Genetic Diversity And Functional Study Of Chalcone Synthase Gene In Citrus

Flavonoids is one of important secondary metabolites in citrus.It is distributed in multiple tissues such as roots,stems,leaves,flowers,fruits and seeds.It has been proved that flavonoids posses the bio-function for the mankind‘s health.Chalcone synthase?CHS?is a key enzyme in the pathway of flavonoids biosynthesis.The objectives of this study are to understand the relationship between the genotypes and flavonoids production,to investigate the genetic diversity of CHS gene,to identify the functional CHSs,and to study the effects of CHS genes to the production of flavonoids.In present study,the 11 flavonoid components were investigated with UPLC method in 37citrus germplasms,the principle of the flavonoids distribution and contents variation among different genetic backgrounds were studied.The CitCHS2s were cloned from 10 citrus germplasms with significant difference of flavonoid contents,the correlation between the flavonoid contents and genetic diversity of CitCHS2s were studied.The 77 CHS or CHS-like sequences were retrieved from citrus genome by homologous sequence alignment and 10 of them were screened as candidates after the phylogenetic analysis.The expression profiles of these CHSs in response to different treatments and the CHS family constitute were studied preliminarily.A novel CHS was found and its function,together with the function of CitCHS1 and CitCHS2 were validated through transgentic experiments.The major results acquired from this study are as follows:1.Temporal and spatial variations of citrus flavonoids productionThe results of the UPLC analysis showed that both of the components and the amounts of citrus flavonoids among 37 germplasms are different from each other,depending on their genetic backgrounds.The amounts of total flavonoids are from 1,918.51 to 17,037.80 mg?kg^-1 Fresh Weight?FW?.The highest amount was found in the peel of Daidai sour orange,which is about 8times of the lowest one that was found in Jindan.The two major features of citrus flavonoids are multi-component and high contents,respectively.All the 11 flavonoids were detected in Zhoupi Gan and Huangpi Suanju.Furthermore,among 37 germplasms,36 were found to have total flavonoids of higher than 3,000 mg?kg^-1 FW except in Jin Dan,which is lower than 2,000 mg?kg^-1 FW.There were 13 germplasms with flavonoids higher than 10,000 mg?kg^-1 FW.The cluster analysis showed that the classification of mandarins and oranges through flavonoid contents are similar to botanical classification.The flavonoid contents of a same tissue changed at different levels through three developmental time points,while the contents varied among different germplasms.Different tissues in a same time point have different flavonoid contents.In a same germplasm,the highest flavonoids was found in young fruits and the lowest one was found in roots,the order from high to low are young fruits,young leaves,young stems,mature stems and roots,respectively.The flavonoid contents were found to be specific among different tissues and organs.2.The genetic diversity of CitCHS2 in citrusAccording to the flavonoid contents among 37 germplasms,10 were selected as materials to study the genetic diversity of CitCHS2 gene.They are C.paradisi Macf.×Poncirus trifoliata L.Raf.,C.aurantium L.,C.sinensis?L.?Osbeck cv Tarroco,C.paradisi Macfad cv?Pink Thamoson grapefruit?and C.daoxianensis S.W.He with high flavonoids phenotype.The other 5 species are C.limon?L.?Burm.f.,C.ichangensis Swingle,C.grandis?L.?Osbeck cv Liangping Pummelo,C.microcitrus Swingle×Sinocitrus cv and C.limonia Osbeck with low flavonoids phenotype.The sequences were cloned from the 10 citrus germplasms,respectively.The GenBank number are KP720583⁷20592.The results showed that the basic structure of CitCHS2 consist of one intron and two exons.The intron is 102bp long.In comparison with the cDNA sequences,the splicing sites of the intron is not the typical type II intron because they do not confirm with the splicing pattern of5'-GT…AG-3'and the polyA tail starts from 1385 bp.The 1313bp cDNA sequence contains an ORF of 1173bp coding 391 amino acids.Although 15 nucleotide sites were found to be changed,resulting into 7 amino acids variations among these CHSs,the main structures of these CHS are highly conservative.The molecular weight of 10 CitCHS2 is from 42,592.1 Da to 42,636.1 Da,the isoelectric point is between 6.28 and 6.47 and the instability index is from 34.25 to 35.95,respectively.The CHS is relatively steady protein and free of transmembrane domain or signing peptide.The analysis of protein secondary structure showed 4 amino acid sites(site1?16¹8 bp?,site2?442⁴44 bp?,site5?961⁹63 bp?and site7?1060¹062 bp?are locating at the?helix structure,and the other 3 were not on any domain regions?3.The variation of CitCHS2 gene expression and their effects on flavonoid contentsThe expression of CitCHS2 was tissue specific.Generally,the higher expression level was detected in young leaves while the lower one was found in mature leaves of the 10 germplasms except Baxi sour orange.In a same tree,the order of CitCHS2 expression level from high to low was found in young fruits,yong leaves,yong stems,mature stems and roots,respectively.The expression trend of CitCHS2 in three germplasms?code 30,14 and 28?druing 6 developmental time points was down-regulated at first and then keep at a relatively stable level.The flavonoid contents analysis showed that there was a positive correlation between the CitCHS2 expression and the flavonoid contents in all tissues except in young fruits.Coefficient analysis showed that the correlation between the expression of CitCHS2 and the flavonoid contents was low?R<0.5?in mature leaves while the correlation was tight?R=0.76?in young leves.There existed a positive correlation between CitCHS2 expression and flavonoid contents in different tissues of a same tree.Furthermore,there were obvious positive correlations between the CitCHS2expression and flavonoid contents in peels of Meyer Lemon,Tarocco?O.L.?and Guangxi Pingxiang Tu Lemon during 6 developmental time points,the coefficient was 0.91,0.99 and 0.84,respectively.4.Identification and expression analysis of CHS family genes and the characterization of CitCHS3The 77 CHS or CHS-like sequences were screened from the citrus genomes available on line by BLAST.The phylogentic analysis showed that except orange1.1t03054.1m,all the other 76sequences were clustered into 3 major groups?I,II,and III?.The annotation showed that the 22sequences from Group III are mostly CHS or CHS related genes.We selected 10 CHS from Group III,each represents a subgroup.Their expression profiles in response to different treatmens were studied in yong seedlings.There were 3 CHSs can be detected after MeJA treatment of young citrus seedlings,they are Ciclev10005133m?CitCHS1?,Ciclev10015535m?CitCHS2?and Ciclev10001405m,respectively.Among these CHSs,Ciclev10001405m was first time characterized as a novel CHS member,termed as CitCHS3 in this study.Of the three CHSs,CitCHS1 was the most active one in response to MeJA.CitCHS2 and CitCHS3 had a similar expression profile in leaves,stems and cotyledons,but not in roots.After the low temperature treatment,three CHSs can be detected in leaves,cotyledons and roots.However,CitCHS3 was not expressed in stems after this treatment.CitCHS2 expressed in all tissues and was rapidly in response to low temperature treatment.The expression of CitCHS2 can be detected in leaves,stems and cotyledons,but not in roots after high temperature treatment.The expression of CitCHS1 was specific among different tissues and the highest expression was detected in roots,4h after treatment.The expression of CitCHS3 can not be detected in stems or roots,and only very weak expression was detected in leaves and cotyledons.CitCHS1 was the only CHS expressed in roots after high temperature treatment.CitCHS1 expressed only in cotyledons but not in the other 3 tissues after dark treatment.However,CitCHS2 can be detected in all tissues but the expression was very low in cotyledons.Furthermore,this gene had a similar expression profile in different tissues.Under darkness,the expression of CitCHS2 was reduced.The expression of CitCHS3 was observed in leaves,cotyledons and stems,but not in roots.This gene also had a similar expression profile in different tissues,with higher expression before darkness,and reduced expression thereafter.Coefficient analysis showed that the sum of 3 CHSs expression was positively correlated with total flavonoid contents.Gene structure analysis showed that the 3 CHSs had a similar structure which was consist of one intron and two exons.Coding 60 amino acids,the first exon was 180bp which was more conservative than the second one.The ORFs of the 3 CHSs were 1170 bp,1176 bp and 1194 bp,respectively.5.Regulating effects of CitCHS1and CitCHS2 on flavonoids biosynthesis in transgenic plantsThe construction of the heterogeneous expression of citrus CitCHS1 was conducted in tobacco plants by transgentic experiment to study the function of CitCHS1.In the positive plants,the highest expression was found in leaves,the lowest expression was found in roots and the medial one was found in stems,respectively.The results of total flavonoids showed that the highest flavonoid contents were detected in leaves,which was 1.33 times and 1.41 times higher than the wild control and vector control plants,respectively.However,in stems and roots,the flavonoid contents of the positive plants were not significant in comparison with control plants,though a little bit higher flavonoids were detected in positive plants.The coefficient analysis showed that in the leaves of positive plants of over-expressing CitCHS1,the expression was highly correlated with flavonoid contents?R=0.73?.In stems and roots the correlation is weak as the coefficient was lower than 0.5.In four CitCHS2 over-expressing positive plants,three of them were up-regulated and 1 was down-regulated.The flavonoid contents showed that the flavonoids increased when the CitCHS2was up-regulated,and flavonoids decreased when the CitCHS2 was down-regulated.Coefficient analysis showed that the expression of CitCHS2 was tightly correlated with flavonoid contents.6.Function validation of CitCHS3The pGBi-CitCHS3-RNAi vector was constructed through RNAi technology and 4 positive transgenic citrus plants were analyzed.Among the 4 positive plants,the average level of down regulation was 76.01%in comparison with the wild control,and 61.71%in comparison with the vector control,respectively.Total flavonoids were recuced significantly in 4 positive plants when the CitCHS3 was dow-regulated,indicating CitCHS3 was important in flavonoids biosynthesis.7.The effect of CitCHS family members on flavonoids biosynthesisThe expression of PAL was detected higher in only one wild plant?wild 1?than in the positive ones,and in all the other plants no significant difference was found.So we cannot figure out the correlation between the PAL gene and CHS3.The CHI,which is next to CHS in the flavonoids biosynthetic pathway and can isomerize the production of CHS.In present study,the expression of CHI shared a similar trend with CHS in positive plants.The 345bp conservative region of CitCHS3gene was used as target for silencing CHSs through VIGS technology.Comparing with the controls,the average expression level of 3 CHSs in silenced plants decreased at 81.03%,79.67%and 76.60%,respectively.Meanwhile,the average amounts of total flavonoids decreased 41.11%.The coefficient analysis showed that the coefficients between the three CHS gene and total flavonoids were 0.90,0.43 and 0.80,respectively.Furthermore,there exist a positive correlation between the sum of CHS expression and the total flavonoid contents.Differential expression profiles were found among the three members of CitCHS family.The results showed these CHSs have different contributions of controlling the biosynthesis of flavonoids.In the leaves of 4 transgenic positive plants,the expression of CitCHS1,CitCHS2 and CitCHS3made up 12.02%,55.52%and 32.47%,of the total CHS expression,respectively.Among all the plants used in present study,the result was 13.29%,58.63%and 28.09%,respectively.The coefficient analysis of gene expression and flavonoid contents showed that CitCHS1 had the greatest contribution to the production of flavanones and flavanonols,CitCHS2 was the biggest contributer to flavones and flavonols,and CitCHS3 had the most effect on total flavonoids.Meanwhile,CitCHS3significantly co-expressed with CitCHS2.