Molecular And Functional Identification Of Limonoids Biosynthesis Genes In Citrus

Limonoids are triterpenoids kind of secondary metabolic products specifically produced in citrus,which possess various bioactive functions,such as anti-tumor,anti-obesity,anti-fungal,and antiviral activities.It is beneficial to human health.So far,there is no much knowledge on genetic control of limonoids biosynthesis.This study aims to investigate the genetic and environmental factors influencing the biosynthesis of limonoids and identify the key genes controlling limonoids biosynthesis processing.The seeds and segment membrane in different development stage of fruit from 12 pummelo varieties was analyzed for nomilin and limonin contents.In order to screen regulatory genes of limonoids biosynthesis,the leaves at three developmental stages,seeds and phloem of Dongfeng early pummelo(Citrus grandis(L.)Osbeck)were analyzed using high-throughput next generation RNA-sequencing.The twenty candidate genes were selected by bioinformation and molecular biology analysis.Two candidate genes Ci SQS and Ci OSC closely related to the limonoids biosynthesis pathway were picked for further analysis to identify their roles in related to the biosynthesis of limonoids.Both genes were cloned from pummelos and used for gene transformation.The main results are as follow:1.Temporal and spatial variations on accumulation of nomilin and limonin in the pummelos.HPLC results showed that the large variations on limonoids concentration were found among different varieties,which ranged from 1233.78 mg/kg FW to 4090.41 mg/kg FW in the seeds at full color stage of the fruit.By ANOVA analysis(the least significant range,LSR),a significant difference was found for the contents of total limonoids among 12 pummelos.Based on the clustering results,12 varieties could be divided into three groups.The varieties of highest limonoids content of Early Siam Pummelo,Menglun Zao You and Yian Wo You were cluster in the same class,which are far away from nine pummelos derived from South China.The results of cluster analysis indicated that the variation of limonoids contents would be greatly affected by the genetic variations.The rapid accumulation or biosynthesis of nomilin and limonoids in the seeds was observed during the physiological maturation of seeds.This result indicated that physiological maturation is a nodal point in the seeds to start the mass accumulation of nomilin and limonin.At 10% of fruit color break,both nomilin and limonoids contents reached the maximal level.From November to January of secondary year,limonin and nomilin contents in seeds and segment membrane were shows rising trend along with falling temperatures.2.Identification of genes involved in limonoids biosynthesis in citrus by RNA-seq analysis.The leaves at three developmental stages,seeds and phloem of Dongfeng early pummelo were analyzed using high-throughput next generation RNA-sequencing.After filtering low quality reads,a total of 14,551,995(LI),13,302,783(LII),14,900,160(LIII),18,151,136(PII)and 14,978,415(SII)clean reads were obtained.Pair-wise comparison of transcriptional profiles between different tissues was carried out according to limonoids content.The numbers of differentially expressed genes(DEGs)were 2,086 between LII and LI,4,082 between LIII and LI,5,837 between PHII and LI,and 9,428 between SII and LI.A total of 924 DEGs were shared across all four sets.Those DGEs were categorized into 30 functional classifications via Map Man.Major functional categories included RNA regulation,protein modification and degradation,signaling,cell,secondary metabolism,hormone metabolism,and cell wall.However,only 14 of 924 genes were involved in terpene metabolism.In order to investigate expression patterns,genes expressed in leaves at different stages(LI,LII,LIII)were clustered into ten groups via K-means cluster,and 924 DEGs sets from LII VS LI,LIII VS LI and LIII VS LII were analyzed to filter the genes at the same expression pattern by MEV software.By analyzing expression patterns of k-means clustered,three conjunctive clusters(including 2,6 and 374 genes)were probably correlated with limonoids contents.Among them,only Ci OSC gene is belonged to terpene biosynthesis.Phylogenetic analysis also revealed that Ci OSC was clustered with Bi and CPQ genes,which were the first committed enzymes of cucurbitacin biosynthesis pathways in cucumber(Cucumis sativus L.)and squash(Cucurbita pepo),respectively.qRT-PCR result showed that the higher expression of Ci OSC associated with higher limonoids content was observed in GXSTY and JC.On the contrary,the lower expression of this gene along with lower limonoids content was also found in HPJG,RAJG,WCZPG,GCMG and DNXY.During seeds development of ESP and GXSTY,the expression pattern of Ci OSC was highly corresponded with the change of limonoids.In addition,19 candidate genes were identified,including five TFs,six CYP450 s,three UGTs,one acyltransferases,one SAUR,one XTR and two transporters.The expression trends of those genes were coinciding with limonoids contents during seeds development.Among CYP450 s family members,significant positively correlations between gene expression level and limonoids were observed in ciclev10000886 m,ciclev10030087m,ciclev10022473 m and ciclev10025448 m genes.And the correlation coefficients of those genes were 0.995 a,0.985 b,0.958 b and 0.981 b,respectively.However,three UGTs genes(ciclev10020010m,ciclev10015042 m,ciclev10001658m)expressed negative correlation with high limonoids contents in ESP during seeds development.In the low limonoids contents variety of GXSTY,UGTs genes expressed positively correlated with limonoids contents during seeds development.Two ABC transporters(ciclev10011147m and ciclev10003388m)also expressed highly coincide with limonoids contents during seeds development,and specifically express in seeds.It is possible that those transporters could be participated in transportion of limonoids from leaves and phloem into seeds.3.Functional characterization and expression analysis of SQS gene in citrus.The length of Ci SQS c DNA sequence is 1813 bp.The CDS region is 1242 bp,which is predicted to encode a putative protein of 413 amino acids with a calculated molecular weight of 47.37 k Da.The deduced amino acids sequence of Ci SQS had 99% homology among 18 citrus varieties.Single nucleotide variations of complete Ci SQS CDS sequences were further analyzed.There are 41 polymorphic sites in CDS sequence.The total frequency of SNPs in all tested citrus varieties was one in every 329 bp.Among these,26 synonymous SNPs and non-synonymous SNPs resulted to the changes of encoded amino acid.There was a C/T nucleotide mutation appeared in Xiaoguo kumquat variety which result in introduced a stop codon(TGA)and produce a pre-terminated protein.Compared with other varieties,Rongan kumquat has longer CDS sequences because of the mutation of six nucleotides at the end of 3'.There has a G/C synonymous mutation at the 14 nucleotide acids of CDS among Xiaoguo kumquat,Rongan kumquat and No.4 Yichang Cheng.Phylogeny analysis shows that low limonoids contents of those three varieties were clustered in the same class,which is divided with other varieties.Suggesting that the 14 nucleotide acids in CDS could be correlated with limonoids contents.qRT-PCR analysis display that expression level of Ci SQS are positive consistent with limonoids content in seeds of most pummelo varieties.But,the higher expression of this gene along with lower limonoids content was observed in GXMY and PSY.During seeds development of those two varieties,the expression pattern of Ci SQS was corresponded with the change of limonoids.However,the highest expression level of Ci SQS associated with lowest limonoids content was observed at ST1 stage in PSY.This suggested that Ci SQS gene may be involved in limonoids biosynthesis,but less significant.Under the abiotic stress of cold,Me JA,SA,GA3 and Na Cl,expression level of Ci SQS gene was significantly induced by cold.4.Importance of SQS gene in limonoids biosynthesis.Plant expression vector of p BI121GSH-SQSOE,p GBi-SQSRNAi and p GBi-SQSRNAi-N were constructed and introduced into epicotyls by Agrobacterium tumefaciens,resulting in 3 SQSOE transgenic lines and 4 SQSRNAi-N transgenic lines.HPLC results showed that total limonoids contents were reduced in 3 SQSRNAi-N interference lines,reach to 72.24~95.12% of control plants.However,limonoids contents in Si N-3 line are 1.24-fold higher than the controls.In all lines,limonin contents were decreased significantly,reach to 35.83~81.56% of control plants.The increasing limonoids contents in silenced lines were caused by higher nomilin content.Content of nomilin in Si N-3 and Si N-4 is increased 1.53-fold and 1.35-fold,respectively.qRT-PCR results revealed that the transcript levels of Ci SQS gene in the 4 Ci SQS interference lines were decreased to from 61% to 79% of control plants.A simultaneous decreasing in the m RNA levels of 123?AS,195ATLUP2,330?AS,632?AS and 951CAS1 genes and rising expression levels of 789ATLUP2,490CAMS1 and 602CAS1 genes of OSCs types were observed in most transgenic lines.The results reveal that Ci SQS gene is positive regulate limonoids biosynthesis.And competitive inhibition could be existed in different types of OSC genes among Ci SQS interference lines.Three lines of over-expressed Ci SQS were obtained.Only SOE-1 line can be grown normally.Nine months after grafted,two little new leaves were observed in SOE-2 line.But SOE-3 line still maintains the status of pre-grafting.HPLC result shows that limonoids contents in SOE-1line are significantly decreased to 68.83% compared with control plants.However,m RNA levels of Ci SQS gene in SOE-1 and SOE-2 lines were increased to 1.45 and 1.31 fold.The rising and decreasing of expression level of OSC genes also observed.The results did not show a clear picture that SQS is significantly involved in biosynthesis of limonoids.In addition,all of the Ci SQS interference lines and over-expressed lines with Ca MV 35 S promoter were appearing to abnormality growth,including necrosis in leaves,growth retardation and even lethal plants.The Ci SQS gene regulates several metabolic pathways with the production of precursors for biosynthesis of phytohormones and steroids,which are essential for the development of the plant.This might be the reason why there were no completely silenced plant generated from RNAi transformation,also very difficulty to geneticly manipulate the SQS gene.5.Use of TRV-mediated VIGS for functional genomics research in citrus.Nicotiana benthamiana was used as model plant in VIGS,which is susceptible to TRV infection.A reporter gene,Magnesium chelatase subunit I(CHll)from C.grandis was constructed into TRV2 vector,because silencing of CHll will lead to photo-bleached leaves.Seven days post infiltration(dpi),silencing symptom appeared on the newly sprout leaves.The photobleaching symptom gradually appeared on the older leaves.About 14 dpi,most of leaves demonstrated the symptom of completely silencing,only a few leaves remained minor green patches.After 27 dpi,the photobleached phenotype developed to the whole plant.The result showed that Ci CHll is an effective marker gene.The VIGS vector with Ci CHll was applied to assess the sensitivity of the citrus varieties.Mexican lemon is the most sensitive one.The chlorosis area per leaf accounted for 65.5% of the whole leaf area.GXSTY exhibited the less sensitivity to VIGS with only tiny patches appeared on treated plants.Several parameters were optimized,such as OD600 value of bacteria concentration,developmental stages of leaves and use of germinated seeds.OD600 of Agrobacterium cell from 0.6 to 1.4 did not show distinct silencing efficiency.Different developmental stages of leaves showed different photobleached phenotypes.Leaves at ST1 and ST2 stages are susceptible to VIGS infection.In germinating seeds of GXSTY,TRV with GFP reporter gene expressed fluorescence.This result indicated that germinating seeds could be an ideal material for VIGS in citrus.Ci OSC gene was silenced by VIGS in germinated seeds of GXSTY.qRT-PCR analysis showed that the expression of Ci OSC gene was notablely inhibited in 6 seedlings,with reduced contents of limonin and nomlin.The expression level of Ci OSC gene in number 2 seedlings was significantly suppressed,reached to 9.09% of the control.Limonin and nomilin contents readuced to 35.08 and 44.49% of the control,respectively.This suggested that Ci OSC gene can regulate limonoids contents,which could be one of the key genes in the upstream of limonoids biosynthesis pathway.