1. Volume 10, Issue 7, Pages 918-929 (July 2017)
Development of “Purple Endosperm Rice” by Engineering Anthocyanin Biosynthesis in the Endosperm with a High-Efficiency Transgene Stacking System 
Qinlong Zhu, Suize Yu, Dongchang Zeng, Hongmei Liu, Huicong Wang, Zhongfang Yang, Xianrong Xie, Rongxin Shen, Jiantao Tan, Heying Li, Xiucai Zhao, Qunyu Zhang, Yuanling Chen, Jingxing Guo, Letian Chen, Yao-Guang Liu 
Molecular Plant 
Volume 10, Issue 7, Pages (July 2017)
DOI: /j.molp
Copyright © 2017 The Author Terms and Conditions

2. Figure 1 The TransGene Stacking II Vector System.
(A) Structural features and the key sequence of the basic acceptor vector. The vector backbone contains a bacterial kanamycin-resistance gene (Kanr), a P1 plasmid replicon for replication in E. coli, a P1 lytic replicon for increasing the plasmid copy number induced by IPTG, and a pVS1 replicon for replication in A. tumefaciens. The loxP and loxP1R sites are located in the T-DNA region (from right border [RB] to left border [LB]) for recombination with a target-gene donor plasmid and deletion of the donor backbone sequence. PI-PspI, I-SceI, and I-Ppo are homing endonuclease sites. A Gateway recipient region containing the SacB gene (which causes lethality in E. coli in the presence of sucrose) flanked by the attP1 and attP2 sites for recombining the excisable marker cassette into the vector after assembling all target genes.
(B) Structural features of the basic target-gene donor vectors. The wild-type and mutant loxP sites, the homing endonuclease sites, and MCSs are located in different arrangements (MCSs are in different orientations) in the donor vectors. The replicon is from pBR322, and Cmr and Ampr denote the chloramphenicol- and ampicillin-resistance genes, respectively. LacZ is used to indicate blue bacterial colonies of the recombinant plasmid with unremoved donor vector backbone.
(C) Structural features of the donor vectors for insertion and excision of selectable markers. The plant selectable marker gene/marker-excision cassette between two loxP sites includes a marker gene (HPT, NPTII, or Bar, for pYLMF-H, pYLMF-N, or pYLMF-B) and the Cre gene (with an intron from the catalase gene) driven by a pollen-specific promoter (Pv4); these components are located between the Gateway attB1/attB2 sites. Pv4 from Villin4 (Os04g ) is expressed specifically in pollen. This cassette can be recombined into the pYLTAC380GW-based constructs by Gateway BP reaction to form a structure that is competent for in planta excision to produce marker-free plants.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

3. Figure 2 Schematic Representation of TGSII for Transgene Stacking.
(A) Multiple assembly cycles for multi-gene stacking. One or more target genes (A–F) are cloned into the donor vectors (Supplemental Figures 6 and 7). For Round I of gene assembly, the acceptor vector and pYL322d1-A (with gene A) are co-transferred into an E. coli strain (NS3529) expressing Cre. The in vivo reversible recombination (i) between the wild-type loxP sites produces an intermediate recombinant plasmid, then an irreversible recombination (ii) between the compatible mutant loxP sites loxP1L (1L) and loxP1R (1R) excises the donor vector backbone (containing two I-SceI sites, a loxP site, and the bacterial selection marker gene), generating the target plasmid Acceptor-A. The irreversible recombination generates an inactive recombinant site 1LR (and 2LR in the even-numbered assembly cycles). After the gene assembly, a digestion with homing endonuclease I-SceI (with PI-SceI for even-numbered cycles) is performed to remove the original acceptor, donor, and intermediate plasmid (which contain one or two of the homing endonuclease sites), recovering the uncut target plasmid that lacks the corresponding homing endonuclease site. For Round II gene assembly, Acceptor-A and pYL322d2-C/B (with genes B and C in this example) are co-transferred into NS3529. The recombination events produce the recombination plasmid Acceptor-C/B/A. A third (and other odd-numbered, with pYL322d1-based vectors) and fourth (and other even-numbered, with pYL322d2-based vectors) round of gene assembly can be conducted to stack additional genes like Round I and Round II, respectively.
(B) When all target genes are assembled, the selectable marker/marker-excision cassette is recombined into the acceptor vector by Gateway BP reaction to generate the final binary construct. In transgenic plants, Cre expression driven by Pv4 (or another suitable promoter) mediates loxP recombination to excise the cassette.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

4. Figure 3
Assembly of the Target Genes in the Binary Transformation-Competent Vector for Engineering Anthocyanin Production.
(A) The structure of the construct containing the eight target genes and the selectable marker/marker-excision cassette. N, NotI site.
(B) Not I-restriction analysis of the acceptor constructs from different rounds of gene assembly with increasing numbers of target genes (1G to 10G). Arrows indicate the newly added gene(s) in each gene assembly cycle. For structural stability testing of pYLTAC380MF-10G in the A. tumefaciens strain EHA105, the plasmid was introduced into EHA105, isolated from colonies, transferred back into E. coli, and isolated for restriction analysis.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

5. Figure 4 Anthocyanin Production in the Rice Endosperm.
(A) Unpolished and polished grains from the black-grained rice (BGR1), ZH11, and the “Purple Endosperm Rice” (PER) or “Zijingmi” (ZJM) lines (T2) of transgenic ZH11. In PER-Z#2 with lighter purple-colored grains, three transgenes (SsCHS, SsCHI, and SsF3′H) were absent (Supplemental Figures 9 and 10).
(B) Cross-sections of unpolished rice grains of a native black-grained rice cultivar (BGR1), ZH11, and a PER/ZJM line (T2).
(C) Frozen sections of developing rice seeds (15 days after pollination), showing specific accumulation of anthocyanins (light brown color) in the endosperm cells of PER.
(D) HPLC analysis with anthocyanin standards detected two kinds of anthocyanins, C3G (cyanidin 3-O-glucoside) and its methyl derivative P3G (peonidin 3-O-glucoside) in polished PER grains and unpolished BGR1 grains.
(E) Quantitative analysis (n = 3) of the C3G and P3G contents in grains of the PER lines and the control materials. gdw, grams of dry weight; nd, not detectable.
(F) TEAC analysis of the unpolished and polished rice grains of the PER lines and ZH11, showing different levels of antioxidant activity. **p < 0.01 and ***p < 0.001, significances between the PER and ZH11 line, respectively (n = 3), with Student's t-test.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

6. Figure 5 Expression of the Transgenes and Endogenous Genes.
Semi-quantitative RT–PCR (22 cycles for all reactions) showed the expression of the transgenes (red) and the activated (or enhanced) endogenous genes (blue) for the anthocyanin biosynthesis pathway and its upstream phenylalanine pathway in developing endosperm (7 and 14 days after pollination) of ZH11 and PER-Z#3 (a homozygous T3 line). Four functional endogenous structural genes, OsF3H, OsANS, OsF3′5′H, and OsLAR, were not activated by the regulatory genes in both ZH11 and PER-Z#3.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

7. Figure 6
Engineering of the Anthocyanin Biosynthesis Pathway in the Rice Endosperm.
The genetically engineered anthocyanin biosynthesis pathway involves the metabolic enzymes expressed from the transgenes (red), the rebuilt MBW transcription factor complex, and some functional endogenous genes (blue) activated (or enhanced) by the MBW complex. The pathway synthesizes cyanidin as the major anthocyanidin, which is further decorated by one and two steps to produce the anthocyanin products C3G and P3G, respectively. The minor proanthocyanidin branch is not shown.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions