Cloning And Function Research Of Pigment Genes From Camellia Nitidissima

Camellia nitidissima, a famous ornamental plant of the world, which has golden flowersand known as "Queen of camellias" is an important parent to breed yellow camellias. Theresearches of cross breeding with C.nitidssima are reported constantly while breeders didn'tgain the hybids which have goledn yellow flowers by many years' work. The reason of thisresult may be that the reasearchers don't uderstand the regularity and biosynthesis pathway ofthe pigmentions. Based on this point, this research was carried out to search the transformatinmechanism and some relative key genes to provide the theory support for breeding the yellowcamellias.To investigate the biosynthesis pathway of the pigments,7genes' cDNA sequences werecloned from the petals of C. nitidissima by the methods of RT-PCR(Reverse transcriptionpolymerase chain reaction) and RACE(Rapid Amplification of cDNA Ends), named CnCHS,CnCHI, CnF3H, CnF3'H, CnFLS, CnDFR and CnNSY respectively. The analysis of thesegenes' bioinformations showed that these are the goal genes we want.The Genbank loadedcodes are HQ269804, HQ269805, HQ290517, HQ290518, JF343560and JF440957, CnDFR isnot loaded.The results of HPLC(High-performance Liquid Chromatography) and the pigmentobservation in epidermal cell of petals showed that the contents of Quercetin(Qu), Quercetin-7-O-β-D-glucoside(Qu7G), Quercetin-3-O-β-D-glucoside(Qu3G), and carotenoids are low andthe accumulation of chromoplasts can't be found in the stage of petal uncolored；during theperiod of petals yellowish, the content of Qu3G and Qu7G are high, the chromoplastsaccumulated in serveal cells; when the petals golden yellow, the contents of Qu7G and Qu3Gare lower than before but the chromoplasts are bigger and in almost all the cells. The resultsindicated that the high amounts of colorless flavonols and no anthocyanidin in the petlas provide the necessary condition for the carotenoids to show its color, and the accumulation oftotal carotenoids in the cells is the determinant factor of the petal color.The results of quantitative Real-time PCR showed that the highest expression quantity ofCnCHS, CnCHI, CnF3H, CnF3'H appears during the period of uncolor petals to yellowish, inwhich the contents of Qu7G and Qu3G reach to highest level; the coperation of these genesmake the production of CnCHS transform to dihydroquercetion,which is the substrate of Qu7Gand Qu3G. The expression of CnFLS is mainly in the stage of uncolored petal and the CnDFRis during the time of full yellow colored indicate that the difference of the two genes makethem avoid competing the same substrate of dihydroquercetin, which restrains the combinationof anthocyanidin and provide enough substrates for the synthsis of Qu7G and Qu3G.We successfully constructed the sense and RNAi expression vectors of CnFLS and CnNSYrespectively by using the expression vector pCAMBIA1300and then the four genes weretransferred to tobacco by the Agrobacterium EHA105, finally,10lines of transgene plants ofevery gene were gained.5were selected in every10transgene plants, after the identifion ofPCR and Southern blotting,3lines transgene tobaccos were obtained of every gene. The resultsof quantitative Real-time PCR showed that the expression of FLS in trans-sense CnFLS is8-13times to the wild types and0.1-0.5times copmared to wild types in trans-interference CnFLSlines. The expression of NSY in trans-sense CnNSY is3-8times to the wild types and0.3-0.5times copmared to wild types in trans-interference CnNSY lines. This results show that theCnFLS and CnNSY can express in the transgens lines normally.The flower color of3lines of trans-sense CnLFS tobaccos appeared shallow yellow-white to shallow pink compared with the color of wild type tobacco. The results of HPLCshowed that the content of Cy3R in the petals of TF1which shows yellowish white color wasalmost0.1times of that of wild tobacco, the content of Qu3G is4times and Qu7G is2times ofthat of wild types. The color of trans-interference CnFLS tobaccos became more red than wildtype, the content of Cy3R in Tf23which shows the reddest color is3times of that of wild type,but the content of Qu3G,Qu7G are lower than that of wild type. These results indicates that the overexpression of CnFLS can make the flower color shallow and repression of FLS willcause the flower color deeper.The flower color of3lines of trans-sense CnNSY tobaccos appeared yellowish to shallowpink compared with the color of wild type tobacco. The results of HPLC showed that thecontent of Cy3R in the petals of TN10which shows yellowish white color was almost0.05times of that of wild tobacco, the contents of neoxanthin, violaxanthin, xanthophyll are2timesof that of wild types. The color of trans-interference CnNSY tobaccos became shallow orangered, the content of Cy3R in the3lines is about0.6times of that of wild type, and the contentsof neoxanthin, violaxanthin, xanthophyll are a little lower than that of wild type. These resultsindicates that the overexpression of CnNSY can promot the synthesis of carotenoids and makethe flower color shallow and repression of NSY will not affect the synthesis of caroteniods butthe overexpression of CnNSY might affect the synthesis of Cy3R.From this research we draw the conclusion that the expression characters of CnCHS,CnCHI, CnF3H, CnF3'H, CnFLS,CnDFR and CnFLS play the decisive role in the synthesis offlavonol and no synthesis of anthocyanidin. The exists of colorless flavonol provide a essentialcondition that the chromoplasts of carotenoids can show its golden color naturally,but the it isthe chromoplasts of carotenoids which accumulated in cells constantly rather than flavonoldecided the color of C.nitidissima. So, we believe that we can obtain the yellow camellias byusing the methods of trans-sense CnFLS or trans-interference CnDFR to decrease the contentof anthocyanidin ans increase the content of carotenoids by the method of trans-sense somecarotenoids synthsis genes such as CnNSY and so on.