Constitutive expression of Asparaginase in Gossypium hirsutum triggers insecticidal activity against Bemisia tabaci

Abstract

Whitefly infestation of cotton crop imparts enormous damage to cotton yield by severely affecting plant health, vigour and transmitting Cotton Leaf Curl Virus (CLCuV). Genetic modification of cotton helps to overcome both the direct whitefly infestation as well as CLCuV based cotton yield losses. We have constitutively overexpressed asparaginase (ZmASN) gene in Gossypium hirsutum to overcome the cotton yield losses imparted by whitefly infestation. We achieved 2.54% transformation efficiency in CIM-482 by Agrobacterium-mediated shoot apex transformation method. The relative qRT-PCR revealed 40-fold higher transcripts of asparaginase in transgenic cotton line vs. non-transgenic cotton lines. Metabolic analysis showed higher contents of aspartic acid and glutamic acid in seeds and phloem sap of the transgenic cotton lines. Phenotypically, the transgenic cotton lines showed vigorous growth and height, greater number of bolls, and yield. Among six representative transgenic cotton lines, line 14 had higher photosynthetic rate, stomatal conductance, smooth fiber surface, increased fiber convolutions (SEM analysis) and 95% whitefly mortality as compared to non-transgenic cotton line. The gene integration analysis by fluorescence in situ hybridization showed single copy gene integration at chromosome number 1. Collectively, asparaginase gene demonstrated potential to control whitefly infestation, post-infestation damages and improve cotton plant health and yield: a prerequisite for farmer's community. L-asparaginases play an important role in bacteria, fungi, plants, and the tissues of some animals excluding humans 1. In plants, asparaginases are key players in regulating asparagine (primary N source) by releasing ammonia and aspartate via the deamidation pathway, which is the preferred route for developing plant organs. The aspartic acid resulting from this pathway is incorporated into the aspartate family of amino acids 2,3. There are two reported categories of asparaginases: plant and microbial. Plant asparaginases are further subdivided into two subclasses based on their dependency on potassium (K +): K +-dependent and K +-independent. These are isolated from various plants including Arabidopsis thaliana, Lupinus albus, Lupinus polyphyllus, Phaseolus vulgaris 4 , Lupinus arboreus, Lupinus angustifolius 5 , Lotus japonicus 6 , Glycine max 7 , Withania somnifera 8,9 , and Pisum sativum 10. K +-dependent asparaginases exhibit asparaginase activity 11,12 whereas K +-independent asparaginases (isolated from Lupinus luteus (LlA)) possess both isoaspartyl peptidase and asparaginase activity and recognize β-aspartyl-His as a substrate 3,13. Under nitrogen-limiting conditions, the stored asparagine is the main source of nitrogen for the growing parts of plants, i.e., developing leaves and seeds 14. A total of 81 % of the global cotton fiber stems from genetically modified cotton; GM cotton helps combat various biotic and abiotic stresses 15 that negatively affect the quality and quantity of fiber 16. In 2017, Rahman et al. (2...

Enhanced Expression of Plasma Membrane Intrinsic Protein 2 Improves Cotton Fiber Length and Potential Economic Viability of Gossypium Arboreum

“…Transgenic cotton plants from the T1 generation were used for CpPIP2gene expression analyses. Young leaves from transgenic cotton plants were used for total RNA extraction according to the protocol of Gul et al (2020) [20]. The cDNA was synthesized using aRevertAidFirst Strand cDNA Synthesis Kit (cat # K1621Thermo Fischer Scienti c) in a reaction mixture comprised of 4 µL 5x reaction buffer, 2 µL 10 mM dNTP mix, 1 µLRiboLock RNase inhibitor (20 U/µL) and 1 µL RevertAid M-MuLV Reverse Transcriptase (200 U/µL), in a nal reaction volume of 20 µL.…”

“…FISH was employed to determine the chromosomal location of the CpPIP2 gene in transgenic plants as described by Gul et al (2020) [20].…”