| The plant architecture mostly affect by the shoot branching, Many factors may be involved in regulating the plant growth and development,and the genetic factor and environment are two main factors. In recent years, Analyses on model plant and mutant phenotypes provide the further information on the mechanism of branch development, Several key genes regulating shoot branch development have been identified and cloned via genetic and molecular methods, which proved that plants can form the gene and hormones regulatory network to control the shoot branching. Based on the research outcome so far, we carried out a series of studies about the relationship between the new type of plant hormone strigolactones and the axillary bud. The main results obtained in this study are as follows:1. According to results of previous research about the Arabidopsis thaliana and tomato. This experiment carry out relevant experiments about strigolactones in tobacco.The full length of NtMAX3, NtMAX4 genes was cloned using RACE technology. The result of amino acid sequence alignment of the NtMAX3 genes of tobacco, tomato and Petunia hybrid, the results show that the NtMAX3 had 84.81% and 61.96% similarity with that of tomato and Arabidopsis at amino acid level. Similarly, the NtMAX4 had 86.51% and 66.01% similarity with that of tomato and Arabidopsis at amino acid level, respectively. And both of the MAX possess the RPE65-domain, which can encode the carotenoids oxygenase and affect the synthetic of strigolactones, they all belonged to CCDs protein family. Results of previous research indicate that in the TCP transcription factors family, the BRC gene may influence the MAX pathways, which means that the BRC gene act as a downstream effector of MAX pathways. We analyze some BRC homologous genes from different species by bioinformatics method to understand the the function of BRC gene and MAX pathway to control the axillary buds2. Based on the specific sequence of NtMAX3、NtMAX4 genes, the primers were designed and synthesized. There are three different RNAi primers in NtMAX3 and Nt MAX4, respectively. And we name the three different product of RNA interference 1, 2, 3 in NtMAX3, the number of 12, 21, 22 represent product of RNAi of NtMAX4. Three interference vectors has been constructed and confirmed by restriction enzyme digestion and DNA sequencing analysis by the Getway technology from the Invitrogen Company. The tobacco K326 was used as material in this study, and we transform the vectors into K326 leaves by Agrobacterium tumefaciens. We obtain 102 strains T0 generation of transgenic plant, Number 1, 2, 3of NtMAX3 include19, 26, 19 strains, respectively.And the number 12, 21, 22 of NtMAX4 contain 9, 16, 13 strains. Compared with control group, the transgenic plants have obvious differences phenotypic in both from seedling to full flowering phase. The greenhouse provides the best grown environment, and all seeds of the transgenic plants were obtained.3. According to screening positive clones and phenotype identification of T0 transgenetic seedlings, we selected the 28 strain and renumbered them to plant in the field. Under the natural conditions, we collect the data of plant height and number of axillary buds. The result of the statistical analysis show that transgenetic seedlings own their special phenotype, and we found that, compared with control plants K326, the RNAi plants appeared the lower bud of branch prematuring. The expression quantity of NtMAX3, NtMAX4 genes in different tissuses of K326 and transgenic plants are assay by the Fluorescent quantitation PCR. The result of the fluorescence RT-PCR showed that the expression of Nt MAX3 and NtMAX4 gene of various plant parts in K326 and transgenic plants have changed. |
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