Effects Of Different Light Quality On The Expression Of Rubisco And ATPase Genes In Chinese Fir Leaves

Cunninghamia lanceolate is the most important timber species in South China and one of the most important timber species in subtropical region of China.China is a big country with wood shortage.In order to meet the demand of wood market and improve the growth quality of Chinese fir plantation,it is necessary for forestry scientists to find a way to achieve rapid growth,high yield and high quality of Chinese fir-to improve the photosynthetic capacity of Chinese fir.All living things depend on photosynthesis.Photosynthesis is the most important chemical reaction on earth.The driving force of photosynthesis is light.Light has a great influence on the growth and development of plants.Plants can increase their volume and dry weight through photosynthesis.However,compared with light intensity and photoperiod,the influence of light quality on plant physiology and morphology is more complex.Light is an important regulator of plant growth and development.Light is not only the direct energy source of plant photosynthesis,but also the key information source of regulating plant growth and development.At present,there are few reports about C3 photosynthetic enzyme gene of Cunninghamia lanceolata,and the research on the function of C3 key enzyme gene of Cunninghamia lanceolata in regulating photosynthesis and stress resistance is even less.Based on the cloning and sequence analysis of C3 photosynthetic enzyme gene of Cunninghamia lanceolata in greenhouse culture and transcriptome data of Cunninghamia lanceolata under different light and mass ratio of red and blue light,this study analyzed the development of new leaves of Cunninghamia lanceolata under different light and mass ratio by means of cell biology,molecular biology,histochemistry,genetics and bioinformatics.The quantitative expression of stage photosynthetic enzyme gene and the analysis of the response of C3 photosynthetic enzyme gene of Chinese fir to different stress,so as to reveal the response of C3photosynthetic enzyme gene of Chinese fir to different light quality and under different stress conditions in theory,the research results can provide theoretical basis and technical support for improving the productivity of Chinese fir and gene engineering research of high light efficiency of crops.The main results are as follows:1.The conservative sequences of Rubisco large subunit（Cl LSMT）gene and ATP synthase complex subunit（Cl F1F0-ATP）gene of Chinese fir were obtained by analyzing the transcriptome data of Chinese fir the specific primers were designed.The leaves of Cunninghamia lanceolata seedlings were used as materials for RNA extraction and reverse transcription,and the first strand of synthetic c DNA was used as template.The target bands were cut and recovered,linked and transformed,sequenced,and the full-length sequences of Cl LSMT and Cl F1F0-ATP gene coding regions of Cunninghamia lanceolata were obtained.The total length of Cl LSMT gene coding region of Chinese fir is 1479 bp,which encodes 492 amino acid proteins.The other Cl F1F0-ATP gene coding region of Chinese fir is 618 bp,which encodes 205 amino acid proteins.2.The bioinformatics software was used to predict and analyze the amino acid sequences encoded by the cloned two Cunninghamia lanceolata photosynthesis gene coding frame sequences of Cunninghamia lanceolata Cl LSMT and Cl F1F0-ATP.The analysis results showed that The chemical formulas are C₂₄₇₂H₃₈₇₃N₆₄₉O₇₅₀S₁₄ and C₉₈₃H₁₅₆₁N₂₅₉O₂₉₁S₆,Cunninghamia lanceolata Cl LSMT is an unstable hydrophilic protein,and Cunninghamia lanceolata Cl F1F0-ATP is a stable hydrophobic protein.By using MEGA 6 to construct a molecular evolutionary tree,it was found that Cl LSMT of Chinese fir has high homology with Selaginella and Eucalyptus grandis,and Cl F1F0-ATP of Chinese fir has high homology with Selaginella,indicating a close relationship.3.The expression of Cl LSMT gene and Cl F1F0-ATP gene in Cunninghamia lanceolata seedlings treated with red and blue light for 1-6months was analyzed by q PCR.The results showed that Cl LSMT gene and Cl F1F0-ATP gene were expressed in untreated leaves and Cunninghamia lanceolata seedlings exposed to white light,red light and blue light for 1:1,1-6 months,respectively,and the relative expression of Cl LSMT gene and Cl F1F0-ATP gene showed different degrees of changes in Cunninghamia lanceolata seedlings exposed to red and blue light.The results showed that the relative expression of Cl LSMT gene and Cl F1F0-ATP gene to white light,red blue light ratio of 1:1,red light and blue light was higher than that of the control group in 1-5 months,and the total relative expression of white light was the highest,but the relative expression of Cl LSMT gene and Cl F1F0-ATP gene decreased significantly after 6 months.Cl LSMT and Cl F1F0-ATP genes of Cunninghamia lanceolata have the highest relative expression of white light,indicating that the response of Cl LSMT and Cl F1F0-ATP genes to white light is closely related to the regulation of Chinese fir wood growth and development.4.The two target genes Cl LSMT and Cl F1F0-ATP were fused with the vector PGWB605,and the over-expressed fluorescent vectors constructed were PGWB-605-35S-Cl LSMT-GFP and PGWB-605-35S-Cl F1F0-ATP-GFP,through Agrobacterium The constructed vector was introduced into tobacco epidermal cells and observed under a fluorescent microscope.Fluorescence observation showed that the target gene Cl LSMT subcellular was localized in the chloroplast,and the target gene Cl F1F0-ATP subcellular was localized in the nucleus.5.The target gene Cl LSMT was constructed into PGWB605-35S-GFP expression vector to combine the target gene with the vector,and the overexpression vector PGWB-605-35S-Cl SMT-GFP of Cl LSMT gene was constructed.The overexpression vector was introduced into Arabidopsis by Agrobacterium infection inflorescence method,and the heterologous overexpression Cl LSMT transgenic Arabidopsis positive plants were obtained through herbicide basta resistance screening and PCR detection.It was observed that the heterologous overexpression Cl LSMT transgenic plants of Cunninghamia lanceolata were dwarf phenotypes.6.The expression of Cl LSMT gene and Cl F1F0-ATP gene in Cunninghamia lanceolata seed buds at different stages after 6h,12h,24h and 48h of temperature stress,drought stress and salt stress were analyzed by fluorescence quantitative analysis.The results showed that the Cl LSMT gene and Cl F1F0-ATP genes all express the seed buds of Cunninghamia lanceolata after temperature stress,drought stress and salt stress,respectively,and the relative expression of temperature stress,drought stress and salt stress showed varying degrees of change,among which Cl LSMT gene and The relative expression of Cl F1F0-ATP gene was the highest when the low temperature,low concentration drought,and low salt treatment of Cunninghamia lanceolata seed buds were obtained after 12h,but Cl LSMT gene and Cl F1F0-ATP gene were obtained after high temperature,high concentration drought,and high salt treatment The relative expression levels of Cunninghamia lanceolata seed buds were significantly down-regulated,and were lower than those of the control group.Therefore,it can be speculated that the Cl LSMT gene and Cl F1F0-ATP gene of Cunninghamia lanceolata play a regulatory role in low tempe-rature stress,low concentration drought stress and low salt stress.