Cloning and Transformation of Sucrose Synthase (SuS) Gene in Cotton (Gossypium Hirsutum L.)

Abstract

Long staple length, optimum micronaire with good strength is the highest demand of the textile industry in Pakistan. Cotton fiber quality is a multifactorial trait controlled by different genes that play a decisive role in different stages of fiber development. However, only a few genes have been explored yet that are crucial in fiber development and some of them have shown a positive effect on fiber yield and quality in transgenic cotton. Sucrose synthase (SuS) is among the important factors involved in inter-conversion of sucrose to fructose and UDP-glucose that serve as a precursor for the synthesis of cell wall cellulose. In the current study, an attempt was made by the introduction of synthetic SuS gene under the control of a CaMV35S promoter. The cassette was cloned in pCAMBIA 1301 vector with BstX1 and Xho1 restriction sites. The recombinant plant expression vector pCAMBIA-SuS was introduced into local non-transgenic cotton (Gossypium hirsutum) variety CEMB-00 through Agrobacterium-mediated transformation method. The transformation efficiency in this study remained 1.02%. Molecular analyses of putative transgenic cotton plants were performed to verify their transgenic nature. Young leaves of T0 plants were used for histochemical GUS assay to confirm the transformation event. Amplification of 813bp fragment by using gene-specific internal detection primers confirmed the successful integration of SuS gene in the cotton genome. Maximum mRNA expression of SuS gene was obtained in MA0023 and MA0034 as compared to other transgenic cotton plants. Four transgenic plants were selected after PCR analysis for molecular and biochemical studies in advanced generations. SuS gene mRNA expression in fiber was significantly higher at 15 DPA and 20 DPA as compared to 8 DPA in all transgenic lines. The Leaf SuS activity in transgenic lines increased up to 54% as in the case of MA0023. SuS activity at the reproductive stage (15DPA) was also increased up to 40% in case of MA0034. In fiber of transgenic lines decrease in sucrose contents while the increase in total soluble sugars contents was evident from elongation (15DPA) towards secondary wall synthesis and maturation (40DPA). Cleavage of sucrose by SuS catalytic activity was found to be higher in transgenic cotton plants as compared to non-transgenic control plants. The mRNA expression was found positively correlated with SuS activity and cellulose contents. Fiber analyses of transgenic plants showed an increase in fiber length up to 11.7%, fiber strength 18.65% and 28% increase in cellulose contents. Improvement in micronaire value up to 3.00 was observed in transgenic line MA0023. SEM analyses revealed that fibers from transgenic plants were smooth, highly spiral and fiber twist number increased per unit length when compared with control. Improvement in fiber length, micronaire value and surface smoothness led to producing better quality fiber for textile purposes. Genetic modification of cotton with SuS gene also resulted in improvement in some morphological and agronomic traits like plant height increased up to 88.76%, number of bolls 87.80% and GOT increased up to 22.08 % as compared to control plants. FISH analysis showed single copy number at chromosome number 9 and no signal was detected in control plants. The results depict that increased SuS activity, fiber properties, morphological and agronomic traits in transgenic lines are caused by genome integration and constitutive expression of SuS gene in cotton. From the results, it is clear that the SuS gene holds the potential to improve fiber quality and also provide more positive effect when combined with other potential fiber trait. Stacking of fiber-related genes into a single plant could be a better way to improve fiber quality in local cotton varieties to meet the standards of the modern textile industry.