Cloning And Analysis Of The Flowering Time Controlling Gene HbFCA In Hevea Brasiliensis

Hevea brasiliensis [(H.B.K) Muell.-Arg.] is the important economic crop and the national strategies resources. At present, the consumption of natural rubber is increasing in China year by year, so, improving rubber output is an urgently problem. However, rubber is the perennial plant, and has the long sexual generation cycle which limited the efficiency of breeding greatly. The transition to flowering is the important physiological processes in higher plants development, and it has always been a hot topic about the plant developmental biology research. Flowering can be divided into three following progressive processes: flowering determination, flower evocation, and floral organogenesis and development. In these processes, the flowering determination is the base of others, and directly controls the flowering time. This flowering determination is genetically controlled by four pathways: photoperiod pathway, vernalization pathway, autonomous pathway and gibberellic acid (GA) pathway.Flowering Locus A (FCA), as a component of the autonomous pathway, regulated flowering time and contained two RNA binding domains (RRMs) and a WW-protein interaction domain. FCA gene functioned in the posttranscriptional regulation of transcripts. It is the functional protein negatively regulates its own expression by the productions of truncated, inactive transcripts at the expense of the full-length FCA transcripts, thus limited the expression of functional FCA protein. In this stdudy, one part of a DNA fragment, from the methylation filtration library in Hevea brasiliensis is the high sequence similarity between with other species FCA cDNA. Based on the conserved sequence (434bp), the author cloned and analyzed the HbFCA gene in order to understand the function and action characters of HbFCA gene. If we can decurtate the sexual generation cycle of rubber tree, it will accelerate the proceeding of rubber tree breeding to a large extent. The results of the research in this paper as follows.1. Comparied with CTAB-LiCl method and CTAB-NaAc method for extracting the RNA of rubber tree, the author decided the CTAB-NaAc method is simple, time-saving and no Li+or Cl-. So, we decided to extract the RNA of different tissues of rubber tree by CTAB-NaAc method.2. Obtained Flowering control locus A(HbFCA) DNA (11691bp), HbFCA promoter (2039bp) and13kinds of cDNA Cutting Modes from rubber tree by RACE and PCR technology. There are8kinds of the cDNA Cutting Modes have the complete open reading frame (ORF) and the conserved functional domain(RRM1、RRM2、WW). The amino acid of them are753-799aa. Analyzed the8kinds of different splicing patterns, the result showed that these proteins are all the Hydrophilic Proteins and the proteins maybe mainly located in nuclear and microbody.3. Downloaded the FCA homologous amino acid sequence of other15kinds of species and used them to construct evolutionary trees. The result shown that:the RNA-binding RRM1、RRM2and WW protein interaction domain of HbFCA protein are highly conservatively; ProLine/glycine-rich regions (P/G) concentrations and glutamine rich regins (Q) are also conservatived; the glycine-rich regions (G) are conserved only in monocots. The genetic distance (GD) from Rubber HbFCA from small to large in order for is Ricinus communis (Euphorbiaceae), grape branch, legume, Cruciferae, Salicaceae, Gramineae, ferns.4. Analyzed the promoter of rubber tree by the search for care software of Plant Care, some other responsive element except TATA-box and CAAT-box like light responsive element, cis-acting regulatory element involved in light responsiveness, cis-acting regulatory element involved in circadian control, protein binding site et al. all found in. So, the author think, these regulatory elements show HbFCA gene is highly sensitivity for light reaction. Otherwise, there are other regulatory elements in the HbFCA promoter, like cis-acting regulatory element involved in the MeJA-responsiveness, cis-acting element involved in heat stress responsiveness, cis-acting element involved in defense and stress responsiveness, cis-acting element involved in salicylic acid responsiveness. It shows that HbFCA maybe also sensitive to MeJA, orthohydroxybenzoic acid, high temperature and so on.5. Analyzed HbFCA-β and HbFCA-y (including HbFCA-γ1、HbFCA-γ2、HbFCA-γ3、 HbFCA-rl, HbFCA-l1、HbFCA-12、HbFCA-r2and HbFCA-θ) by Real-Time PCR, we conclude:HbFCA-y and HbFCA-β are antagonistic in the same tissue, or synergy in the different tissues. HbFCA expression levels in leaf blade/inflorescence/root were more abundant in bark/somatic embryo/rubber latex, with the levels changed in the different stages of leaf blade. HbFCA-y in rubber maybe played a part in promoting the starting of flower bud development.6. After the transformation of HbFCA-γ1excessive eukaryotic expression vector pXCSHAStrep-35S:HbFCAγ1into Arabidopsis thaliana, T1is not showing early flower characters like other FCA of Arabidopsis thaliana and Brassica napus transgenic plants. It looks like the OsFCA transgenic plants, showing late flower characters. So, the author inferred the over-expression of HbFCA has toxic effects on Arabidopsis.7. In this paper, the author also built the HbFCA-γ1excessive eukaryotic expression vector pCAMBIA2301-HbFCA-MCS, HbFCA promotor excessive eukaryotic expression vector pCAMBIA2300-HbFCA promotor-GUS-NOS and the HbFCA excessive RNA interference eukaryotic expression vector. Now, we have successfully infected rubber tree callus germs with these vectors by agrobacterium-mediated transformation. And the HbFCA promoter has promoted the GUS gene expression in rubber embryo.