatory genes affecting anthocyanin biosynthesis.MYB transcription factor is a crucial regulator of plant anthocyanin biosynthesis.MYB regulates anthocyanin accumulation by activating downstream structural genes of anthocyanin biosynthesis.However,the upstream genes of MYB transcription factor are less studied.Here,we obtained all of NAC members containing NAC conservative domain from Litchi genome database and target 6 NAC genes which potential associated with accumulation of anthocyanin in litchi through technology of homologous comparison and expression profiling.Dual luciferase report gene experiment and yeast single hybridization assays were employed to test if these NAC transcription factors can interact with key MYB promoter of anthocyanin biosynthesis in litchi.Hopefully,NAC transcription factors involved in regulating MYB expression of litchi anthocyanin biosynthesis will be targeted.These results will provide a new idea and research direction for the improvement the coloration of litchi.The main findings were as follows:1.Based on N-terminal of NAC family genes containing highly conserved NAC domain,fifty putative genes with characteristics of the NAC family were identified in the genome database of litchi cultivar ?Feizixiao?.The phylogenetic analysis indicates these putative NAC genes can be classified into 6 subgroups.Furthermore,comparing them with NAC genes regulating anthocyanin biosynthesis in peach and Arabidopsis and basing on transcriptome data of the three stages of litchi pericarp development,we obtain six NAC genes which maybe play an important role in regulating anthocyanin synthesis.2.Total RNA of litchi pericarp were extracted and then transcribed into c DNA.Six litchi NAC members with high similarity to anthocyanin biosynthesis related NAC from other species were successfully cloned from c DNA.These NACs were named Lc NAC1,Lc NAC2,Lc NAC3,Lc NAC4,Lc NAC5 and Lc NAC6,respectively.3.Expression levels of 6 important putative NAC genes in different tissues and development stages were analyzed by q PCR.The expressions of Lc NAC4,Lc NAC5 and Lc NAC6 were observed paralleling with the maturation and coloration of pericarp.These three genes may be the key members involved in anthocyanin biosynthesis.4.The transgenic vector pcambia1300-Ca MV35S-Lc NAC1-6 and the experimental vector p Green II 0800-Lc MYB1 promoter-LUC were constructed to the regulatory role of NAC transcription factors on Lc MYB1 expression in tobacco(Nicotiana benthamiana).Lc NAC4,Lc NAC5 and Lc NAC6 were found significantly activate the expression of Lc MYB1.5.pAb Ai-Lc MYB1 promoter and p GADT7 AD-Lc NAC1-6 yeast vector were constructed,and the interaction between NAC transcription factor and Lc MYB1 promoter was further verified by yeast single hybridization experiment.The results showed that only candidate gene Lc NAC5 can interact with the promoter of transcription regulator Lc MYB1. |
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