Cloning And Functional Analysis Of A Somatic Embryogenesis Receptor-like Kinase Gene In Cotton

Cotton is one of the important economic plants worldwide. As its generative organs are associated with practical value. So the developmental mechanism in its reproductive tissues, such as flower, fiber,especiallly anther, is necessary for breeding high quality cotton. Meanwhile it will also accelerate the development of cotton industry.Somatic Embryogenesis Receptor-Like Kinases (SERKs) form a sub-family of the LRR-RLK family. It contained three conserved domains: an extracellular domain, a transmembrane domain and an intracellular catalytic kinase domain, sharing the same characteristic structures observed in receptor kinases. Some studies had found that SERK proteins were involved in many plant developmental processes, such as somatic embryogenesis and male sporogenesis, etc.Now we found two novel Somatic Embryogenesis Receptor-like Kinase (SERK) genes in upland cotton (Gossypium hirsutum), named GhSERK1 and GhSERK4 according to the protein blast. The genomic sequence of GhSERK1 was 6,920 bp in length, and it consisted of 11 exons interspersed with 10 introns. The full-length cDNA of GhSERK1 was 2,502 bp (GenBank Accession no HQ621831), comprised of a 424-bp 5'UTR (untranslated region), an ORF of 1,884- bp, and a194-bp 3'UTR. It encoded a protein of a 627amino acid with a predicted molecular weight of a 69-kDa and a pI of 5.55.The predicted results using SignalP 3.0 Server tool indicted the existence of a signal peptide at the N- terminus of GhSERK1, with a possible cleavage site between amino acid residues 28 and 29. The extracellular domain was between residues 1-241, and the TM domain was between residues 242 and 264, with intracellular domain residues 265-627, according to the prediction of the TMHMM program. Sequence analysis of GhSERK1 revealed high levels of similarity to other reported SERKs, as the protein structure started with a signal peptide at the N terminus, followed by a Leu-zipper domain, five Leu-rich repeats (LRRs), a Pro-rich domain called the Ser-Pro-Pro motif, a single TM domain, three conserved subdomains of a Ser/Thr kinase, and a C-terminal Leu-rich domain. Southern-blot hybridization resulted in only one band, when cotton genomic DNA digested with HindIII and EcoRI separately. Expression analysis showed that GhSERK1 mRNA was present in all organs of cotton plants and at different developmental stages. So GhSERK1 might have a broad role in the development of plant. However, its transcripts were most abundant in reproductive tissues, especially in floral buds, anther and ovule, compared to a low level of other tissues, such as root, stem, leaf and fiber. Meanwhile it was expressed increasingly with the development of the floral buds. Furthermore, compared with the male-fertile line, the level of GhSERK1 mRNA was lower while the formation of the tapetum in the male-sterile cotton line P30A, the pollen of which was defected. At the open flowers stage, the abundance of GhSERK1 in anthers was much lower in the sterile than in the fertile types but was similar in the sepals, petals and ovules in both types. For further functional study, RNAi vectors were constructed and transformed into the cotton. Downregulation of the GhSERK1 gene with different interference fragements resulted in different phenotype. The transgenic plants showed normal if only the GhSERK1 gene was downregulated. While the transgenic plants with downregulation of the GhSERK family showed unnormal anther compared with the wildtype, such as quanlity and energy of the pollen decreased,some anthers indehiscent or without pollen. Taken together, these findings demonstrated that GhSERK1 play a critical role during anther formation, and GhSERKs had functional complementary on male development. Meanwhile,The paper focused on cloning and functional analysis of the GhSERK. The study showed that GhSERK1 mainly had function on anther development, and play roles on other aspects of the plant development.