Identification, Molecular Cloning And Expression Analysis Of Gene Families Involved In Gibberellin Metabolism In Salvia Milotiorrhiza

Gibberellin is a classical phytohormone. It is a large group of tetracyclic diterpenoid compounds with more than 136 members. GAs are synthesized from trans-geranylgeranyl diphosphate (GGPP), an important intermediate diphosphate precursors produced mainly from pyruvate and glyceraldehyde 3-phosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in the plastids. The formation of GGPP during diterpernoid biosynthesis also depends on cross-talk between the MEP pathway and the mevalonate (MVA) pathway operated in the cytoplasm and mitochondria. GA metabolic pathway from GGPP to end-products can be generally divided into three stages and various gene families, including CPS, KS, ent-kaurene oxidase (KO), ent-kaurenoic acid hydroxylase (KAO), gibberellin 20-oxidase (GA20ox), gibberellin 3-oxidase (GA3ox) and gibberellin 2-oxidase (GA2ox), are involved. In this study, we computationally identified 22 SmKO, SmKAO, SmGA20ox, SmGA3ox and SmGA2ox genes from the whole genome sequence database of Salvia miltiorrhiza. The open reading frames (ORFs) of these genes were cloned using the PCR technology. Sequence features of cDNA and deduced amino acid were analyzed computationally. Subcellular localization of deduced protein was predicted using TargetP. Phylogenetic trees were constructed. The expression levels of identified genes in roots, stems, leaves and flowers of S. miltiorrhiza were analyzed using qRT-PCR. The responses of SmCPS, SmKSL, SmKO, SmKAO, SmGA20ox, SmGA3ox and SmGA2ox to GA3 treatment were analyzed. Moreover, SmJDI1 and SmIDI2 involved in the upsteam of gibberellin biosynthesis were cloned from S. miltiorrhiza. Artificial microRNA constructs used for down-regulation of SmIDI1 and SmIDI2, respectively, were constructed. Transgenic S. miltiorrhiza plants were produced. The results are as follows:(1) A total of 22 genes, including a SmKO, two SmKAOs, six SmGA20oxs, two SmGA3oxs and 11 SmGA2oxs were identified by BLAST analysis of Arabidopsis and rice homologus genes against the current assembly of the S. miltiorrhiza genome. The coding regions of all 22 identified genes were cloned.(2) Sequence features of cDNA and deduced amino acid were analyzed using computational approaches. The results showed that the length of cloned 22 ORFs varies from 933 to 1557bp, the theoretical isoelectric point varies between 5.38 and 9.43, the length of deduced proteins varies from 311 to 519 amino acids with the molecular weight varied between 34.36 and 57.73 kDa. Subcellular localization of deduced proteins were predicted using TargetP. SmKO, SmKAO1 and SmKAO2 contain the secretory pathway signal peptides at the N-terminal, suggesting these proteins are most possibly present in the endoplasmic reticulum. SmGA20ox4, SmGA2ox1 and SmGA2ox5 contain the chloroplast transit peptide at the N-terminal, suggesting these proteins are most possibly located in the plastids. No signal peptide and transit peptide were identified in the other 11 proteins, indicating they are probably located in the cytoplasm.(3) Phylogenetic trees were constructed for plant KO, KAO, GA20ox, GA3ox and GA2ox proteins. SmKO was clustered with Sesamum indicum and Erythranthe guttata. SmKAO1 and SmKAO2 were clustered together, suggesting they are paralogous. Plant GA20ox proteins could be divided into four clades, of which two included SmGA20ox proteins. Plant GA3ox proteins could be divided into three clades. SmGA3ox1 and SmGA3ox2 were separated into two clades. Plant GA2ox proteins could be divided into four clades, of which three included SmGA2ox proteins.(4) The expression of 22 identified genes in roots, stems, leaves and flowers of S. miltiorrhiza were analyzed using the qRT-PCR method. The results showed that genes involved in GA metabolism were tissue-specific. SmKO showed ubiquitous expression in all the tissues analyzed, with the highest in flowers, followed by stems and leaves, and the lowest in roots. SmKAO1 and SmKAO2 were expressed in all tissues examined. The expression of SmKAO1 was higher in stems, roots and leaves than flowers, while the expression of SmKAO2 was higher in roots, flowers and leaves and less in stems. Tissue-specific expression was also observed for SmGA20ox, SmGA3ox and SmGA2ox genes.(5) The expression levels of the majority of SmCPS, SmKSL, SmKO, SmKAO, SmGA20ox, SmGA3ox and SmGA2ox genes were significantly altered in roots, stems and leaves of S. miltiorrhiza treated with GA3 for 12,24 and 48 hours. Some of them, such as SmCPS1, SmCPS3, SmCPS5、SmKSL1、SmKSL2、SmGA2ox4、SmGA2ox5, SmGA2ox6 and SmGA2ox7 were significantly up-regulated in roots, stems and leaves. Some of them, such as SmGA20ox2, SmGA3ox1, SmGA2ox1、SmGA2ox8、SmGA2ox10 and SmGA2ox11 were significantly down-regulated. The expression of the other genes was complex with up-regulation in a tissue or different time-point while down-regulation in the other tissues or diverse time-point.(6) The cDNAs of SmlD11 and SmID12 involved in the upstream of gibberellin metabolism pathway were cloned. No intron was found in the cloned SmIDI1 cDNA, while the cloned SmIDI2 contained introns.(7) Four artificial microRNA constructs were made for down-regulation of SmIDI1 and SmIDI2, respectively. Genetic transformation of S. miltiorrhiza was performed. Transgenic plants are ready for further analysis and identification.These results provide useful information for demonstrating the function of SmIDI1 and SmIDI2 and shed light on gibberellin metabolism and regulation in S. miltiorrhiza.