Cloning And Functional Characterization Of IbPSY And IbSGR From Ipomoea Batatas

Ipomoea batatas is an improtant plant.The colors of the tuber are varied.Purple tuber contains abundant anthocyanidins,and orange tuber is rich in carotenoids.?-carotene is one of the carotenoids which plays an important role in human body.?-carotene can be converted into vitamin A.when lack of vitamin A,?-carotene can relieve vitamin A deficiency.?-carotene is also a good antioxidant,which can slow down the ageing.In this research,we cloned the first rate-limiting enzyme gene IbPSY(Phytoene synthase)from the orange Ipomoea batatas named YuShu11-10-97.The coding sequence(CDS)of IbPSY is 1320 bp and encoding 439 amino acids.The length of plastidial transit peptide is 47 amino acids.Subcellular location analysis revealed that IbPSY was located in chloroplasts,which is the same with other PSY proteins.carotenoids engineering bacteria was used to analysis the function of IbPSY.The results showed that IbPSY had the same function of crtB(crtB is a homologous gene of PSY).Overexpression of IbPSY in Arabidopsis thaliana,the content of carotenoids was significantly improve,it means IbPSY is phytoene synthase.We cloned the gene IbSGR from Ipomoea batatas.which is relevant with chlorophyll degradation.The coding sequence(CDS)of IbSGR is 801 bp and encoding266 amino acid.The length of plastidial transit peptide is 52 amino acids.Subcellular localization results showed that Ib SGR protein was located in chloroplasts which is the same with Ib PSY protein.Using qPCR to analysis the relative expression of IbSGR in Ipomoea batatas.Four tissues(young leave,mature leave,young stem and old stem)were used to do this research,finally we found that the expression level in leaves were higher than stems.IbSGR had a high expression level in mature leave.The lowest expression level was in the young stem.It is reasonable,in the process of leafsenescence,SGR plays an important role in this process,so the expression pattern of IbSGR is consistent with the function of SGR.Overexpression of IbSGR in Arabidopsis thaliana showed that the chlorophyll content had a significantly decreased,compared with the wide type.This suggest that IbSGR is the protein that relevant with chlorophyll degradation.BiFC expriment proved that Ib PSY protein and IbSGR protein had an interaction in vivo.In this research we use an engineering bacteria that can produce ?-carotene to test if the IbSGR can influence the content of ?-carotene in Escherichia coli.The color of the engineering bacteria which contains IbSGR was white,but the the color of the engineering bacteria was orange.It means Ib SGR can inhabit the function of IbPSY.High-pressure liquid chromatography(HPLC)test showed no ?-carotene in the engineering bacteria which contains IbSGR.This suggests that IbSGR can suppress crtB in Escherichia coli,also implies Ib SGR can inhibit Ib PSY.Arabidopsis thaliana that overexpression of IbSGR had less content of carotenoids in leaves than wild type.The results showed that overexpression of IbSGR in Arabidopsis thaliana could decrease the content of carotenoids.This research proved that IbPSY is the gene that encode phytoene synthase,IbSGR we cloned is the gene that relevant with chlorophyll degradation.Finally,we can draw a conclusion that IbSGR can interact with Ib PSY,and then regulate the levels of carotenoids in the plants.