Overexpression of a Sucrose Synthase Gene Indirectly Improves Cotton Fiber Quality Through Sucrose Cleavage

Abstract

The study aims to improve fiber traits of local cotton cultivar through genetic transformation of sucrose synthase (SuS) gene in cotton. Sucrose synthase (SuS) is an important factor that is involved in the conversion of sucrose to fructose and UDP-glucose, which are essential for the synthesis of cell wall cellulose. In the current study, we expressed a synthetic SuS gene in cotton plants under the control of a CaMV35S promoter. Amplification of an 813-bp fragment using gene-specific primers confirmed the successful introduction of SuS gene into the genome of cotton variety CEMB-00. High SuS mRNA expression was observed in two transgenic cotton plants, MA0023 and MA0034, when compared to the expression in two other transgenic cotton plants, MA0035 and MA0038. Experiments showed that SuS mRNA expression was positively correlated with SuS activity at the vegetative (54%) and reproductive stages (40%). Furthermore, location of transgene was found to be at chromosome no. 9 in the form of single insertion, while no signal was evident in non-transgenic control cotton plant when evaluated through fluorescent in situ hybridization and karyotyping analysis. Fiber analyses of the transgenic cotton plants showed increases of 11.7% fiber length, 18.65% fiber strength, and up to 5% cellulose contents. An improvement in the micronaire value of 4.21 was also observed in the MA0038 transgenic cotton line. Scanning electron microscopy (SEM) revealed that the fibers of the SuS transgenic cotton plants were highly spiral with a greater number of twists per unit length than the fibers of the non-transgenic control plants. These results determined that SuS gene expression influenced cotton fiber structure and quality, suggesting that SuS gene has great potential for cotton fiber quality improvement.

Keywords: Agrobacterium-mediated transformation; cotton fiber; genetic modification; overexpression; sucrose synthase gene.

FIGURE 1

Schematic diagram of a CaMV35S-sucrose synthase (SuS) construct.

FIGURE 2

Karyotyping and location of SuS gene in the cotton genome through fluorescent in situ hybridization (FISH) technique. (A) Arrow indicates fluorescent signal at chromosome 9 in the transgenic plant. (B) Non-transgenic control plant.

FIGURE 3

Relative expression of SuS gene mRNA (A) from subtending leaves of transgenic plants and (B) from fiber during elongation [8, 15 days post anthesis (DPA)] and early secondary wall synthesis stage (20 DPA). SuS activity in subtending leaves and fibers of transgenic and control lines. (C) SuS activity in leaves. (D) SuS activity in cotton fibers at 8, 15, and 20 DPA. Sucrose and total soluble sugars in fibers at different DPAs. (E) Sucrose contents in transgenic and control plants. (F) Total soluble sugars. Values represent the average of three replicates from each transgenic line. Asterisks indicate a significant difference between transgenic and control (^∗P ≤ 0.05, ^∗∗P ≤ 0.01; ns, non-significant using ANOVA).

FIGURE 4

Cellulose contents in mature cotton fibers of transgenic and control plants. Values represent the average of three replicates from each transgenic line. Asterisks indicate a significant difference between transgenic and control (^∗∗P ≤ 0.01; ns, non-significant using one-way ANOVA).

FIGURE 5

(A) Fiber lengths in transgenic and control plant lines. Photographs of cotton fibers in transgenic (MA0023, MA0034, MA0035, and MA0038) and control plant lines. (B) Fiber characteristics of transgenic and control plant lines. (A) Fiber strength. (B) Micronaire value. (C) Uniformity index. (D) Maturity ratio. (E) Fiber length. Values represent the average of three replicates from each transgenic and control line. Asterisks indicate a significant difference between transgenic and control (*P ≤ 0.05, **P ≤ 0.01; ns, non-significant using one-way ANOVA).

FIGURE 6

Observation of surface microstructure of transgenic and control fibers by SEM. In each row left-hand image is control fiber, while the others are transgenic fibers. Top Row: Surface of mature fiber at 4,000x left-hand image is control fiber, while others are transgenic fibers. The surface of control fiber is rough with wrinkles compared to the control. Middle Row: Surface of mature fiber at 1,000x showing twists in a single fiber. Bottom Row: Fibers observed at 400x; transgenic fibers highly spiraled with increased fiber twist number compared to control.

FIGURE 7

Phenotypic characteristics of transgenic and control plants. (A) Plant height of transgenic and control plant lines. (B) Number of bolls per plant line. (C) Average boll weight of transgenic and control plants (D). Leaf area of transgenic and control cotton plants. Values represent the average of three plants from each transgenic and control line. Asterisks indicate significant difference (^∗P ≤ 0.05, ^∗∗P ≤ 0.01 using one-way ANOVA; ns, non-significant).