Isolation And Function Identification Of The Key Genes Related To Shoot Branching In Chinese Fir (Cunninghamia Lanceolata (Lamb) Hook)

Shoot branching is important for the establishment of plant architecture. The major source of plant morphogenesis variation derives from variation in the behaviors of AMs. The study of molecular mechanism of shoot branching has scientific significance. It is more important to production practice, such as, Agriculture, Forestry, Pomology, Olericulture, Floriculture. Shoot branching is one of the important agronomic traits that effect yield by effecting the setting rate. Shoot branching is responsible for the yield of grasses such as rice, wheat and barley.A Chinese fir (Cunninghamia lanceolata (Lamb) Hook) no-branching mutant -monostem was skillfully used in this study. The key gene fragments related to shoot branching in Chinese fir were isolated using optimized cDNA-AFLP technique. We constructed a gene regulatory network of branching in Cunninghamia lanceolata, which can offer important reference for the understanding of the mechanism of branching. Four gene's full-length cDNA sequences were cloned using RACE. The expression patterns of these genes were identified in different tissues by using real-time fluorescent quantitative reverse transcriptase PCR (FQ RT-PCR). The over-expression vectors were constructed and then transformed into Arabidopsis thaliana mediated by Agrobacterium tumefaciens for function identification. These results provide some theoretical reference for the molecular mechanism of shoot branching and apical dominance.1.The phenological observation of Chinese fir"020"cloneFor a better understanding of the development of Chinese fir"020"clone, preliminary analysis was done on its phenological pattern and periodicity of cambium activity. By the end of April ( or at the beginning of May) , axillary buds for the first branches are formed. Axillary buds for the second branches are formed by the end of May ( or at the beginning of June) . And July is the time for forming the third branching axillary buds. The Chinese fir"020"clone has two fast growth periods. May is the first period, and from the middle of June to the beginning of July is the second fast growth period. The cambium of the Chinese fir"020"clone reactive early and become dormant lately. This can partly explain the rapid growth property of this clone. It reactivates in the middle of February, which has four to five layers of flat cambium cells. From the middle of April to the end of June, the cambium activity is the most vigorous of the year. The cambium increased to 7 or 8 layers. The cambium entered into dormancy during the first 10 days of November.2.The optimization of cDNA-AFLP reaction system.In order to ensure stability and efficiency of the differently display, we establish and optimize cDNA-AFLP reaction system for Chinese fir"020"clone. According to the optimized reaction system, the polymorphous strips in 6% PAGE electrophoresis were clear and stable. 3.The key genes related to branching were isolated and analyzedThe key genes related to branching were isolated from Chinese fir"020"clone SAMs that are not initiating AMs, Chinese fir"020"clone SAMs that are initiating AMs and the branching mutant"monostem"SAMs using optimized cDNA-AFLP reaction system. 43 gene fragments that regulate branching positively were obtained. 22 gene fragments that regulate branching negatively were obtained. And the mechanisms that they were involved in shoot branching were discussed.By systemly analysis of these key regulating genes, we find out that obviously different physiological processes are involved in regulation the initiation and outgrowth of AMs. Negative regulatory genes are mostly involved in IAA synthesis, hormone signal transduction, vesicle trafficking, electron transfer, chlorophyll synthesis. Positive regulatory genes are mostly involved in cell division control, the transfer of glycosyl, sugar transport, transcriptional regulation and translation regulation. According to these results, we constructed a gene regulatory network of branching in Cunninghamia lanceolata, which offerred important reference for studying the genetic mechanism of branching.Then we discussed in detail the mechanism that they are involved in branching regulation.4.Full-length cDNA sequences cloning and function identification of four key genesWe cloned the full-length cDNA sequences of PAE,RAB11C,RNaseâ…¢and DELLA that regulate branching using RACE. The expression patterns of these genes were identified using FQ RT-PCR. The over-expression vectors were constructed and then transformed into Arabidopsis thaliana for function identification. And the mechanisms they regulate branching are discussed.(1)ClPAE regulate branching by hormone signal transductionThe full-length cDNA sequences of ClPAE is 1031bp, with 711bp ORF encoding 237 amino acids. Conserved domain database (CCD) search indicates that it encodes 20S proteasome subunit alpha type E. The allignment of deduced amino acid sequene with other homolog genes indicates that the ClPAE gene is highly conserved among species. The expression pattern of ClPAE in different tissues was identified using FQ RT-PCR. The ClPAE gene is expressed rather differently in different tissues. It is expressed preferentially in inflorescences and leaves. The over-expression of ClPAE gene in Arabidopsis thaliana promotes branching. The apical meristem is divided into two parts. Dichotomous branching appeared in the Arabidopsis thaliana. It is supposed that there is a precise regulation of expression of this gene. Inhibit the expression of ClPAE gene could promote branching, but over-expression of ClPAE gene may could change the regulation network that result in increasing branches.(2)ClRab11C regulate branching by hormone transportationThe full-length cDNA sequences of ClRAB11C is 1133bp with 639bp ORF that encoding 213 amino acids. The identity of the deduced amino acid between Chinese fir RAB11C and populus trichocarpa RAB11C is 82.57%. Conserved domain database (CCD) search indicates that it encodes a number of Ras-like GTPase super family-RAB11C protein. It has the conserved domains of RAB proteins. On the homology tree, Chinese fir, populus trichocarpa and Arabidopsis thaliana are divided into one group. FQ RT-PCR analysis showed that there is no obvious difference of ClRAB11C gene expression between different tissues. It might be widely involved in regulating many physiological activities. The over-expression of ClRAB11C gene in Arabidopsis thaliana caused death after the outgrowth of the four to six leaves. We proposed that ClRAB11C may regulate plant body plan, including branching by effecting the positioning of auxin efflux carriers through vesicle trafficking.(3)ClRNaseâ…¢effect branching by transcriptional and translational regulationThe full-length cDNA sequences of ClRNaseâ…¢is 1045bp with 705bp ORF that encodes 235 amino acids. The allignment of the deduced amino acid sequence with other homolog genes showed that there are great differences among species. Conserved domain database (CCD) search indicates that it encodes a E. coli RNaseâ…¢, with five active sites, three metal binding sites, thirteen dimerization interfaces. FQ RT-PCR analysis shows that it is expressed great differently in different tissues. That means the function of ClRNaseâ…¢might be specialized. Because it is only a component of the gene regulatory network of branching in Cunninghamia lanceolata, not the"molecular switch", Arabidopsis thaliana that over-expression ClRNaseâ…¢gene grows slowly with dark green leaves.(4) ClDELLA promote branching by regulating GA signal transduction negativelyThe full-length cDNA sequences of ClDELLA is 2656bp with 2085bp ORF that encoding 695 amino acids. Because the N-terminal of DELLA is variable, the allignment of its deduced amino acid sequene with other homolog genes showed that its identity with populus trichocarpa is only 47.08%. Conserved domain database (CCD) search indicates that it encodes ClDELLA protein. It has conserved domains of DELLA superfamily, DELLA domain,VHYNP domain, LERLL domain, NLS domain, SAW domain. On the homology tree, it is divided into one group as Gymnosperm. We propose that DELLA proteins repress the height growth of Cunninghamia lanceolata"020"and promote its shoot branching by regulating GA singnal transduction negatively.