The cloning and genetic transformation of Chinese fir phytochrome A (phyA) gene
Dugan fir is a special type of Chinese fir that was isolated from the perennial progeny test of Yangkou state own forest farm in Fujian province, and is severely defective in shoot branching and flower organ formation, as well as the radial growth in stems. The previous researches in related to the defection in shoot branching of Dugan fir showed that the failure of axillary bud activation is the cellular basis that results in the deficient in lateral branch formation, and the defection in the perception/signal transduction of red/far-red light, as well as it-induced changes in endogenous hormones (including auxin/IAA, cytokinins, strigolactones and gibberellins et al) are the potential regulatory signals that inhibit axillary bud activation of Dugan fir. However, the lack of flower organs and wildtype material makes the related studying works very difficult to carry out. For this purpose, this study is to take the Dugan fir as the ‘ mutant’, and to take the normally branching clone of Yangkou 020 as the ‘wildtype’, and hope to campare their difference in the genetic composition, genetic structure and functions of phytochrome A (phyA), thus providing a basic clue for analyzing the mechanism that controlling the photomorphogenesis of Dugan fir. The primary conclusions of this study are as follows:
1) According to the full-length cDNA sequence of Chinese fir phyA gene in GenBank transcriptome databases, we designed and synthesized the specific sense and antisense primers, and amplified a 2500bp target band from the apical stems of Dugan fir and Yangkou 020 fir respectively; the PCR reaction and sequencing of gene clones all showed the sizes of theses two phyA genes are all 2529bp, and are similar to the size of ORF of the known Chinese fir phyA gene, which indicated that the coding sequence of phyA from the different varieties and types of Chinese fir is conservative;
2) The clustering results of the two Chinese fir phyA genes and homologous genes from other species of plants including Arabidopsis thaliana and populous based on the encoding sequences showed that the genes of Dugan fir and Yangkou 020 fir are all clustered together with the homologs of pine, while their phylogenetic distance with the corresponding members in Arabidopsis and popolar is very far, which is coordinated with the evolutionary history of taxodiaceae and Pinaceae;
3) The results of multiple sequence comparison between phyA genes of Dugan fir and Yangkou 020 fir showed that they share 99.26 similarity in sequence excepting for the base changes of A to G at the location of 2239bp, which not only led to the changes in amino acid composition, but also resulted in a significant change in the 3D construction of this two proteins. All these results suggested that the single base change of phyA genes in these two kinds of Chinese fir, as well as its-triggered change in protein spacious structure should connect with the abnormal phytomorphogenesis of Dugan fir through influencing the stability and function of phyA.
4) To test the functional changes of these two kinds of phyA genes caused by the single base mutant, we constructed the eukaryotic expression vector through integrading the ORF sequences of phyA genes into the location of pBI121 between BamH I and Sma I, and transformed into the wildtype plants of Arabidopsis through the trivia infection method. Now, we have got some T1 generation seeds, and the screening of positive transgenic plants are underway; in addition, we also tested the expression efficiency of recombination by using of the tobacco transient expression system, and the dying results of GUS reporter gene showed that they work well, which provide a basis for revealing the functional variances of these two kinds of phyA genes in Arabidopsis.
Key words：Chinese fir; phytochrome A (phyA) gene; Gene cloning and genetic transformation.