Cloning And Expression Pattern Analysis Of Promoters Of LjBIO Homologous Genes AtKIX8 And AtKIX9 In Arabidopsis

BIO ORGANS (LjBIO) gene plays a role in dorsoventral differentiation, organ internal asymmetry and floral organ size control in Lotus japonicas, the function and applications of which remains unclear at present. AtKIX8 and AtKIX9 are the homologous genes of LjBIO in Arabidopsis, which code the KIX-domain proteins and predicted to participate in regulation of plant organ size and shape. However, their functions and mechanism remains unclear. The regulation of plant organ size has broad and prospective applications, especially in the impact of genetic characteristics as plant appearance, reproduces and stress tolerance. Promoter is an important cis-element in regulating gene expression, which determines its specific expression time, position and quantity. Therefore, the study of plant promoters can help to understand the genes expression patterns and their regulation mechanism. In this study, we used arabidopsis as plant materials, cloned the promoters of LjBIO homologous genes AtKIX8 and AtKIX9, and studied their expression patterns and potential regulatory mechanism. It can help us to better understand these genes’ roles and mechanism in organ size control. Further, it can provide more clues to elucidate the function and the mechanism of LjBIO gene. The main results are as follows:1. The promotor sequences of AtKIX8 and AtKIX9 were analyzed using online software PlantCARE. Results showed that these sequences contain auxin (IAA) response element, abscisic acid (ABA) response element, gibberellin (GA) response element or cold stress response element.2. Two promotors differed in length were cloned for each gene of AtKIX8 and AtKIX9 in Arabidopsis, named pKIX8L, pKIX8S, pKIX9L and pKLX9S with the lengths of 2851 bp,954 bp,2982 bp and 1379 bp. Expression vectors of pKIX8L:GUS, pKIX8S:GUS,pKIX9L:GUS and pKIX9S:GUS were generated, and were successfully introduced into agrobacterium tumefaciens GV3101.3. The four vectors of PKIX8L:GUS, pKIX8S:GVS, pKIX9L:GUS and pKIX9S:GUS were transformed into Arabidopsis by agrobacterium tumefaciens.Using hygromycin for homozygous plant selection, we finally obtained 7 pKIX8L lines,6 pKIX8S lines,6 pKIX9S lines and 6 pKIX9S lines respectively.4. GUS staining results showed that the expression of GUS gene driven by AtKIX8 promoter were focused in shoot apical meristems, vascular tissues of hypocotyls, new leaves and primary roots in 10-day-old seedlings, and were detected in primary veins of leaves and stems in 4-week-old plants. The expression of GUS gene was undetectable with the At KIX9 gene promoter.5. Plants with positive GUS staining results were selected for and real-time PCR. Results displayed that the expression of GUS gene were upregulated after auxin (10 μmol/L IAA) and low temperature (4℃) treatments. However, expression of GUS gene remained stable after the ABA (200 μmol/L) and GA (50μmol/L) treatments.