Studies On Ponkan BAC Library Based Isolation Of LEC1-LIKE Promoter And Genetic Transformation Of LEC1-LIKE In Citrus

Citrus which ranks the first in both acreage and yield over the years, is one of the most common ever green fruit crops throughout the tropical and subtropical regions of the world. Embryonic callus, as an ideal material for in vitro germplasm preservation of citrus, could produce somatic embryos which then develop into whole plant (a phenomenon termed somatic embryogenesis). Moreover, embryonic callus is widely used in genetic transformation, somatic mutation induction, physiological and biochemical researches of citrus. However, embryonic callus of many citrus species and varieties would gradually decrease or even lost their embryogenic capability during long-term subculture. Consequently, it is necessary to study on the mechanism of citrus somatic embryogenesis. LEC1-LIKE(L1L) gene is one of the key genes of plant zygotic and somatic embryo development. In-depth study on citrus L1L gene(CsL1L) may explore somatic embryogenesis mechanism from the perspective of a single gene, meanwhile provide theoretic basis to manually enhance citrus somatic embryogenesis capability. In the present study, the BAC library of’Egan No.1’Ponkan (Citrus reticulata Blanco) was screened using specific primers for CsL1L gene. The promoter sequence of CsL1L was then obtained by walking-sequencing. Moreover, CsL1L gene was introduced into epicotyl of Olinda orange (C. sinensis Osbeck) and callus of Satsuma mandarin (C. unshiu Marc.)’Guoqing No.1’which had lost somatic embryogenesis capability, to testify its function. The main results were as follows:1. From Ponkan’Egan No.1’BAC library, two positive single clones "42-14-P" and "52-8-I" were successfully screened, in which the length of inserted sequences were 120 Kb and 90 Kb respectively as shown by PFGE. Then the full genome sequence of CsL1L gene was obtained through PCR and BAC end-sequencing.2. Primers of walking-sequencing were designed according to the known sequence of CsL1L gene, by which 1071 bp promoter sequence of CsL1L was acquired in clone "42-14-P". Promoter analysis via prediction software (PlantCare and PLACE) demonstrated that CsL1L promoter contained most of the conservative elements of higher plant promoters, indicating its potential promoter activity; there were multiple cis-acting elements, including basic transcriptional elements TATA-box and CAAT-box; many seed-specific promoter elements were also found, such as RY repeat element, E-box, ACGT element, SEF4, W-box and GT1, etc, indicating the possibility of its being a seed-specific promoter, which was in accordance with the previous result that CsL1L gene was related to citrus embryo development.3. An overexpression plasmid carrying CsL1L gene was transformed into epicotyl of Olinda Valencia Orange and callus of Satsuma mandarine’Guoqing No.1’. Embryoid-like structures were observed on transformed epicotyl of Olinda Valencia Orange at the wounds which normally generate buds. However, in the transformed callus of Satsuma mandarin, no significant phenotypic changes were observed. Results demonstrated that over-expression of exogenous CsL1L gene can promote the conversion of somatic cells of citrus vegetative organs into embryonic structures, while may not be able to induce somatic embryos from callus without somatic embryogenesis capability.