Cloning And Genetic Transformation Of PaGLK Gene In Populus Alba × P.berolinensis

Golden2-like(GLK)transcription factor is a kind of transcription factors widely present in plants,and belong to the GARP transcription factor family.GLK mainly regulates chloroplast development and leaf color of plants,and also participates in plant biological stress and abiotic stress,plant aging and hormone signal transduction.Studies have shown that the loss or inhibition of the expression of BpGLK gene can lead to gold leaf birch.Populus alba × P.berolinensis is one of the main broad-leaved tree species in Northeast China.Its trunk is straight,its bark is gray and green,and its beautiful posture is widely used in urban roads,parks,courtyards,and residential areas.The creation of golden leaf Populus alba× P.berolinensis will enhance the role and application prospect of this tree species in landscaping.Therefore,the experiment used wild type Populus alba × P.berolinensis as material,cloned the PaGLK gene,and constructed a plant expression vector.Agrobacterium-mediated leaf disc method was used to transform silver middle poplar.The leaf color,growth characteristics and photosynthetic characteristics of transgenic silver middle poplar were analyzed.Analysis and development of PaGLK gene function.The findings are as follows:(1)The PaGLK gene consists of 1269bp,the start codon is ATG,and the stop codon is TGA,encoding a total of 422 amino acids.The molecular formula is C3769H6271N1269O1549S316,and the relative molecular mass is 104278.78.Phylogenetic analysis shows that the amino acid sequence of PaGLK has the highest homology with the amino acid sequence of PtrGLK1.PaGLK was expressed in the root,stem,and leaf tissues of Populus alba × P.berolinensis,and the gene expression was highest in the leaves.(2)The overexpression and inhibition expression vector of Populus alba × P.berolinensis was constructed by using enzyme digestion and ligation technology,and the genetic transformation of Populus alba × P.berolinensis was carried out by Agrobacterium-mediated leaf disc method.The obtained transgenic plants were detected by PCR and fluorescent quantitative PCR technology.The results showed that three PaGLK overexpression lines and three PaGLK-RNAi suppressed expression lines were successfully obtained.(3)Investigation of the plant height of the transgenic Populus alba × P.berolinensis showed that the seedling height of PaGLK over-expressing lines was significantly lower than that of WT lines,while PaGLK-RNAi inhibiting expression lines were not significantly different from WT lines.The comparison of leaf color and photosynthetic pigment showed that the leaves of PaGLK overexpressing lines were darker in green than WT lines,and the leaf color parameters L*and b*were lower than those of WT lines,and the chlorophyll a(Chla),chlorophyll b(Chlb),total chlorophyll content(Chl)and carotenoid(Car)indicators were significantly higher than the WT line,the net photosynthetic rate was lower than the WT line.The leaves of PaGLK-RNAi-ihibited expression lines showed yellow-green,the leaf color parameters L*,b*were significantly higher than those of WT lines,and chlorophyll a(Chla),chlorophyll b(Chlb),total chlorophyll content(Chl),and carotenoids(Car)and other indicators were significantly lower than the WT line,and the net photosynthetic rate was higher than the WT line.(4)Real-time PCR analysis showed that,compared with WT strains,the genes involved in chlorophyll synthesis(PaCHLH,PaCHLD1,PaCHLD2)and photosynthesis-related genes(PaLhcb1,PaLhcb2,PaLhcb4,PaLhca3)were up-regulated in the leaves of PaGLK overexpressing lines and inhibited by PaGLK-RNAi The expression lines were all down-regulated.In summary,the low expression of PaGLK gene in Populus alba × P.berolinensis can down-regulate key genes for chlorophyll synthesis.Chlorophyll a(Chla),chlorophyll b(Chlb),total chlorophyll content(Chl),and carotenoid(Car)photosynthetic pigments are significantly lower than WT and The over-expressing strains have yellow-green leaf color,and the PaGLK-RNAi strain of Populus alba × P.berolinensis has potential application value in urban landscaping.The results of this study prove that the GLK gene is a candidate gene for the design and breeding of leaf color molecules in forest trees.