| Anthocyanin is a kind of water-soluble pigments in nature,which is one of the main components of flavonoids.It has been attracted much attention because of its strong free radical scavenging ability.The eggplant with purple peel is riched in anthocyanins.In recent years,there are more and more studies on the molecular mechanism of anthocyanin biosynthesis in eggplant,but most of them are functional verification through ectopic expression in tobacco or Arabidopsis.Based on our previous work,transcription factor Smb HLH1 was screened as a candidate gene that was related to anthocyanin biosynthesisin eggplant.However,Smb HLH1 and Sm TT8 with high homology were cloned from eggplant cultivar ‘76’ according to the eggplant genome datas published in 2014 and 2019.The main results are as follows:1.The c DNA and amino acids sequences of Smb HLH1 and Sm TT8 in eggplant are highly homologous.The the N-terminus(amino acids 1-157 and amino acids 187-191)and C-terminus(amino acids 634-659)of Smb HLH1 is significantly different from Sm TT8.The Smb HLH1 could not interact with Sm MYB113.The expression patterns analysis of the two genes in six varieties with different anthcyanin content in peel of eggplants showed that the expression level of Smb HLH1 in the peel of eggplant with high anthocyanin content is lower than those with low anthocyanin content,while the expression pattern of Sm TT8 was opposite.The above results suggested that the function of Smb HLH1 and Sm TT8 might be different.2.Eight Smb HLH1 overexpression eggplant lines were obtained by Agrobacterium mediated method.Compared with the WT,the expression level of Smb HLH1 was significantly increased in the Smb HLH1 overexpression eggplant lines.The anthocyanin content in leaves of the Smb HLH1 overexpression eggplant lines were not changed significantly,but significantly decreased in stems.At the same time,the chlorophyll content in leaves and stems of the Smb HLH1 overexpression eggplant lines were increased significantly.Moreover,the expression level of anthocyanin biosynthesis structural genes including Sm CHS,Sm F3 H,Sm F3’H,Sm DFR,Sm ANS,Sm3 GT in the Smb HLH1 overexpression eggplant lines were significantly decreased comparing with the WT.3.The cis-elements in the promoters of anthocyanin biosynthesis structural genes were predicted,and the G-box(5′-CACGTG-3′)or E-box(5′-CANNTG-3′)sequences recognized by b HLH transcription factors were found in all the anthocyanin biosynthesis structural genes.And Sm CHS,Sm CHI,Sm F3 H,Sm F3’H,F3’5’H,Sm DFR,Sm ANS,Sm3 GT and Sm5 GT contain 3,4,5,6,3,2,8 and 2 cis-acting element respectively.The results of dual luciferase activity experiments showed that Smb HLH1 can directly inhibit the expression of Sm DFR and Sm ANS,thereby,which inhibited the accumulation of anthocyanins in tobacco leaves.4.RNA-seq analysis found that 126 genes were upregulated and 181 genes were downregulated simultaneously in the two Smb HLH1 overexpressing eggplant lines compared with the WT.GO and KEGG pathways enrichment analysis showed that the co-upregulated genes in the Smb HLH1 overexpression eggplant lines were mainly enriched in "starch and sucrose metabolism" and "carbon fixation in photosynthetic organisms" pathways,while the co-downregulated genes were mainly enriched in "flavonoid biosynthesis" and "anthocyanin biosynthesis" pathways.Based on the above results,the photosynthetic rate and carbohydrate content of stems were examined.As expected,because of the chlorophyll content in stems was significantly increased,the photosynthetic rate was significant increases by 165% and100% in stems,and the content of glucose,fructose and sucrose were found in the two Smb HLH1 transgenic eggplant plants compared with WT. |
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