【Objective】 The CRISPR/Cas9 ribonucleoprotein (RNP) system is an efficient gene editing technology that is simple and accurate and has been widely used in animal and plant gene editing research. Introducing the Cas9 protein and gRNA RNP complex into recipient cells by particle bombardment to obtain marker-free gene-edited plants provides an effective approach for the rapid plant mutant creation. In this study, the BpGLK1 gene of birch (Betula platyphylla × B. pendula) was used as the target gene for editing, and CRISPR/Cas9 RNP technology was used to edit the BpGLK1 gene without T-DNA insertion. 【Method】 A target site was designed for the first exon of birch BpGLK1. The target fragment of BpGLK1-E1 was amplified by PCR. The recombinant plasmid pAbAi-BpGLK1-E1 was constructed by enzyme digestion and ligation, whereafter the plasmid was linearized and reacted with Cas9 protein to detect the CRISPR/Cas9 RNP activity and gRNA accuracy in vitro. Zygotic birch embryo-induced calluses were used as receptor for BpGLK1 directed mutagenesis via particle bombardment. 【Result】 The in vitro CRISPR/Cas9 RNP activity assay indicated that the Cas9 protein and gRNA could effectively cleave specific BpGLK1 target sites. The mature birch embryos were bombarded with gold particles coated with Cas9 protein and gRNA (RNP) at a distance of 12 cm and 7 584.5 kPa after 25 d of culture without light. After differentiation and subculturing, a glkc mutant with yellow-green leaves was obtained. Sequencing results showed that the mutant had a homozygous deletion of 18 bp in BpGLK1 gene. 【Conclusion】 A precise birch BpGLK1 mutation without T-DNA insertion was achieved using CRISPR/Cas9 editing technology.
【Objective】 Betula pendula ‘Dalecarlica’ is an European white birch species found in the northeast of China. After entering the reproductive growth stage, the pistil development is normal and can pollinate with other European white birch or white birch species to produce offspring; however, the stamens are aborted and cannot produce pollen. Because of the beautiful tree posture, clean white bark, and notable leaf edges with cracks, B. pendula ‘Dalecarlica’ has high ornamental value and is gradually being used in street greening to enrich plant species in urban landscaping. However, with improvements in the living standards of urban residents, diverse and colorful forms of greenery have become favored. Therefore, changing the leaf color of these tree species will enhance the application prospects in landscape architecture. The golden2-like (GLK) gene belongs to the GARP transcription factor superfamily of Myb transcription factors. GLK transcription factors are involved in regulating plant chloroplast development and hormone signaling, and play important roles in plant disease resistance, nutrient synthesis and leaf senescence, indicating that GLK transcription is the key gene for reforming plant leaf color. GLK transcription factors mainly regulate light capture and chlorophyll biosynthesis-related gene expression during plant chloroplast development as well as fruit skin color, thereby affecting chlorophyll synthesis and chloroplast development. Therefore, to cultivate colorful tree species for urban landscaping and scenery preferences, a molecular-based breeding method was used to create the golden leaf B. pendula ‘Dalecarlica’. 【Method】 B. pendula ‘Dalecarlica’ stem segments were used. The previously constructed 35S::BpGLK1-RNAi vector was introduced into the B. pendula ‘Dalecarlica’ genome via the agrobacterium method. DNA and RNA were extracted from wild type (WT) and resistant transgenic lines and were measured. The BpGLK1 interfering expression lines of B. pendula ‘Dalecarlica’ were expanded and transplanted. The leaf color, photosynthetic pigments, photosynthetic parameters, plant height growth, and gene expression characteristics of the BpGLK1 interfering expression lines of B. pendula ‘Dalecarlica’ were determined. 【Result】 Resistant calluses were obtained after co-cultivation on a selective medium for 30 days. The calluses were differentiated and cultured to obtain resistant adventitious buds, which were inoculated into a rooting medium. Adventitious roots were grown and transgenic B. pendula ‘Dalecarlica’ mutants were obtained. Eight herbicide-resistant regenerated transformation lines (RE1-RE8) were obtained following transplantation into the seedling trays. Using the total DNA of eight transgenic B. pendula ‘Dalecarlica’ leaves as templates and the pFGC5941-GLK1 plasmid as positive control, PCR amplification was performed on the forward target sequence and complementary sequence of the BpGLK1 gene, indicating that the interfering fragment of BpGLK1 gene and Bar gene have been integrated into the genome of the transgenic B. pendula ‘Dalecarlica’. The relative BpGLK1 expression in these transgenic lines was downregulated, with RE1-RE4 being the largest, whereas the RE6-RE8 decrease was relatively small. Among the eight transgenic lines, RE1-RE5 were yellow-leaf lines and RE6-RE8 were green-leaf lines. The leaf color parameters and relative chlorophyll content of 1-year-old transgenic lines were investigated in the transplanted fields, and the results indicated that compared with the WT and green-leaf lines RE6-RE8, the yellow-leaved strains RE1-RE5 showed significantly higher leaf color parameters L* and b* and lower chlorophyll a and b contents (P<0.05); however, the ratio of chlorophyll a to b exhibited an increasing trend. This result showed that low BpGLK1 expression significantly improved the leaf brightness of the transgenic lines. The Fv/Fm value of the transgenic strains was higher or significantly higher than that of the WT strain, with the yellow line RE2 having the highest Fv/Fm value, which was 1.39% higher than that of the WT strain. However, there was no significant difference in net photosynthetic rate (Pn) between transgenic lines and WT plants. Plant heights of the transgenic lines in the year of transplantation were surveyed. Plant height analysis showed that the four transgenic lines RE1, RE2, RE4 and RE6 were significantly higher than those in the WT, and there was no significant difference between the three transgenic lines RE3, RE5, RE7 and the WT. Only the height of line RE8 was significantly lower than that of WT. Based on RNA-seq, four genes, BpCOL, BpLCHⅡ, BpPDS, were significantly down-regulated. qRT-PCR analysis of these four genes indicated that they were significantly downregulated in RE1-RE3 expression. 【Conclusion】 The introduced GLK1 interfering target sequence reduced the BpGLK1 expression in transgenic B. pendula ‘Dalecarlica’. The obtained yellow-leaved B. pendula ‘Dalecarlica’ lines have potential applications in landscaping.
【Objective】The formation of wood, one of the most important raw materials for pulp and energy, depends on a complex and precise transcriptional regulation process in which transcription factors figure prominently. In Populus trichocarpa, PtrLBD12 (lateral organ boundaries domain 12) is a transcription factor lying downstream of PtrbHLH186, a key regulator of wood formation. Hence, PtrLBD12 was studied in depth here to investigate its function in tree growth and wood formation.【Method】 To determine the role of the PtrLBD12 transcription factor in the growth and wood formation of poplar trees, we analyzed its expression characteristics, generated PtrLBD12 overexpressing plants of P. trichocarpa, and assessed the growth and wood formation traits of transgenic plants. (1) Xylem, phloem, and terminal bud and leaf samples of wild-type P. trichocarpa plants, cultivated in a greenhouse, were collected to extract their respective RNA. Next, transcriptome sequencing was done to analyze the patterns of differentially expressed genes in those distinct tissues, as well as their expression levels of PtrLBD12. (2) To clarify the expression pattern of the PtrLBD12 protein, its subcellular localization was investigated by using the transient transformation system of stem-differentiating xylem protoplasts of P. trichocarpa. (3) To create PtrLBD12-overexpressing plants, the Agrobacterium tumefaciens-mediated transformation system of P. trichocarpa was used, with transgenic plants identified at both the DNA and RNA level. (4) Plant stem height, ground (basal) stem diameter, number of stem nodes, and length of the 8th stem node of transgenic and wild-type plants were measured at 30, 60 and 90 days after planting. (5) The 2nd, 4th, 6th and 8th stem segments of overexpressing and wild-type greenhouse plants cultivated for 4 months were paraffin-sectioned. Their stem characteristics were then respectively observed via Safranin O/Fast Green and Toluidine Blue staining. LAS X V2.0 software was used to calculate the number of vessel and fiber cells, as well as average lumen area of vessel. (6) The relative expression levels of 22 monolignol biosynthetic pathway genes in the PtrLBD12-overexpressing plants were determined by using quantitative PCR and applying the 2△△Ct calculation.【Result】 (1) Transcriptome analysis of the xylem, phloem, and terminal bud and leaf tissues of P. trichocarpa revealed a higher expression level of PtrLBD12 in both the xylem and phloem. (2) Subcellular localization showed that PtrLBD12 was expressed in the nucleus, where transcription factors in general were found. (3) Three PtrLBD12 overexpressing transgenic lines of P. trichocarpa were obtained, whose relative expression levels were 40.91, 79.51 and 102.19. (4) Overexpressing PtrLBD12 adversely affected the normal growth and development of P. trichocarpa: plant height, ground stem diameter, number of stem nodes, and length of the 8th stem node all decreased significantly in transgenic plants vis-à-vis the wild type. (5) However, overexpression of PtrLBD12 did lead to significantly more vessel and fiber cells per unit of area in the stems of transgenic plants, but these vessels had smaller lumen area. Furthermore, the augmented expression of PtrLBD12 enhanced lignification of the plant stem. (6) Finally, PtrLBD12’s overexpression bolstered the expression levels of multiple genes in the monolignol biosynthetic pathway, namely PtrPAL1, PtrC4H1, PtrC4H2, PtrHCT6, PtrCSE, PtrCSE2, PtrCCoAOMT1, PtrCCoAOMT2, PtrCCoAOMT3, PtrCCR2 and PtrCAld5H1.【Conclusion】As the gene downstream of PtrbHLH186, a key regulator of wood formation in poplar, the LBD12 transcription factor is able to govern the expression of monolignol biosynthetic genes, change the mode of lignin deposition, alter the morphology of xylem cells, and thereby affect plant growth and development.
【Objective】 As a variety of Betula pendula, B. pendula ‘Dalecarlica’ exhibits lobed shaped split leaves, which have significant application value in horticultural ornamental, breeding, and plant developmental biology research. Understanding the mechanisms of leaf shape variation in B. pendula ‘Dalecarlica’ has academic value in the field of genetics, and identifying the genes corresponding to this trait is important for further research on this topic. This study aimed to preliminarily locate and identify the genes regulating the leaf shape of B. pendula ‘Dalecarlica’, and screen and annotate potential candidate genes, thus laying the groundwork for subsequent validation experiments and mechanistic research. 【Method】 A total of 24 offspring individuals from a single B. pendula ‘Dalecarlica’ maternal tree were selected as experimental materials. This half-sibling family included the offspring of individuals with different leaf types, including normal, week-lobed, lobed and strong-lobed leaves. Five leaves were collected from each plant and scanned. Leaf shape irregularity degree (Dli) was measured as Dli=4πS/C2, and which for each individual was calculated. The significance of Dli differences among individuals was analyzed. RNA-Seq data were obtained for each individual, and SNP/InDel information was called. Using the degree of leaf shape irregularity as a phenotype, an association analysis was performed using a generalized linear model (GLM). The association area were delineated based on SNPs/InDels with significant association signals. Furthermore, gene expression levels of the entire genome in the experimental materials were analyzed based on RNA-Seq data. The significance of gene transcription level differences was calculated, and differentially expressed genes (DEGs) within the association area were identified. SNPs/InDels with association P values smaller than the Bonferroni threshold were considered associated SNPs/InDels. Genes affected by associated SNPs/InDels and differentially expressed genes were considered candidate genes. Gene functional annotations were performed using online databases. 【Result】 The degree of leaf irregularity showed extremely significant differences among different individuals within the experimental population (P < 0.01). The coefficient of variation for leaf shape irregularity in the experimental population was 60.51%, which met the requirements for association analysis. A total of 331 Gb of RNA-Seq data was generated, of which 3 303 530 SNPs/InDels were detected. After filtering, 1 367 359 high-quality polymorphic SNPs/InDels were retained. Using the degree of leaf shape irregularity as the phenotype, association analysis was performed on these SNPs/InDels using a GLM. Association analysis identified 66 SNPs/InDels with significant P values below the Bonferroni threshold. By closely linking these 61 SNPs/InDels, the association area containing the leaf shape-regulating genes of B. pendula ‘Dalecarlica’ was defined, spanning a length of 9.18 MbP and containing 400 genes. Within this area, 61 associated SNPs/InDels were found to affect the coding of 17 known genes. Analysis of gene expression levels revealed that of the 400 genes within the association area, 25 genes showed extremely significant differences (P < 0.01) between B. pendula, B. pendula ‘Dalecarlica’. Functional candidate gene annotation identified three transcription factors, three proteins involved in cell composition, six proteins involved in protein translation processing, nine protein kinases involved in signal transduction, five enzymes involved in biochemical reactions, three proteins involved in substance transport, one protein involved in the post-transcriptional modification of RNA, one DNA endonuclease/exonuclease, and one protein involved in stomatal regulation. Additionally, eight genes could not be found in the existing online databases, and their types and functions remain unknown. Finally, three transcription factors and one auxin carrier protein were considered as possible key genes regulating the leaf shape of B. pendula ‘Dalecarlica’. 【Conclusion】 The gene locus responsible for regulating the splitting trait of B. pendula ‘Dalecarlica’ leaves was mapped to the interval of chromosome 7:7466344-16651477, with a total of 40 candidate genes located within this association area. Based on the gene annotation results, future studies should focus on the three identified transcription factors and one auxin carrier protein.
