Isolation and functional characterization of GhLIM6 promoter in transgenic tomato and cotton plants

Functional Genomics

poster

Zou, Chuang; Ye, Shue; Li, Fang; Zhai, Yunlan; Wei, Ting; Pei, Yan; Luo, Ming

Zou, Chuang

Luo, Ming

Screening and functionally charactering a promoter expressed in specific tissue or organ and different developmental stages is an important field in plant molecular biology. Cotton fibers are single-celled seed trichomes originated from the epidermis of the outmost integument of ovules. Fiber cell development includes four discrete but partially overlapping stages: initiation, elongation, secondary cell-wall accumulation, and maturation. In order to express target gene only in fiber cell or certain developmental stage of fiber cell, study on the gene and its promoter specifically or preferentially expressed in fiber cell is a major area of molecular biology in cotton. The GhLIM6 gene was expressed preferentially in cotton fiber and pollen. It played important roles in the quality and yield of fibers. Overexpressing GhLIM6 can promote the elongation of fiber. On the contrary, suppressing GhLIM6 can inhibit the elongation of fiber. Either overexpression or suppression of the GhLIM6 in transgenic cotton, the pollen fertility was impacted. The percentage of setting boll and the yield of per plant were lower compared with wild type. For further study the regulatory mechanism of the gene in cotton fiber development and screen the appropriate promoter expressed in fiber, we have cloned GhLIM6 promoter (L6p) and constructed plant expression vectors in which the reporter gene GUS was controlled of L6p through 5'-end deletion mutants. Through transgenic tomato plants and cotton plants, we clarified that L6p specifically expressed in pollen and fiber. The 1.7 kb fragment of L6p has been cloned from upland cotton genome by genomic DNA walking (YADE walking). There are many cis-acting regulatory elements in the fragment such as pollen-specific expression elements AGAAA, light regulatory elements (G-Box and I-box), hormone response cis-acting elements (GA-responsive elements GARE, ABA-responsive element ABRE), low-temperature-responsive element and so on. In transgenic tomato plants, GUS activity was highest in pollen and seed hair. Weak GUS signal also be detected in fruit vascular and endosperm. The result was confirmed by quantitative real-time RT-PCR. These revealed that L6p expressed preferentially in pollen and seed hairs. In transgenic cotton plants, the GUS activity presented in pollen and fiber cell while the other detected samples did not display any GUS signal. The highest expression level of GUS was in pollen and the moderate level was in fiber cell. At various developmental stages of fiber cell, the GUS activity was detected after 10 DPA. The higher activity was in the fiber of 15 DPA. The result from quantitative real-time RT-PCR was similar with that from GUS stain except for a certain transcript of GUS was detected in ovules after 10 DPA. This might result from fiber cells contained in the ovule sample. Furthermore, GUS stain showed there was no significant difference between 1.5 kb and 0.5 kb sequence of L6p. It suggested that the 0.5 kb sequence is a core sequence of L6p. These results indicate that GhLIM6 promoter expressed specifically in cotton pollen and fiber.