摘 要

独杆杉是于20世纪90年代从福建省洋口国有林场的多年生杉木子代测定林中发现的一株分枝和花器官完全缺失、且径向生长受到显著抑制的特殊杉木类型。针对独杆杉分枝发育缺陷的前期研究表明腋芽生长激活受限是造成独杆杉分枝形态建成的重要细胞学基础，而红光/远红光信号的识别和信号转导缺陷及其介导的内源激素（包括生长素、细胞分裂素、独角金内酯和赤霉素等）水平变化很可能是抑制独杆杉腋芽生长激活的重要信号调节基础。由于独杆杉不开花、且缺乏必要的野生型材料，导致相关的研究尚且没有得到深入细致的开展。为此，本课题拟以分枝缺失的独杆杉为“突变体”，以正常分枝的洋口020杉为“野生型”，通过比较两者光敏色素A基因（phyA）在遗传组成、结构和功能上的差异，为解析独杆杉光形态建成缺陷的信号调节机制提供必要的线索。本课题的研究研究结论如下：

1）根据GenBank转录组数据库中已知的杉木phyA基因的全长cDNA序列，设计合成引物，分别从独杆杉和洋口020杉顶端茎段组织中扩增出一条2500bp左右的目标条带；经基因克隆、阳性克隆子的菌液PCR检测以及测序，证实两条目标条带的长度均为的2529bp，与已知杉木phyA基因预测的开放阅读框（ORF）大小相一致，表明该基因的遗传组成在不同杉木品种和类型中是比较保守的；

2）基于phyA基因编码区序列的聚类分析表明，洋口020杉和独杆杉的phyA与松科植物的同源基因聚类在一起，而与拟南芥和杨树等物种中同源基因的亲缘关系较远，这与松科、杉科植物的系统进化过程相一致；

3）基于对独杆杉和洋口020杉phyA基因的序列比较，发现两者除了在2239bp位置上存在A、G碱基水平上的变化以外，其它位置上均保持一致，相似度达到99.26%；根据预测蛋白质序列的3D建模和比较发现，单碱基突变导致两者蛋白在空间结构呈现出显著的变化，这很可能是造成其蛋白稳定性和功能变化的重要结构基础；

4） 通过构建含有独杆杉和洋口020杉phyA基因完整ORF的过量表达载体及遗传转化拟南芥的哥伦比亚野生型植物，已经达到部分T1代的种子，阳性转基因苗的筛选工作正在进行中；针对烟草叶肉细胞的瞬时表达分析，证实构建的真核表达载体能够高效地表达GUS报告基因，这为利用拟南芥来验证两种不同杉木来源的phyA基因的功能创造了条件。

关键词：杉木；phyA基因；基因克隆和遗传转化

The cloning and genetic transformation of Chinese fir phytochrome A (phyA) gene

ABSTRACT

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.