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Construction of Yeast Two-hybrid cDNA Library and Screening of the Proteins Interacting with ARC1 from the Stigma of Ornamental Kale

Author: YangJia
Tutor: LiYuHua
School: Northeast Forestry University
Course: Developmental Biology
Keywords: Kale Self-incompatibility and ARC1 Yeast two-hybrid cDNA library
CLC: S681.9
Type: Master's thesis
Year: 2009
Downloads: 205
Quote: 2
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Abstract


Plant self-incompatibility (Self-incompatibility, SI) is the pistil on their own and alien pollen recognition, intracellular signaling cascade, and ultimately inhibit the growth of pollen, hinder the fertilization process. SI is the kind of plants taken to promote outcrossing, avoid self strategy. The main part of the genus Brassica self-incompatibility reaction is to inhibit the growth of self-pollen to the stigma mastoid cells by intracellular signaling cascades. Have been identified stigma papillae cells the SI signal transmission factor ARC1 (Arm repeat containing 1) ARC1 contains a U-Box function domain protein with E3 ubiquitin ligase activity via the ubiquitin / 26S proteasome pathway the degradation of its downstream substrate, resulting in the occurrence of SI. Currently, for the ARC1 the downstream substrate SI response mechanisms is unclear. In this paper the kale (Brassica oleracea var.acephala) S the 13b / sub> S 13b self-incompatible lines for materials, extract the stigma of total RNA isolated by affinity chromatography purified mRNA using CytoTrap XR building a database kit to build kale stigma cDNA library used in the yeast two-hybrid system. The original library storage capacity of 2.5 × 10 5 , amplified library storage capacity of about 4 × 10 8 96% of the recombination rate of insert size of 0.4 -3 kb between the average length of about 0.8 kb. Constructed ARC1 full-length yeast bait the expression vector conversion cdc25H of yeast cells to self-activity detection, proven not activate phenomenon. Yeast cytoplasm CytoTrap two-hybrid system as the operating platform, the use of co-transformation method the kale stigma yeast two-hybrid cDNA library screening, screening a total of 5 × 10 5 a yeast clones, preliminary verification and sequencing 2 protein coding sequence was obtained. A protein containing 71 amino acids, similar to the Arabidopsis Nucellin protein, up to 79% consistency, protein Nucellin is a new class of plant aspartic proteases may be involved in the process of barley fertilized ovule nucellus programmed cell death; another protein contains 288 amino acids, similar to the Arabidopsis DnaJ protein, up to 88% consistency, DnaJ is a molecular chaperone protein, heat shock protein Hsp70 is an auxiliary protein, protein translation, folding, unfolding, play an important role in the transport and degradation processes. These studies provide new clues to explore the ARC1 the function and SI molecular mechanisms.

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CLC: > Agricultural Sciences > Gardening > Ornamental Horticulture ( flowers and ornamental trees) > Annual and biennial flower class > Other
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