| Limonoids are secondary metabolites widely distributed in the Rutaceae and Meliaceae,with biological and pharmacological activities such as anti-oxidant anti-allergy,anti-obesity,anti-osteoporosis,anti-cancer,anti-bacterial,anti-viral.The genetic modulation of limonoids biosynthesis is still poorly understood.So the investigation of the genes regulating the limonoids biosynthesis and study of their functions will help to understand the regulatory mechanism of limonoids biosynthesis and provide a theoretical basis for breeding high metablilites citrus variety.In this study,limonoids contents and expression of CclSAUR49 in the leaves of different developmental stages from two genotypes of Shatianyou pomelos(Citrus grandis(L.)Osbeck)varied in limonoids content were analyzed with HPLC and Real-time q RT-PCR.The relative expression of five candidate genes associated with limonoids biosynthesis derived by previous transcriptomic analysis in relation to the expression pattern of Ci OSC and the change of limonoids in two Shatianyou pomelos was examined.The promoter of CclSAUR49 was isolated from precocious trifoliate orange(Poncirus trifoliata L.Raf.)by PCR amplification.The cis-acting element of the promoter was predicted through the Plant CARE website.In order to analyze the characteristics of the promoter,p SAUR49::GUS fusion expression vector was constructed and transformed into precocious trifoliate orange.Uninfested precocious trifoliate orange was used as a control.The whole positive transgenic plants were stained chemically with GUS(beta-glucuronidase)and then observed and photographed.The coding regionr of CclSAUR49 was isolated from Shatianyou pomelo.Subcellular localization of CclSAUR49 protein was carried with transient expression of 35S::SAUR49-GFP fusion protein in the leaf of Nicotiana benthamiana and observed by confocal microscope.Overexpression vector of p SAUR49::GFP fusion protein was constructed and transformed into precocious trifoliate orange.Positive transgenic shoots were grafted onto the rootstock and grown outside of the door.The contents of limonoids,chlorophylls and expression of CclSAUR49 gene in transgenic plants were analyzed.The length of leaf and internode and circumference of stem were determined at three months after grafting.The effect of CclSAUR49 gene on citrus limonoids content and its mode of action were initially investigated.The main findings are as follows.(1)Screening for the genes related to citrus limonoids biosynthesisHPLC results showed that the limonoids content of two Shatianyou varieties is significantly different.STY2 contains significantly higher limonoids than STY1.The contents of nomilin and limonin were continuous decreasing along with the growth and development of leaves,while the gene expression level of CclSAUR49 was rising.The correlation analysis of the relative expression of candidate genes with the expression pattern of Ci OSC and limonoids content showed that the expression patterns of CclSAUR49 and Ci OSC were highly significantly and positively correlated in two Shatianyou pomelos;the expression patterns of 257P450 and 632P450 were also significantly and positively correlated with Ci OSC.However,the expression of CclSAUR49,257P450 and 632P450 showed significant negative correlation with the limonoids content.In comparison of above results,CclSAUR49 was choosen for the further study.(2)Cloning and expression analysis of CclSAUR49 promoterA 1600 bp fragment upstream of the start codon(ATG)of CclSAUR49 gene was cloned from citrus.The results of bioinformatic analysis predicted several types of cis-acting elements in the promoter of CclSUAR49 gene,but no Aux REs-responsive elements have been found.Six transgenic seedlings carrying fused CclSUAR49 and GUS were obtained by citrus genetic transformation.Results of GUS staining transgenic seedlings indicated that GUS was mainly expressed in stems,roots and old leaves,but not in young leaves.(3)Cloning and overexpression analysis of CclSAUR49The CDS sequence of CclSAUR49 was cloned from STY1 c DNA.The full length of sequence is 553 bp,which has only one exon and no intron.The coding region of this gene is 303 bp,which encodes a polypeptide of 101 amino acid residues.Compared to the sequence of SAUR49 in C.clementina genome,the similarity between the two sequences was 99.01% with three synonymou mutations existed in CclSAUR49.Subcellular localization analysis indicated that CclSUAR49 protein is located in the plasma membrane and nucleus.Two CclSUAR49 overexpression transgenic seedlings were obtained.The leaf length,stem thickness,internode length and chlorophylls content of the transgenic plants were measured and analyzed.Comparing the growth performance of transgenic and control plants grown under the same growth conditions,significant morphological difference was observed.The transgenic plants had larger size of leaves,longer internodes,thicker stems and higher contents of chlorophylls.Compared to control,the leaf length,stem thickness,and internodes length were increased by 28.71% and29.71%,18.45% and 14.34%,18.19% and 26.93% in two transgenic plants,respectively,while chlorophylls content was decreased by 39.38% and 10.53%,respectively.Contents of nomilin and limonin were significantly reduced in transgenic plants.Compared to control,nomilin content was 44.81% and 22.60% lower,and limonin content was 23.74% and 67.36% lower in OE-1 and OE-2,respectively.q RT-PCR results showed that expression of CclSAUR49 gene was highly significantly increased in overexpression transgenic plants with 657 and 151 fold of increases in OE-1 and OE-2,respectively.Analysis of expression of CclSAUR49 and limonoids content in transgenic plants revealed that expression of CclSAUR49 was significant negative correlated to the limonoids content with correlation coefficients of-0.983(P<0.01)and-0.975(P<0.01)in OE-1 and OE-2,respectively.In order to clarify the role of CclSAUR49 gene in the limonoids biosyntheses,expression analysis was also performed on six genes associated with limonoids synthesis identified by previous transcriptomic analysis.Overexpression of CclSAUR49 affected the expression of those genes,suggesting that CclSAUR49 may interact with some of those genes to regulate the biosynthesis or accumulation of limonoids in citrus. |
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