陆地棉GhSBP3基因功能研究和调控棉花纤维发育的分子机理

SBP (Squamosa Promoter Binding Protein) genes are transcription factors which have so far been found only in plants, controlling a number of fundamental aspects of plant growth and development, including vegetative phase change, flowering time, branching, and developing trichome. In our recently study, SBP family gene was involved in cotton fiber development. GhSBP3 gene expression reduced with in the fiber elongation, otherwise miR156d was increased as the same time. GhSBP3 was the possible target genes of miR156d by analysis of miRNA and RNA-seq on 8 DPA and 20 DPA fiber of Ji228. This study analyzed GhSBP3 in different time and space expression patterns in the process of different quality cotton fiber development. GhSBP3 and miR156 gumming region should be found by using 5´RACE. The promoter should be cloned and the expression of the promoter specificity should be tested in Arabidopsis. To provide more direct evidence that decreased levels of GhSBP3 were the cause of fiber elongations in cotton fiber development, we examined transgenic plants over-expressing the GhSBP3 and miR156d by 35S promoter and the specific promoter in fiber or trichome. Phenotype and fiber quality of transgenic Coke 312 plants were determined. We test the fiber transcriptome sequencing in different development periods of the transgenic positive strains and wild type controls. GhSBP3 gene function should be demonstrated, and the molecular mechanisms of miR156d –relgulated GhSBP3 define a development fibers elongation pathway. The results may not only enrich mechanism of cotton fiber, may also help using miRNAs genes control technology to improve the qualities of cotton fiber.

SBP (Squamosa Promoter Binding Protein )是植物特有的一类转录因子，该家族成员参与叶的形态建成、开花调控、表皮毛伸长等多个生理过程。项目组前期研究发现GhSBP3参与棉纤维发育，其表达量随着纤维的伸长而减少，且是miR156d可能的靶基因。本研究要明确GhSBP3在不同品质品种棉纤维发育过程中的表达模式；确定GhSBP3和miR156d的靶位点和启动子区及启动子的表达特异性；构建不同启动子启动的GhSBP3和miR156d的过表达载体，转化陆地棉，进而对转基因植株的表型和纤维品质进行测定，对转基因阳性株系和野生型对照不同发育时期的纤维进行转录组测序，最终明确GhSBP3的基因功能，探讨miR156d调控GhSBP3表达影响纤维发育的分子机制。本研究不但能够丰富棉纤维发育机制的相关理论，还将有助于利用miRNAs调控技术，加速优质棉种质资源创新。

SBP (Squamosa Promoter Binding Protein )是植物特有的一类转录因子，该家族成员参与营养生长次级组织如分支、绒毛等的发育调节等多个生理生化过程。. 本研究对优质纤维品种冀228、优质海岛棉和非优质品系3056纤维发育各阶段纤维长度进行了动态检测，发现优质品种（系）纤维发育非优质品系在纤维发育后期有所不同。通过构建冀228纤维均一化cDNA文库、转录组测序和mRNA-miRNA联合分析，发现GhSBP3在纤维发育中与miR156有相互调控关系，从而影响纤维发育。. 结果表明，GhSBP3基因在优质陆地棉冀228中在20DPA和25DPA优势表达，在纤维发育过程中呈现先升后降的趋势。miR156在20DPA优势表达，而后降低，说明miR156与GhSBP3表达趋势是一致的。而纤维伸长过程中是20DPA达到伸长高峰，而后伸长速度减慢，长度增加不大。优质陆地棉与海岛棉表达趋势一致，不同的是，海岛棉中miR156表达量继续升高至25DPA的3倍。由此可见，优质陆地棉与优质海岛棉中都miR156正调控GhSBP3表达，并与纤维伸长一致，而海岛棉长度优于优质陆地棉可能与miR156的持续优势表达有关。. 构建了GhSBP3基因的基因沉默（VIGS）TRV2载体，接种TRV2-GhSBP的棉花植株长势正常，与对照相比没有明显差异，但基因的表达水平进行检测发现，该基因的表达水平是明显降低的。. 构建了GFP载体，亚细胞定位的结果表明，GhSBP3定位在细胞核里。构建了GhSBP3的过表达载体、基因编辑载体和miRNA156的过表达载体，获得了胚性愈伤组织和基因编辑载体的转基因T0代植株。. 综上所述，GhSBP3基因的沉默能够引起叶片中GhSBP3基因表达水平降低，优质陆地棉与优质海岛棉中都miR156正调控GhSBP3表达，并与纤维伸长一致，GhSBP3基因可以作为棉纤维伸长的基因资源加以利用。

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