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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 fiber developmental mutants are excellent resources for cloning cotton fiber development key genes and clarifying their developmental molecular mechanisms. The upland cotton Li 1li1 mutant is a dominant single-gene mutant, which is characterized by lethal dominant homozygote (Li1Li1), phenotype of dominant hybrid (Li1li1) with stem twisting, leaf curl and fiber As short as 6 mm, while recessive homozygotes (li1li1) showed normal plant type and fiber development. In this study, mRNA differential display reverse transcription PCR (DDRT-PCR) analysis was performed on the mixture of li1li1 and Li1li1 ovule fibers 10 days after flowering, Development of normal materials up-regulated expression of the different fragments. Bioinformatics analysis of DNA sequencing and sequencing showed that the difference fragment has higher homology with the genes encoding glutamate decarboxylase and protol pyrophosphatase, respectively. The full-length cDNA of glutamate decarboxylase (GhGAD) and protolysin pyrophosphatase (GhVP1) in cotton was cloned by electron splicing, 5’RACE and full-length cDNA sequence analysis, and its function and chromosome location were further analyzed . Transcriptional level analysis showed that these two genes were constitutively expressed in cotton roots, stems, leaves and fibers and predominantly expressed in cotton fibers. In this study, 140 BC1 mapping populations were cultivated in TM-1 and Qingdao 7124, and GhGAD and GhVP1 were mapped on chromosome 12 and 8, respectively.