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BioMed Research International
Volume 2016, Article ID 4275904, 8 pages
Research Article

Functional Characterization of 9-/13-LOXs in Rice and Silencing Their Expressions to Improve Grain Qualities

1Department of Horticulture, Washington State University, Pullman, WA 99164, USA
2State Key Lab of Rice Biology, International Atomic Energy Agency Collaborating Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
3Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China
4Agricultural Extension Extending Stations, Shaoxing & Zhuji Agricultural Bureau, Shaoxing, Zhejiang 312000, China
5Institute for Wheat Research, Henan Academy of Agricultural Sciences, Zhengzhou, Henan 450002, China

Received 5 December 2015; Accepted 24 April 2016

Academic Editor: Sudhir Sopory

Copyright © 2016 Moytri RoyChowdhury et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Figure S1: Amplification of lipoxygenase fragments in rice.

Figure S2: Structure of the pANDA vector and procedure for RNAi vector construction. (A) The sequence of a gene used for inverted repeats (IR) is amplified by PCR using primers. The CACC sequence is added to 5′ end of forward primer for providing the correct direction to the PCR product. (B) The overhang in the the pENTR/D-TOPO cloning vector (GTGG) invades the 5′ end of the PCR product, anneals to the added bases, and stabilizes the PCR product in the correct orientation. The entry clone containing the PCR product is completed. (C) The final RNAi vector was produced by an LR clonase reaction between the entry clone and pANDA. PANDA vectors contain the promoter and IR regions. The LR clonase reaction site attR is located at both sides of the gus linker in the antisense and sense orientations. The pANDA vector is the binary vector for Agrobacterium mediated transformation and has kanamycin and hygromycin resistance marker genes.

Figure S3: Transformation into pENTR/D TOPO and verification of inserted DNA fragments by PCR and restriction enzymes. The transformation is checked using gene specific primers for (A) CDS-r9-LOX1, CDS-L-2, and CDS-RCI-1. The enzymes digested the inserted DNA (B) CDS-r9-LOX1, (C) CDS-L-2, (D) CDS-RCI-1. In this case NotI and AscI are the special sites.

Figure S4: Transformation of r9-LOX1 insert in pANDA: Complete transformation of r9-LOX1 insert in pANDA vector was verified by PCR and restriction digestion using Kpn1 and Sac1.

Figure S5: (A) Regeneration control; (B) GUS positive control (N. xanthae); (C) construct EHA105+pWHNG; (D) EHA105+pANDA r9-LOX1; (E) GUS possitive control (N. xanthae) roots.

Figure S6: Transformation of insert r9-LOX1 in Agrobacterium (EHA105 strain). (A) PCR was used to confirm r9-LOX1, Gus linker (GL) and neomycin phosphotransferase (NPTII) in pANDA (B) PCR was used Transformation of rice using pWNHG vector, Rice (c.v. Taipei-309 and c.v. LaGrue) was transformed with a construct (pWNHG) that carried genes coding for nptII, hygromycin phosphotransferase (Hyg), and p-glucuronidase (GUS). This served as the positive control (C) The regenerated plant.

Table S1: Primer sequences of q-PCR for three LOX genes.

  1. Supplementary Material