High Expression of Cry1Ac Protein in Cotton (Gossypium hirsutum) by Combining Independent Transgenic Events that Target the Protein to Cytoplasm and Plastids

Abstract

Transgenic cotton was developed using two constructs containing a truncated and codon-modified cry1Ac gene (1,848 bp), which was originally characterized from Bacillus thuringiensis subspecies kurstaki strain HD73 that encodes a toxin highly effective against many lepidopteran pests. In Construct I, the cry1Ac gene was cloned under FMVde, a strong constitutively expressing promoter, to express the encoded protein in the cytoplasm. In Construct II, the encoded protein was directed to the plastids using a transit peptide taken from the cotton rbcSIb gene. Genetic transformation experiments with Construct I resulted in a single copy insertion event in which the Cry1Ac protein expression level was 2-2.5 times greater than in the Bacillus thuringiensis cotton event Mon 531, which is currently used in varieties and hybrids grown extensively in India and elsewhere. Another high expression event was selected from transgenics developed with Construct II. The Cry protein expression resulting from this event was observed only in the green plant parts. No transgenic protein expression was observed in the non-green parts, including roots, seeds and non-green floral tissues. Thus, leucoplasts may lack the mechanism to allow entry of a protein tagged with the transit peptide from a protein that is only synthesized in tissues containing mature plastids. Combining the two events through sexual crossing led to near additive levels of the toxin at 4-5 times the level currently used in the field. The two high expression events and their combination will allow for effective resistance management against lepidopteran insect pests, particularly Helicoverpa armigera, using a high dosage strategy.

Fig 1. Construct I and its state of integration in the genome of event Tg2E-13.

(A) Schematic representation of the T-DNA region of Construct I; (B) Integrated pattern of T-DNA cassettes in the genome in the transgenic event Tg2E-13 as discerned by genome walking experiments. Only two of the transgene cassettes contained in the construct–namely nptII gene and cry1Ac gene have integrated in the plant genome.

Fig 2. Construct II and its state of integration in the genome of event TM-2.

(A) Schematic representation of the T-DNA region of Construct II; (B) Pattern of integration of the T-DNA region in the transgenic event TM-2 as discerned by genome walking experiments. Two copies of the transgene cassettes (T-DNA region) are present in an inverted manner.

Fig 3. Levels of the Cry1Ac protein in μg/g fresh weight (Mean±SD) in different tissues of the two transgenic events of cotton developed in this study, Tg2E-13 and TM-2, and their comparison with two commercial lines—BioCot-1 containing cry1Ac gene of the event Mon 531 in a hemizygous condition and BioCot-2 containing the cry gene in a homozygous condition; data is from the growing season of 2014.

(A) Cry1Ac expression in the leaf tissues of 20, 60, 90 and 120 day old plants; (B) Cry1Ac expression in the flower bud and bract tissues of 70 day old plants; (C) Cry1Ac expression in the root and cotyledonary tissues of seedlings. Significantly different expression values (P >0.05) are denoted with letters above each of the bar. Letter “a” “b” and “c” denote significantly different values as compared to the values in BioCot-2, TM2 and Tg2E-13 lines, respectively.

Fig 4. A comparative study of expression levels of the Cry1Ac protein in μg/g fresh weight (Mean±SD) in the leaf and other tissues of transgenic events Tg2E-13, TM-2 and their F1 (Tg2E-13 x TM-2); results are from the growing season of 2015.

(A) Cry1Ac expression in leaf tissues of 30, 60, 100 and 120 day old plants; (B) Cry1Ac expression in the flower bud and bract tissues of 70 day old plants. Significantly different expression values (P>0.05) are denoted with letters above each of the bar. Letter “a” “b”, “c” and “d” denote significantly different values as compared to the values of BioCot, TM2, Tg2E-13 and F1 plants, respectively.

Fig 5. Transcript levels of the cry1Ac gene and the encoded protein in the events Tg2E-13 and TM-2.

Comparative mRNA levels of cry1Ac gene in the cotyledon and root tissues of seedlings of the two transgenic events Tg2E-13, TM-2 and commercialized lines BioCot-1 and BioCot-2. Five biological replicates were used for each of the sample tissue. Similar age tissues from the same plants were used for quantification of the mRNA and the protein encoded by the cry1Ac gene. The fold change in transcript levels are represented in box and whisker plot while the protein levels (Mean±SD) are represented as red dots. As expected, no mRNA expression was recorded in the control tissues. Expression of the cry1Ac encoded mRNA was high in the root tissues of event TM-2 but the amount of Cry1Ac protein was zero like in the control plants.

Fig 6. Insect bioassays using leaf tissues of l20 day old control and transgenic lines of cotton.

Four day old insect larvae were released on the leaf tissues and pictures were taken after 3 days of feeding. (A) Consumed surface area in (a) the control plant (b) BioCot-1 (event Mon 531 in a hemizygous condition) (c) BioCot-2 (event Mon 531 in a homozygous condition) (d) eventTM-2 (e) eventTg2E-13 and (f) F1 (TM-2 x Tg2E-13); (B). Percent mortality of larvae fed on leaf tissues of different transgenic lines.