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Analysis of genes differentially expressed during initial cellular dedifferentiation in cotton

Chinese Science Bulletin 2008年第23期53卷 3666-3676页

作者： zhu huaguo tu lili jin shuangxia xu li tan jiafu deng fenglin zhang xianlong National Key Laboratory of Crop Genetic Improvement Huazhong Agricultural University Wuhan 430070 China

The early phase of phytohormone induction is a vital stage of somatic embryogenesis. This phase includes a key process for acquiring cellular totipotency through cellular dedifferentiation. To unravel the molecular mechanism of cellular dedifferentiation in cotton, we constructed a cDNA library using the suppression subtractive hybridization method. A total of 286 differential cDNA clones were sequenced and identified. Among these clones, 112 unique ESTs were significantly up-regulated during the early phase of phytohormone induction, and 40.2% of the ESTs were first identified. GST was highly expressed from 6 to 24 h after induction with phytohormone treatment. PRPs were predominantly expressed and exhibited distinct expression patterns in different treatments, suggesting that they are closely related to cellular dedifferentiation in cotton. Putative GhSAMS, GhSAMDC, GhSAHH and GhACO3 involvement in SAM metabolism was identified in this library. The analysis of qRT-PCR showed that two remarkable increased expressions of the four SAM-related genes happened during the early phase of phytohormone induction, and that a highly positive correlation existed between GhSAMS and GhSAHH. The highest expression level of GhSAMS might be associated with its reentry into the cell cycle. The histological observations further showed that some cells accomplished cellular dedifferentiation and division within 72 h in 2,4-D treatment, and that cellular dedifferentiation might be regulated through two alterations in SAM-dependent transmethylation activity in cotton. In addition, the expression patterns of differential genes in different treatments disclosed the complicated interaction between 2, 4-D and kinetin.

关键词：细胞去分化棉花脯氨酸蛋白质转甲基作用

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Suitable internal control genes for qRT-PCR normalization in cotton fiber development and somatic embryogenesis

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Chinese Science Bulletin 2007年第22期52卷 3110-3117页

作者： tu lili zhang xianlong liU DiQiu jin shuangxia CAO jinglin zhu LongFu deng fenglin tan jiafu zhang CunBin National Key Laboratory of Crop Genetic Improvement Huazhong Agricultural University Wuhan 430070 China

The mechanisms of cotton fiber development and somatic embryogenesis have been explored sys-tematically with microarray and suppression subtractive hybridization. Real-time RT-PCR provides the simultaneous measurement of gene expression in many different samples,with which the data from microarray or others can be confirmed in detail. To achieve accurate and reliable gene expression re-sults,normalization of real-time PCR data against one or several internal control genes is required,which should not fluctuate in different tissues during various stages of development. We assessed the gene expression of 7 frequently used housekeeping genes,including 18S rRNA,Histone3,UBQ7,Actin,Cyclophilin,Gbpolyubiquitin-1 and Gbpolyubiquitin-2,in a diverse set of 21 cotton samples. For fiber developmental series the expression of all housekeeping genes had the same down tendency after 17 DPA. But the expression of the AGP gene(arabinogalactan protein) that has high expression level at the later fiber development stage was up-regulated from 15 to 27 DPA. So the relative absolute quanti-fication should be an efficient and convenient method for the fiber developmental series. The expres-sion of nonfiber tissues series varied not so much against the fiber developmental series. And three best control genes Histone3,UBQ7 and Gbpolyubiquitin-1 have to be used in a combinated way to get better normalization.

关键词：棉花纤维胚胎测量模式

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