Study On Gene Functions In Response To A Decrease Of CO₂ Concentration In Cucumber

Solar greenhouse is the main type of greenhouses for counter-season production of vegetables in northern China.Due to the tightness of solar greenhouse structure,the carbon dioxide?CO₂?in greenhouse consumed by vegetables after sunrise for photosynthesis is not supplemented,and CO₂ in greenhouse is chronically deficient.Long-term growth of greenhouse vegetables in low-concentration CO₂ environment can not effective utilize the light and heat resources in greenhouse,resulting in low photosynthetic efficiency,low yield and low quality.In order to explore the feasibility of using genetic engineering methods to cultivate high-photosynthetic cucumber varieties,in this study,we cloned the inorganic carbon transporter genes: Cs?CA1 and Cs?CA4 and the key enzyme genes of photosynthetic carbon assimilation: Cs FBA and Cs SBP from cucumber inbred line 3407.Simultaneously we synthesized five key enzyme genes of E.coli glycolic acid metabolic pathway.We constructed single gene and multigene overexpression vectors and then introduced the vectors into cucumber using Agrobacterium-mediated method and obtained Cs?CA1,Cs?CA4,Cs SBP,Cs FBA,14 SF,GT and DEF transgenic plants.By measuring the morphological indices,photosynthetic indices and enzyme activities of different transgenic plants in atmospheric environment and greenhouse environment,we analyzed the effects of different genes on cucumber response to low-concentration CO₂,aiming to provide a theoretical basis for high photosynthetic efficiency breeding of greenhouse cucumber.The main results obtained are as follows:1.Bioinformatics analysis showed the full-length open reading frame?ORF?of Cs?CA1 is 993 bp,and the amino acid sequence identity with other plants ranges from 60.4% to 92.3%,the ?-helix,extended chain,?-turn and random curl of the secondary structure of Cs?CA1 account for 26.89%,20.54%,9.77% and 42.90%,the protein tertiary structure formed a more symmetrical “mouth-shaped” structure;The ORF of Cs?CA4 is 777 bp,and the amino acid sequence identity with other plants ranges from 60.4% to 92.3%,the ?-helix,extended chain,?-turn and random curl of the secondary structure account for 38.37%,16.67%,7.75% and 37.21%,the protein tertiary structure formed a more symmetrical “Mouth-Shaped” structure;The ORF of Cs SBP is 1167 bp,and the amino acid sequence identity with other plants ranges from 77.5-94.46%,the ?-helices,extended chains,?-turns and random coils of the protein secondary structure accounted for 35.57%,22.68%,10.05% and 31.70%,the protein tertiary structure formed a more symmetrical “X-Shaped” structure;The ORF of Cs FBA is 1194 bp,and the amino acid sequence identity with other plants ranges from 74.88-90.19%,the ?-helices,extended chains,?-turns and random coils of the protein secondary structure accounted for for 43.15%,17.77%,11.42% and 27.66% respectively.,the protein tertiary structure forms a more symmetrical “X-Shaped” structure.2.q RT-PCR analysis of Cs?CA1,Cs?CA4,Cs SBP and Cs FBA genes in different tissues of cucumber showed that Cs?CA1,Cs?CA4,Cs SBP and Cs FBA were expressed in the growing point,leaves and female flowers,and the tissue of the highest expression of these genes is the leaf.3.The results of expression analysis of the co-expressing transgenic plant showed that when using “2A” as a linker to construct a multigene co-expression vector for genetic transformation,the expression levels of different genes in the vector were different,the expression of different genes is related to the connection order,the closer to the 35 S promoter,the higher the gene expression.4.The analysis of protein content and Ru BP carboxylase activity of the corresponding genes in the transgenic plants overexpression of Cs?CA1,Cs?CA4,Cs SBP,Cs FBA could increase the protein content of the corresponding genes,the content of Cs?CA,Cs SBP and Cs FBA.Overexpression Cs?CA1,Cs?CA4,Cs SBP,Cs FBA,14 SF,GT and DEF could increase the Ru BP carboxylase activity of transgenic plants in atmospheric environment?CO₂ concentration is 400 ?mol·mol-1?,and the highest Ru BP activity was observed in 14 SF transgenic plants.The analysis of protein content and Ru BP carboxylase activity of showed that overexpression of Cs?CA1,Cs?CA4,Cs SBP and Cs FBA could increase Cs?CA1,Cs?CA4,Cs SBP,Cs FBA,14 SF,GT and Overexpression of DEF increased the Ru BP carboxylase activity of transgenic plants in atmospheric environment,and the activity of Ru BP carboxylase was highest in 14 SF transgenic plants.5.The analysis of photosynthetic,morphological and biomass indexes of different transgenic plants in a low-concentration CO₂ greenhouse environment?CO₂ concentration ranged from 100 to 300 ?mol·mol-1?revealed that the photosynthetic rate,plant height,stem diameter,and the dry weights of aboveground and underground of Cs?CA1,Cs?CA4,GT,DEF,and 14 SF transgenic plants were significant higher than control,indicating that the cucumber inorganic carbon transporter genes Cs?CA1,Cs?CA4 and photorespiration metabolism genes Glc DH,GCL and TSR have positive effects on cucumber responding to low concentrations of CO₂;Although the overexpression of Cs SBP and Cs FBA can increase the activity of Ru BP carboxylase in atmosphere,the photosynthetic rate and biomass did not increase significantly in greenhouse environment,indicating that activities of Cs SBP and Cs FBA were limited in low concentrations of CO₂;The photosynthetic,morphological and growth indexes of 14 SF transgenic plants were significantly higher than those of Cs?CA1,Cs?CA4,Cs SBP and Cs FBA,indicating that there is a genes cumulative effect for expression of Cs?CA1,Cs?CA4,Cs SBP and Cs FBA simultaneously in 14 SF ransgenic plants.The expression of GT and DEF in cucumber could significantly increase the photosynthetic and biomass of cucumber under low CO₂ concentration,which indicated that it was an effective way to improve the photosynthetic efficiency of cucumber in low CO₂ environment through the introduction of exogenous photorespiration branch.