Construction and Application of a Reference Plasmid Suitable for Determination of CpTI and cry1A Gene Dosages in Genetically Modified Cottons

Abstract

A reference plasmid, pMD-CCS, was constructed to detect multiple target genes in the insect resistant cotton (Gossypium hirsutum L.) varieties in China. This plasmid contained cowpea trypsin inhibitor (CpTI), Bacillus thuringiensis insecticidal crystal protein (cry1A), and cotton endogenous gene Stearoyl-acyl carrier protein desaturase (Sad1) sequences targeting the key exogenous gene types. The real-time quantitative PCR methods for CpTI and cry1A were established using pMD-CCS as the reference material (RM). The dosagess of CpTI and cry1A from 9 insect resistant cotton varieties were determined. The average CpTI dosages were 0.018–0.020 copies per genome and the average cry1A dosages were 1.377–2.136 copies per genome in the 3 insect resistant cotton varieties including Kemian 3. The average cry1A dosages were 0.887–2.564 copies per genome in 6 varieties including Ezamian 1-F₁. The standard deviations of the quantitative measurement ranged from 0.001 to 0.049. These results demonstrated that pMD-CCS could be used as the RM for the quantitative measurement of CpTI and cry1A genes in insect resistant cotton varieties.