两个棉纤维发育相关基因的克隆与特征分析(英文)

Molecular Cloning and Characterization of Two Fiber Elongation Genes Using a Cotton Fiber Developmental Mutant (Gossypium hirsutum L.)

棉纤维发育突变体是克隆棉纤维发育关键基因和阐明其发育分子机理的优异资源。陆地棉李氏超短纤维突变体(Li1li1)是显性单基因突变体,表现为显性纯合体(Li1Li1)致死,显性杂合时(Li1li1)表型为茎秆扭曲、叶片卷曲和纤维短至6mm,而隐性纯合体(li1li1)则表现为株型和纤维发育都正常。本文对开花后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2条在李氏纤维发育正常材料中上调表达的差异片段。测序及DNA序列的生物信息学分析表明该差异片段分别与编码谷氨酸脱羧酶和质子焦磷酸酶的基因有较高同源性。通过电子拼接,5′RACE和全长cDNA序列验证,克隆了棉花的谷氨酸脱羧酶(GhGAD)和质子焦磷酸酶(GhVP1)基因全长cDNA,进一步对其功能和染色体定位进行了初步分析。转录水平分析表明,这两个基因在棉花根、茎、叶和纤维中组成性表达,在棉纤维中优势表达。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GhGAD和GhVP1分别定位在第12条染色体和第8条染色体。

Cotton fibers are single-celled seed trichomes of major economic importance. Many important genes are expressed during cotton fiber development and fiber developmental mutants can be used to preferentially detect the genes controlling fiber development. The Ligon lintless mutant （Zi1li1） 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 （li1li1） 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 Li1li1 and li1li1. RNAs isolated from 10 days post anthesis （DPA） fibers and mixtures in Li1li1 and li1li1 were used to screen differential gene expression in fiber development using differential display reverse transcriptase polymerase chain reaction （DDRT-PCR）. Two differential expression cDNA segments were isolated, the corresponding full-length cDNAs were cloned and their primary function was analyzed. The two genes encoded 542 and 667 amino acid residues and functioned as glutamate decarboxylase （GhGAD） and vacuole-pyrophosphatase （GhVP1）, respectively. Transcriptional level assays showed the two genes were constitutively expressed in tested tissues with higher expression levels during the fiber elongation stage. Furthermore, a BCI mapping population derived from hybridization between G hirsutum acc. TM-1 and G barbadense cv. Hai 7124, and TM-1 as the recurrent parent, was used for the location of GhGAD and GhVP1 on chromosomes 12 and 8, respectively, using cleaved amplified polymorphic sequences （CAPs）.