Gene Clone And Genetic Transformation Of Cinnamoyl-CoA Reductase Gene1in Betula Platyphylla

Lignins, one of the most abundant biopolymers on earth, accumulate in the secondary cell wall of xylem cells. And it is very necessary for the growth and development of the vascular plants,which is a very important factor in the evolution of terrestrial plants. Cinnamoyl-CoA reductase, encoded by CCR gene, is the key enzyme of the lignin-specific pathway. The reaction that regulated by the CCR is rate-limiting reaction of lignin’s synthesis, which has a regulatory role of regulating the flow of carbon into the lignin biosynthesis pathway. It’s of great significance that use the genetic engineering techniques of regulating of lignin biosynthesis and nurture new varieties of trees, which the lignin content change.We, using Agrobacterium-mediated, get the birches those are overexpression of BpCCR1and down-regulation of it. Through measuring the seedling growth traits, chlorophyll fluorescence parameters, lignin content, etc. of transgenic plants, we want to know the reveal function of BpCCR1and the synthesis mechanism of birch lignin The results are as follows:The results of bioinformatics analysis show that the length of BpCCR1’s coding sequence region is1089bp, which encoding362amino acids. In BpCCRl the conservative CCR protein sequence KNWYCYGK changes into KLWYALGK, but it still has the function binding sites. BpCCR1has N-glycosylation site, Protein kinase C phosphorylation site, Casein kinase II phosphorylation site and N-myristoylation site. It also contains3-beta hydroxysteroid dehydrogenase site and NAD(P)-binding domain. Phylogenetic analysis show that BpCCR1and CCR of Populus trichocarpa have high similarity.We get58transformants that19of them are overexpression of BpCCR1and39of them are down-regulation ones. Real-time PCR analysis,that measures the BpCCRl expression in some transgenic ones, shows that the BpCCR1’s expression of either overexpression or down-regulation ones are significantly higher than the control. It shows that the exogenous gene is able to express at the RNA level, and the suppressor of CCR1in down-regulation ones may occur in the post-transcriptional level. In some transgenic plants, phenotypic changes are found, such as abnormal leaves, leaves turning into yellow, some of tissue culture not rooting, etc.Analysis the transgenic plants’growth trait, fluorescence parameters and lignin content at seedling stage. The results show as follow:No matter the overexpression or down-regulation ones are lower than the WT.Chlorophyll content of cr3, fcr8, crl7, crl, cr10is significantly higher than the WT, while cr6fcrl, fcr2, fcr15cr3h is significantly lower than the WT; Chlorophyll fluorescence parameters show:the transgenic ones’average Y (Ⅱ) is1.08times of the WT, and the ability of photoprotection (NPQ) is only63.3%of the WT. The photosynthetic activity (qP) of some transgenic birches, overexpression ones-cr3h cr6, cr8, cr9, cr11, crl3, crl4, cr19and down-regulation ones-fcr2, fcr3, fcr4, fcr8, fcrll fcr12, fcr13, fcr14, fcr15, fcrl6, fcr18, fcrl9, is enhanced comparing with WT,and the conversion efficiency of primary light energy (Fv/Fm) decrease. What’s more, the vast majority of potential maximum photosynthetic capacity increasing ones are overexpression. Analyze the BpCCR1overexpression birches’leaves lignin content and down-regulation ones’.The result shows that:the leaves lignin content of fcrl9、 cr3、cr18、crl is higher than the control, while fcr15, fcr2fcr11, fcrl significantly lower than the control. Take part of the selected lines’petioles to have histochemistry staining, and measure the xylem diameter ratio. The results of overexpression lines are higher than the down-regulation lines, which preliminary indicates BpCCRl having relations with the birch’s lignin synthesis.