Cloning, Expression And Functional Analysis On Regulatory Region Of LATERAL SUPPRESSOR Gene From Nervilia Genus

The plants with different shapes in the nature possess their unique plant architecture, which directly determine the crop yields. Formation of higher plant architecture involves various organogenesis that relevant to plant morphology. The polymorphic of shoot branching determines the diversity of plants because it is one of the most pivotal components in plant architecture. In 1968, Donald CM put forward that greater economic benefits can be achieved by the breeding of crop ideotypes, because the reasonable structure of plants can make full use of environmental resources and increase their growth and production.Herba Nervilia Plicatae is one of famous and valuable herbal medicine in Lingnan Area that comes from Nervilia fordii (Hance) Schiltr. from Nervilia Genus of Orchidaceae family. The growth characteristics of N. fordii are unique, with the corms as the asexual reproductive organ and its rigorous requirements of growing environment; hence the proliferation rate is very low. The clinical and food demand for this herb is very strong in China and Southeast Asia because of the remarkable therapeutic effects on treating lung diseases. The wild production of N. fordii has long been facing a shortage due to excessive harvesting. Nervilia aragoana Gaud, is one of the close relative species of the N. fordii in the Nervilia Genus. These two species are very similar in morphology and growth habit, but it is common that one plant with more than one corm or leaf for N. aragoana. Therefore, to compare the regulatory of branching and development mechanism between these two species will provide theoretical basis for promoting the reasonable branches and improve the yield of N. fordii through functional genes regulation.LATERAL SUPPRESSOR (LS) is a transcription factor belongs to GRAS family which regulates the branching and development in plants. We had previously cloned the NfLS gene from N. fordii. In the current thesis the genomic DNA sequences of LS genes and their promoters were isolated by homology cloning and PCR-based chromosome walking techniques from N. aragoana and N. fordii genome. The expression and function pattern of the LS genes and their regulator regions have been preliminary studied; these results can deepen the understanding of the regulation mechanism of branching development on N. fordii and lay the foundation of getting the ideotype of N. fordii that have more branches or more bulb. The main progresses of this study are as follows:(1) The genomic DNA sequences were identified by homology cloning from N. aragoana (gNaLs) and N. fordii (gNfLs). The genomic sequences of NaLS and NfLS have no introns. The sequence similarities between NaLS and NfLS are both 99% in nucleic acids and amino acids. The results of Wolf PSORT prediction showed that the proteins NfLS and NaLS are both mainly located on cytoplasm and nucleus; according to the prediction analysis of ProtFun2.2 these two LS genes may have the function of signal transducer, stress response and growth factors.(2) The LS genes promoters and 3’UTR sequences were obtained using PCR-based chromosome walking techniques and their putative cis-elements were predicted by searching the databases of PLACE and PlantCARE and other bioinformatics databases. The identity of nucleic acid of these two promoters and 3’UTR sequences were 96% and 88%, respectively; the two promoters both contained a lot of putative upstream components and promoter core elements, such as TATA BOX、CAAT BOX、GATA box and DOFCOREZM, indicating the conservation of LS gene promoter among these species is at very high level. The different type and number of some special components between them revealed the inter-specific differences.(3) Real-time quantitative PCR was used to analyze tissue-specific expression pattern of NaLS gene in the N. aragoana in different growth period. Results showed that NaLS gene expressed the highest level in the growth period 2, however it expressed the lowest in the period 3. And the expression patterns were almost the same in the rhizome, petiole and leaf. The expression levels are higher in the leaf and rhizome than in the petiole in these four different periods, and the expression levels in corms were decreasing with the growth of the above ground parts of the plant.(4) The EGFP gene was fused to the NfLS gene from N. fordii and the NaLS from N. aragoana, respectively, by touchdown-overlap extension PCR (TD-OE PCR). The resulted fusion genes were then cloned into plant binary expression vector pRI 101-AN DNA, resulted in two recombinant expression vectors pRI 101-NfLS/NaLS-EGFP by restriction and ligation. The recombinant plasmids were then successfully transformed into Agrobacterium strain EHA105, transient expression of the fusion genes protein in onion epidermal cells t showed that the location of the NaLS-EGFP fusion gene was in cytoplasm, while NfLS-EGFP was in nucleus, which indicated that NfLS was nucleus located protein, but NaLS was not.(5) Using GUS gene as reporter, the plant binary expression vectors with NfLS and NaLS gene promoters and 3’UTR were constructed and introduced into tobacco leaves via impregnation method mediated by Agrobacterium. According the results of transient gene expression, we speculated that there may be enhancer elements existing in the 5’region sequences from+1 bp to +72 bp of NfLS cDNA. The core region of NfLS promoter possibly locate in the region from-1 bp to-133 bp, and the negative regulatory region may locate between nucleotide-134 bp to-963 bp. Similar distribution of regulatory regions in the promoter of NaLS gene was also observed. The NaLS gene 3’UTR sequences obtained in this study included enhancer elements, which are absent in NfLS 3’UTR.