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Functional analysis of the cellulose synthase-like D (CSLD) gene family in Physcomitrella patens.

机译：Physcomitrella patens中的纤维素合酶样D（CSLD）基因家族的功能分析。

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摘要

The moss Physcomitrella patens is an attractive model organism, due to its small genome, dominant haploid phase, and high rate of homologous recombination. P. patens has eight genes that form a family known as the Cellulose Synthase-Like Ds (CSLDs). They may be involved in the biosynthesis of crystalline or non-crystalline cellulose and polarized tip growth, but their direct role is still unknown. In order to study the functions of CSLDs, RT-qPCR was preformed, and knockout (KO) mutants for PpCSLD1, PpCSLD2, and PpCSLD8 were developed, but showed no obvious phenotype. As a result, PpCSLD4, PpCSLD5, and PpCSLD6 KO vectors were created to produce double KO mutants. For global silencing of the entire CSLD family, an RNAi vector (pUD8D8i) was developed containing both sequences that are highly similar among all P. patens CSLDs and sequences containing beta-glucuronidase. This construct has been placed in a P. patens line that expresses a nuclear localization signal (NLS) for green fluorescent protein fused to beta-glucuronidase, in order to monitor active silencing. The P. patens colonies silenced by pUD8D8i had a significantly smaller area and greater circularity and solidity (P 0.001) than P. patens silenced by the control vector pUGi. Preliminary Carbohydrate Binding Module (CBM) labeling of crystalline cellulose in csld1 KO mutants and wild-type moss showed an unexpected increase in crystalline cellulose deposition in cells grown on mannitol. As a result, a comprehensive polysaccharide labeling study was performed with various monoclonal antibodies and CBMs. We have identified six cell wall carbohydrates that exhibit increased deposition and one that exhibit decreased deposition in cells grown on mannitol
机译：苔藓小孢子菌（Physcomitrella patens）是一种有吸引力的模型生物，因为它的基因组较小，优势单倍体相和同源重组率很高。彭氏疟原虫具有八个基因，这些基因形成称为纤维素合酶样Ds（CSLD）的家族。它们可能参与结晶或非结晶纤维素的生物合成和极化的尖端生长，但是它们的直接作用仍是未知的。为了研究CSLD的功能，进行了RT-qPCR，并开发了PpCSLD1，PpCSLD2和PpCSLD8的敲除突变体，但没有明显的表型。结果，创建了PpCSLD4，PpCSLD5和PpCSLD6 KO载体以产生双KO突变体。为了使整个CSLD家族整体沉默，开发了一种RNAi载体（pUD8D8i），其中包含在所有彭定康氏菌CSLD中高度相似的两个序列以及包含β-葡萄糖醛酸苷酶的序列。为了监测活性沉默，已将该构建体置于P.patens品系中，该品系表达与β-葡萄糖醛酸苷酶融合的绿色荧光蛋白的核定位信号（NLS）。被pUD8D8i沉默的P. patens菌落比被控制载体pUGi沉默的P. patens菌落具有显着更小的面积和更大的圆形度和坚固性（P <0.001）。 csld1 KO突变体和野生型苔藓中结晶纤维素的初步碳水化合物结合模块（CBM）标记显示，甘露醇上生长的细胞中结晶纤维素的沉积出乎意料的增加。结果，使用各种单克隆抗体和CBM进行了全面的多糖标记研究。我们已经鉴定出六种在甘露醇上生长的细胞中表现出增加的沉积的细胞壁碳水化合物和一种表现出减少的沉积的碳水化合物

著录项

  • 作者

    Dimos, Christos Sotirios.;

  • 作者单位

    University of Rhode Island.;

  • 授予单位 University of Rhode Island.;
  • 学科 Biology Molecular.;Biology Plant Physiology.;Biology Cell.;Biology Botany.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 97 p.
  • 总页数 97
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:37:01

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