Construction And Functional Verification Of Two Vectors Containing Hps-phi Gene And Establishment Of Antirrhinum Majus' S Regeneration System

Formaldehyde is one of the main pollutants after the indoor renovation and has a impact on human health.Phytopurification as an eco-friendly means of clean indoor air quality,has been of concern.Studies have shown that using genetic engineering to enhance the HCHO metabolism of transgenic plant could increase the absorption rate of exogenous HCHO and detoxification capacity.HPS and PHI are the key enzyme in many methylotrophic bacteria RuMP of formalin-fixed.Our work:(1)To clone a gene RbcS3 c promoter and signal peptide sequence which can be located in chloroplast of tomato plant,and carry on the elemental analysis and function prediction;(2)Analyze the codon preference of Codiaceae codonopsis and analyze the target gene hps-phi(3)Construction of RbcS3 c gene signal peptide and hps-phi target gene into the transient expression vector to observe whether it was localized to chloroplast expression;(3)Construction of RbcS3 c gene two different lengths of the promoter to guide hps-phi(4)To establish the regeneration system of snapdragon,to obtain a large number of transgenic plants,to obtain a large number of transgenic plants,to obtain a large number of transgenic plants,Transformed plants.The RbcS3 c gene promoter and signal peptide sequences were amplified from the genome of the tomato genome respectively and named RbcS3 c PS1 and RbcS3cPS2,respectively.The sequence of the RbcS3cPS1 fragment was 738 bp.Sequence analysis and comparison with published sequence of RbcS3 c gene(X05986)showed that there were no alterations in the important functional regions such as TATA box and CAAT box.Fragment is the upstream regulatory sequence of tomato RbcS3 c gene.Plant CARE analysis showed that the promoter contained more conserved sequences and more conservative in the region of the initiation codon.The promoter sequence contained the CAAT and TATA boxes necessary for the promoter,I-box,box I,box II,ATC-motif and other 12 kinds of light response-related components.In addition,also contains a MeJA response element,an ethylene response element.The length of RbcS3cPS2 fragment was 1086 bp after elongation of the promoter of the gene.This promoter was found to have a number of responsive elements related to plant abiotic stress,such as ABRE,G-BOX,HSE,GARE-MOTIF,TATC-BOX,MBS,in a 300 bp sequence extending upstream from the RbcS3 c gene promoter 738 bp.Compared with X05986 in GenBank,the promoter and signal peptide sequences of two different RbcS3 c genes cloned in this study did not change base in TATA-box,CAAT-box,G-box and other important functional regions,There were 9 base differences in the region,including 8 base substitutions and 1 base deletion,all of which were present in the 738 bp sequence.And 98% and 98.7% homology with the published sequences of RbcS3 c gene(X05986),respectively.This indicated that the promoter sequence of RbcS3 c gene was different among different tomato cultivars.The results of the second part of the work showed that the ENc value of S.angustifolia was 61,which was too large,which indicated that the codon usage was not strong.In the chloroplast,the codon usage was relatively uniform,except for the stop codon,the smallest was 2.5,which was threonine ACG and serine AGC,the largest is phenylalanine UUU,the usage rate was about 53.5 per thousand,followed by Asparagine 37.0 per thousand.According to the calculation results of biological software,the RSCU values of 30 GCC,GAC,GAG,TTC and other C or G bases were all greater than 1,and the Frac value was also high,which was hps-phi Preferred codon.Based on the codon degeneracy,the hps-phi fusion gene sequence in methyltrophobacteria was optimized,and the similarity between the optimized sequence and original sequence was about 76%.Transient expression of tobacco,we can see in the signal peptide under the guidance of carrying the target gene successfully located in the chloroplast.This indicated that the signal peptide of our clone had the same amino acid composition as that reported in NCBI,but its signal peptide function did not change,which was consistent with the prediction of biological software.We cloned the signal peptide and NCBI signal peptide cleavage sites reported in different,but the function can play the role of signal peptide.The results showed that hps-phi and pC1301-RbcS3cP1 S :: hps-phi-Nos(small vector)and pC1301-RbcS3cP2 S :: hps-phi were respectively constructed by two different length promoters and signal peptide of tomato RbcS3 c gene.Hps-phi-Nos,and transgenic tobacco plants were obtained by Agrobacterium tumefaciens-mediated transformation.Quantitative PCR was used to detect the expression level of the target genes in transgenic tobacco plants containing large and small vectors,respectively.The results showed that the average expression level of the target gene in transgenic plants with small vector was more than 20 times higher than that in transgenic plants containing large vectors.Transgenic tobacco plants showed that transgenic plants with small veins were very strong,about 14-20 times higher than wild type,which was consistent with quantitative detection of target genes.Quantitative analysis of target genes and anti-formaldehyde test results showed that the elongation of the promoter did not increase the expression of exogenous gene,but the expression of the promoter was lower than that of the non-elongated promoter.We hypothesize that some of these functional elements may inhibit the efficiency of this promoter on the elongated promoter sequence,resulting in a decrease in the expression level of the target gene to almost no expression level.Finally,we established the regeneration system of the snapdragon,and the results showed that IBA could promote rooting of snapdragon better than NAA,and the rooting rate of IBA or NAA was higher than that of IBA or NAA.The rooting rate was 100%,and the average number of roots per root was significantly higher than that induced by NAA in the medium supplemented with IBA.When IBA was 1.5 mg / L,the average number of rooting reached 24,which was consistent with the suitable rooting concentration promoted by Japanese scholars.The transformation methods of the two vectors constructed in this study need further exploration and improvement.The study laid the foundation for the further cultivation of high formaldehyde decomposing transgenic plants.