Cloning Of Receptor-like Kinase Coding Sequences And Functional Analyses Of RLCK561 In Arabidopsis Thaliana

Receptor kinases play critical roles in both animal and plant signaling pathways regulating cell division, cell differentiation, cell death and pathogenic defense responses, and finally control plant growth and development. In Arabidopsis thaliana, there are at least 610 receptor-like kinases (RLKs) forming a super gene family. However, the functions of the most members have not been elucidated. To facilitate the functional analyses of these RLKs, the Gateway technology was employed to clone the coding sequences of 377 RLKs (not including the LRR family members). This study also investigated the biological functions of RLCK561, a gene of the RLCK VIII subfamily, by means of genetics and molecular biology.The coding sequences of 227 RLKs were cloned successfully into the Gateway donor vector pDONR/Zeo and analyzed by DNA sequencing. Among them,179 RLKs showed sequences identical to the predictions of the TAIR database, and the other 48 CDS sequences showed different structures possibly because of alternative splicing of pre-mRNA. Some predicted intron sequences were proved as exon sequences in 21 cloned RLK CDS sequences; a total of 15 cloned RLK CDS sequences missed predicted exons; the other 7 cloned RLK CDS sequences had both exon and intron changes.In this study, we investigated preliminarily the biological functions of the RLCK VIII subfamily members. The loss-of-function mutant rlck561 produced defective embryos. Results from genetic cross, microscopic observation of cleared embryos, and pollen staining suggested that the processes of sperm-egg recognition or fusion may had problems. This study provided materials for functional analyses of the other RLKs, and the study of RLCK561 may contribute to further reveal the molecular mechanisms regulating double fertilization and embryo development in Arabidopsis.