Functional Analysis Of CHS Genes And R2R3 MYB Repressors Related To Flavonoid Biosynthesis Pathway In Nicotiana Tabacum

Flavonoids are a major class of secondary metabolites in plants.These compounds function in key roles in multiple processes,including in plant growth and development,in production of anthocyanin pigments,and in plant defense reaction.Chalcone synthases are key enzymes that catalyze the first committed step in flavonoid biosynthesis.However,the mechanism of CHS gene function in regulating the flavonoid biosynthesis was not clear.Sub 4 group R2R3 MYB memmbers were reported involved in negative regulated the flavonoid biosynthesis pathway in Arabidopsis thaliana,while little information about MYB repressor in tobacco.Tobacco is one of the most model plants in engineering research,and play important roles in identifying the gene function.In this study,we constructed the CHS overexpression or RNAi silence or R2R3 MYB overexpression transgenic tobaccos,and investigated their functions in regulating the flavonoid biosynthesis.In this study,we investigated the CHS gene and R2R3 MYB repressors related to flavonoid biosynthesis pathway and their molecular regulatory mechanisms in Nicotiana tabacum.The study provides the molecular mechanism of high quality and resistance in crop breeding and a new way in molecular breeding.1.We identified and characterized seven CHS superfamily genes from Nicotiana tabacum,named NtCHS1-NtCHS7,based on the sequences of the Honghuadajinyuan(HD)common tobacco genome from the Chinese tobacco genome database.The characters of NtCHS1-NtCHS7 as follows:(1)NtCHS1-NtCHS7 share high homology and similar structure with other plant CHSs.High amino acid level homology was found with other Solanaceae CHS genes and among the seven family members.As with the other reported plant CHS genes,seven tobacco NtCHSs contained multiple representative active sites.Changes in NtCHS5-NtCHS7 at conserved amino-acid sites were detected,while NtCHS1-NtCHS4 did not vary at these sites.(2)Our phylogenetic analysis suggested two characters:Firstly,the seven tobacco NtCHS genes have diverged into three subgroups,including the classical CHS genes for NtCHS1-NtCHS4,the CHS-Like genes for NtCHS5,and the uncharacterized CHS genes for NtCHS6,NtCHS7.There is a large genetic distance between NtCHS6,NtCHS7 and CHS or CHS-like group proteins.Secondly,Solanaceae CHSs diverged into different subfamilies prior to the divergence of the genera.(3)NtCHS1-NtCHS7 show different spatial and temporal expression patterns.NtCHS1-NtCHS4 were widely expressed in pigmented or/and non-pigmented vegetative tissues and floral organs.NtCHS5 was expressed strongly in the calyx and corolla organs at the full-bloom stage.NtCHS7 was highly expressed in calyx at the squaring stage,and NtCHS6 expressed in all tissues at a low level.(4)NtCHS1-NtCHS7 show diverse expression patterns under abiotc stress.The expression of NtCHS1 and NtCHS3 increased markedly under drought and high salt stress.NtCHS2 and NtCHS4 expression were obviously induced by ABA and MeJA treatment,and NtCHS4 expression was specifically induced by 2,4-D.NtCHS6 was induced by the light,drought and high salt.While NtCHS5 and NtCHS7 expression were not induced by any of these treatments.2.In order to identify the function of NtCHS1 gene in the tobacco flavonoid biosynthesis,we generated the transgenic tobacco plants overexpression or RNAi interference NtCHS1 gene.(1)NtCHS1 was involved in regulating the accumulation of the total flavonoid,chlorogenic acid,rutin,anthocyanin and proanthocyanidin.The results showed that NtCHS1 overexpression significant increased those compounds,while those compounds were decreased in the interference NtCHS1 interference transgenic tobacco leaves.NtCHS1 interference also disturbed the development of the flower,decreased the length of receptacle and chapiter,and caused deformities in the development of chapiter.(2)It was notable that NtCHS1 interference increased the resistance to CMV infection.The CMV infection caused accumulation of phytoalexins,such as proanthocyanidin,caffeic acid and chlorogenic acid in NtCHSl interference lines and directly defending against viral infection.Meanwhile,the flavonoids induce the expression of resistance related genes,such as PAL,PR1 and LOX3,increased the activity of SOD and POD,activated the SA and MeJA signal pathway to resistant the CMV infection.(3)NtCHS1 overexpression increased the tolerance to high salt stress.The results showed that NtCHS1 overexpression increased the accumulation of chlorogenic acid and rutin,increased the activity of SOD and CAT activity.High antioxidant ability in NtCHS1 overexpression lines decreased the damage of oxidative stress to plant cells caused by high salt stress,increased the tolerance to the high salt stress.3.We identified and characterized three MYB repressor genes from Nicotiana tabacum,named NtMYB1,2,4 based on the sequences of the Honghuadajinyuan(HD)common tobacco genome from the Chinese tobacco genome database.NtMYB1,2,4 contained the[D/E]Lx2[R/K]x3Lx6LX3R and LIsrGIDPxT/SHRxI/L domains conservative in R2R3 MYB transcription factors and pdLNLD/EIxiG/S and CX1-2CX7-12CX2C domains especially exist in sub4 MYB repressors.The result indicated that the NtMYB1,2,4 were the R2R3 MYB repressors.(1)NtMYB1,2,4 have different spatial and temporal expression patterns.NtMYB1,2,4 were dominant expressed in floral organs.Meanwhile,the expression of NtMYB4 was dominant in plants.Moreover,NtMYB1,2,4 were induced by dark treatment,while NtMYB4 was specially induced by drought and high salt stress(200 mM).(2)There may be a mutual regulation exists in NtMYB1,2,4.There may be a synergisticeffect between NtMYB1 and NtMYB2,NtMYB1 and NtMYB4,and a supplementary effect between NtMYB2 and NtMYB4.(3)There was interaction between NtMYB1,2,4 and structure genes related to flavonoid biosynthesis.Overexpression of the NtMYB1,NIMYB2,and NtMYB4 gene inhibit the expression of the PAL,CHS and ANR1 gene.4CL and F3H were induced by the overexpression of NtMYB1 and NtMYB2,but were inhibited by the overexpression of NtMYB4.DFR and FLS were inhibited by the overexpression of NtMYB and NtMYB2,but were induced by the overexpression of NtMYB4.(4)To identify the function of NtMYB,2,4 in regulating the flavonoid biosynthesis pathway,we generated the NtMYB1,NtMYB2 and NtMYB4 overexpression genetic tobacco plants,respectively.The overexpression the NtMYB1,NtMYB2,and NtMYB4 gene,respectively,decreased the accumulation of total flavonoid,caffeic acid,chlorogenic acid,neochlorogenic acid and rutin in transgenic tobacco leaves.Furthermore,there may be an interaction between NtMYB1,2,4 and bHLH transcription factors.The results indicated that NtMYB1,NtMYB2,and NtMYB4 gene might negative regulated the tobacco flavonoid synthesis pathway by regulated the expression of the bHLH transcription factors.