棉花阿拉伯半乳糖蛋白基因的克隆、表达与染色体定位分析

Cloning,Expression and Chromosome Mapping of Arabinogalactan Protein Gene in Cotton

陆地棉李氏超短纤维突变体(Li1li1)是显性单基因突变体,表现为茎秆、叶片卷曲,纤维短至6mm,其隐性纯合体(li1li1)则表现为株型和纤维发育都正常。对开花后10d的李氏纤维发育正常材料(li1li1)和超短纤维突变体(Li1li1)胚珠纤维混合体进行mRNA差异显示反转录PCR(DDRT-PCR)分析,获得1条在李氏纤维发育正常材料中上调表达的差异片段。进一步通过5'RACE技术获得全长为621bp的cDNA,测序及DNA序列的生物信息学分析表明该cDNA片段与拟南芥编码阿拉伯半乳糖蛋白基因AGP14有52%相似性,故命名为GhAGP。表达特征分析表明,该基因在陆地棉根、茎、叶和纤维中组成性表达,在棉纤维中优势表达。基因组序列分析显示,该基因在二倍体棉种非洲棉和雷蒙德氏棉中各有1个拷贝,在四倍体棉种陆地棉TM-1和海岛棉海7124中分别存在2个拷贝,表明A、D亚组中各有1个拷贝。利用本实验室陆地棉遗传标准系TM-1和海岛棉海7124培育的含140个单株的BC1作图群体,将GhAGP基因的2个拷贝分别定位在四倍体棉花部分同源转化群染色体6和染色体25上。

The Ligon lintless mutant （Li1li1） is a fiber elongation developmental mutant with a dominant monogenetic mutation characterized by short fibers and distorted leaf, stem and flower growth, and the recessive pure line （lili1） exhibits normal fiber developmental characteristics. The objectives of this study were to isolate genes preferentially or specifically expressed in fiber elongation stage by comparing gene expression differences between Li1Ii1 and li1li1. RNA isolated from 10 days post anthesis （DPA） fibers and ovules mixture in Li1Ii1 and li1li1 were used to screen differential gene expression in fiber development using differential display reverse transcriptase polymerase chain reaction （DDRT-PCR）. One differential expression cDNA segment was isolated and the corresponding full-length cDNA was cloned by 5＇ RACE method. Sequencing and bioinformatics analysis showed the protein had 52% homology with arabinogalactan protein from Arobidopsis, therefore, this gene was temporarily designated GhAGP. RT-PCR and quantitative real time RT-PCR assays all indicated that the transcript of GhAGP is constitutively expressed in root, stem, leaf and fiber, with higher expression levels in fiber cells as fiber developments. Genomic sequence analysis showed there was one copy of GhAGP in diploid cotton species, G. herbaceum and G. raimondii, and two copies in tetraploid cotton species, G. hirsutum and G. barbadense respectively, indicating that the sub-genome A and sub-genome D contain each of them. Using the BC1 mapping population derived from the hybridization between the upland cotton standard line TM-1 and G. barbadense cultivar Hai7124, and TM-1 as recurrent parent, two copies of GhAGP were located on the homoelogous chromosomes 6 and 25, respectively.