Expression Regulation Of EuSRPp1 Gene On Rubber Synthesis In Eucommia Ulmoides Oliv

Eucommia ulmoides Oliv.is a second-class protected plant in China,distributed in tropical and warm temperate regions,Eucommia ulmoides Oliv.is a perennial deciduous trees,and a single family,single genus,single species of plants.E.ulmoides is a unique and excellent economic forest tree species in China.It is not only a traditional and precious Chinese medicinal material,but also a gum-producing plant with development potential.Its percha,bark and leaves contain white gum,also known as gutta-percha.However,the biosynthesis mechanism of E.ulmoides gum has not yet been fully understood.It has been found that the extension of the rubber chain is related to the rubber elongation factor(REF)and the small rubber particle protein(SRPP),both of which have the same conserved domain.it is distinguished by the different lengths of N-terminal and C-terminal outside the conservative structure.REF has a variable-length N-terminus,and SRPP has a variable-length Cterminus.In this study,18-year-old Eucommia ulmoides adult plants grown in the experimental base of Guizhou University(Guiyang Huaxi)were used as materials,based on the genome of E.ulmoides,Eu REF8,EuSRPP2 and EuSRPP3 were cloned,their expression patterns were studied,and the mechanism of action of EuSRPP1 gene was mainly studied.The following research results were obtained:1.Accumulation regularity of gum during the development of Eucommia ulmoidesAfter the flowering period of E.ulmoides,from April to November,the leaves,stem bark and pericarp of the male and female E.ulmoides were taken each month to determine the gum content of each tissue.The results showed that the gum content of leaves,stem bark and pericarp of male and female plants increased with the development of tissues and organs.The content of gum was the lowest in April,and the period from June to September was the rapid period of gum accumulation.After September,gum accumulation gradually stabilized,and reached the highest in November.The gum content of female plant ranged from 0.51% to 2.23% in leaf,0.76% to 12.38% in stem and 2.93% to 14.67% in pericarp.The leaves and bark of male plant ranged from 0.33% to 2.57% and 0.74% to 11.05%.The amount of gum in the leaves of female plants was higher than that of male plants in May,and the amount of gum in leaves of male plants was higher than that of female plants in June,July,August,October and November,and the differences were all significant(P<0.05).From April to November,the gum content of each organ of E.ulmoides was pericarp>stem bark>leaf,the differences were extremely significant(P<0.01).This shows that with the growth and development of E.ulmoides,the gum content of male leaves is gradually higher than that of female leaves,and the gum content of female stem bark is gradually higher than that of male stem bark.2.Changes of molecular weight of gum during the development of Eucommia ulmoidesThe average molecular weight(Mw)of gum in different organs was analyzed.The results showed that the Mw of gum in different organs increased with time from April to September,but the increase was different,and the difference was not significant from September to November.It was lowest in April,the average Mw of gum in leaves of female and male was 3.59×106 Da and 7.38×106 Da,respectively,the average molecular weight of gum in stem bark of female and male was 2.51×106Da and 0.84×106Da,respectively,the Mw of fruit was 2.53 x 106 Da.In September,the average molecular weight of gum in each organ reached the maximum,the average Mw of gum in leaves of female and male were 12.43×106 Da and 16.57×106 Da,respectively,the average molecular weight of gum in stem bark of female and male was 5.29×106 Da and 6.23×106 Da,respectively,and 8.31×106 Da in fruit of female.From April to November,the average Mw of gum in leaves ware higher in male than in female.The average molecular weight of gum in stem bark was higher in female than in male in April and May,but from June to November,the male was higher than the female,the differences reached significant level(P<0.05).During this period,Mw is leaf > pericarp > stem bark.It shows that although the gum content in the blade is the least,the Mw of the gum is the largest.From April to November,in different organs of E.ulmoides females and males,the molecular weight distributions showed different trends with the development of organs.The distribution of small molecular weight(less than 100×104 Da)gum showed a trend of first decreasing and then increasing,and the distribution was the most in April.The leaves of female and male plants were 27.86% and 17.08%,respectively,the stem bark was 44.61% and 75.15%,and the pericarp was 38.86%;the leaves of the male plant were the least in July(2.44%),the leaves of the female plant were the least in August(2.60%),the stem bark of the female plant was the least in September(9.62%),and the stem bark of the male plant was the least in August,It is 9.65%,and the percha is the least in July,which is 2.57%.The distribution of high molecular weight gum(1300×104)showed a trend of increasing first and then decreasing,and it was the least in April.The male and female leaves were 3.60% and 17.10%,respectively,the stem bark was 1.97% and 0.93%,and the pericarp was 4.76%;The most in September,the leaves of the female and male plants were 39.79% and 33.06%,respectively,the stem bark of the female and male plants was 20.14% and 20.39%,and the pericarp was 20.13%.It indicated that the molecular weight of gum in each organ tissue increased with the growth of the organ from April to August,while the long-chain polyisoprene did not increase or the short-chain polyisoprene increased slowly from August to September.3.Cloning and expression analysis of EuREF/SRPPs gene12 REF/SRPPs genes of E.ulmoides fragments were screened from the transcriptomic and genome database constructed by the project team,and 3 EuREF/SRPPs genes with complete c DNA were cloned,with sequence lengths of 267 bp,630 bp and 651 bp,respectively.After the analysis of the conserved region and N-terminal and C-terminal,they were named EuREF8,EuSRPP2 and EuSRPP3,respectively.Phylogenetic tree analysis showed that EuREF8,EuSRPP2 and EuSRPP3 were closely related to the REF/SRPP of Hevea brasiliensis and Taraxacum kok-saghyz.EuSRPP1 has been obtained in our laboratory in the early stage,and the sequence length is 678 bp,encoding a total of 225 amino acid residues.The relative expression levels of EuSRPP1,Eu REF8,EuSRPP2 and EuSRPP3 in each tissue of E.ulmoides at different stages were analyzed by q RTPCR,and the molecular weight and content of gum in the tissues and organs of E.ulmoides in the same period were analyzed.The results showed that from April to November,EuSRPP1,Eu REF8,EuSRPP2 and EuSRPP3 were all expressed in leaves and stem bark of male and female plants at different stages,and the expressions first increased and then decreased.The expression of EuSRPP1 in leaves reached the maximum in September;the expression of EuREF8 and EuSRPP2 reached the maximum in July;the expression of EuSRPP3 reached the maximum in May.The expression of EuSRPP1 in stem bark reached the maximum in September;the expression of EuREF8 and EuSRPP2 both reached the maximum in July;the expression of EuSRPP3 reached the maximum in September.The expression of EuSRPP1 in the percha showed a downward trend,and it was the highest in April.The expression of EuREF8 in the percha showed a trend of first increasing and then decreasing,and was the highest in May;the expressions of EuSRPP2 and EuSRPP3 in the percha showed a trend of first decreasing and then increasing,and the lowest in September.During the period from April to September,the expression characteristics of EuSRPP1 in leaves and stem bark were positively correlated with the growth rule of gum content and the change rule of molecular weight(P<0.05).The expression characteristics of EuREF8 in female and male stem skins were significantly positively correlated with the change of gum molecular weight(P<0.05).The expression of EuSRPP3 gene in the stem skin of female and male plants was significantly positively correlated with the growth of gum content(P<0.01),the expression of EuSRPP3 gene in female was positively correlated with the change of gum molecular weight(P<0.05).The expression characteristics of EuSRPP1 had the highest correlation with the growth of gum content and molecular weight change,indicating that EuSRPP1 played an important role in the formation of E.ulmoides gum.4.EuSRPP1 location and the effect of gene expression on gum content and molecular weightThe prokaryotic expression vector PET-28A-EuSRPP1 was constructed to prepare monoclonal antibody against EuSRPP1.Using 10 nm gold particles as the marker,the EuSRPP1 protein in the phloem glial cells of Eucommia ulmoides was localized.The results showed that the protein was localized on the surface of the gum particles.It shows that EuSRPP1 plays a role in the presence of gum particles.The EuSRPP1 gene overexpression vector p GM626-Act1-EuSRPP1 and RNAi interference plant expression vector p GM626-Act1-EuSRPP1-RNAi were constructed with Actin1 as the promoter.After verification,EuSRPP1 and EuSRPP1-RNAi transgenic E.ulmoides seedlings were obtained respectively.The expression characteristics of EuSRPP1 in the two resistant seedlings were analyzed.Compared with the wild type,the expression of EuSRPP1 in OE plants was 2.53 and 3.68 times that of WT,the expression of E.ulmoides gum synthesis-related gene Eu IPI,Eu FPS and Eu GGPPS were significantly higher than WT.In RNAi interference plants,the expression levels of EuSRPP1 were only 3.92% and 42.38% of those of the WT,while Eu IPI,Eu FPS and Eu GGPPS were also significantly lower than those of WT.This indicated that the expression of EuSRPP1 in E.ulmoides could feedback regulate the expression of upstream gum synthesis precursor-related genes.The gum content of the two transgenic callus was measured,and the results showed that: The gum content of OE callus was significantly higher than that of WT,and the gum content of RNAi-interfered callus was significantly lower than that of WT.The molecular weight analysis of gum showed that the Mw of gum was 1.29×106Da in WT callus,2.31×106Da in OE callus,and 0.36×106Da in RNAi-interfered callus.Analysis of the molecular weight distribution of gum in the three callus showed that there was no gum with a molecular weight greater than 300×104 Da in the RNAi-interfered callus,and the distribution of the molecular weight greater than 500×104 Da was OE callus>WT callus.It indicated that EuSRPP1 expression could increase the gum content and gum molecular weight.The gum particles in the phloem of transgenic and WT E.ulmoides leaves were observed by transmission electron microscopy.The electron density of the gum particles was deeper in OE plants than in WT.In the RNAiinterfered plants,the diameter of the gum particles was smaller than that in the WT plants.It indicates that EuSRPP1 may be involved in promoting the biosynthesis of gum.5.The effect of GA on Gum Content and Molecular WeightThe gum of E.ulmoides seedlings treated with 300mg/m L GA3 was extracted,and the gum content and molecular weight were determined.The results showed that the gum content in the treatment group was 1.40%,the control group was 0.72%,the gum Mw in the treatment group was 2.72×106Da,and 2.04×106Da in the control group,and the difference reached a very significant level(P<0.01).In the treatment group,the distribution of gum Mw less than 1×104 Da was greater in the control group than in the GA3 group.The distribution of gum Mw greater than 300×104 Da was smaller in the control group than in the treatment group,indicating that GA3 treatment can promote the increase of gum synthesis and molecular weight.The expression levels of EuSRPP1,EuSRPP2 and EuSRPP3 in E.ulmoides seedlings treated with GA3 were 1.34,1.27 and 2.12 times higher than those in control group,respectively.Only EuREF8 expression decreased,which was 65% of the control group.These results indicate that GA3 can regulate the expression of EuREF/SRPPs and cause the change of gum content and molecular weight.