Preparation and micro-dissection method of wheat alien substitution line chromosome specimen suitable for SLmuCUT (molecular machine industry, MMI) system

Abstract

The invention relates to a preparation and micro-dissection method of a wheat alien substitution line chromosome specimen suitable for a SLmuCUT (molecular machine industry, MMI) system. The preparation and micro-dissection method comprises the following steps: (1) preparing a chromosome specimen to obtain a target chromosome; (2) carrying out micro-dissection and separation on the target chromosome (segment), dripping absolute ethyl alcohol on the processed separation film of the target chromosome, covering a clean glass slide, and putting on an objective table to cut, searching dispersed target chromosome under a low-power lens before cutting, then transferring to a high-power lens to cut, and collecting the separated chromosome by a viscous Eppendorf pipe. The method disclosed by the invention has the characteristics of simple material source, fast preparation, easiness of identification, and low preparation cost, and greatly reduces damage of acid, alkali and the like to the chromosome. The exogenous chromosome (monovalent) in hybrid F1 PMC MI is cut and collected by using the system so as to provide reference to micro-dissection of the exogenous chromosome in a wheat alien substitution line and establish the foundation for follow-up research work such as building of a chromosome library.

Description

Technical field [0001] Wheat alien replacement chromosome specimens preparation methods and micro -cuts suitable for the SL μCUT system are in the field of molecular cell genetics. Background technique [0002] Melanogaster Polytene Chromosome [J] .Chromosoma, 1981, 82: 205-216) a new molecular cell genetic technology.This technology first cuts the fruit flies chromosome, and successfully obtained 80 clones, and then used for rats and humans (Rohme Dan, Howard Fox, Bernhard Herrmann, ET Al.Molecular Clones of the Mouse Complex Derived from MicrodisseCted MetaphaseSeomoseSeomos] .Cell, 1984, 36: 783-788.)., 1989, 338: 348-350).Since Sandry M J, Et Al.isolation of Sequence Common to A and B Chromosomes of Rye (Secale Cereal L.) by Micro Cloning [J] .plant Molecular Biology ReporterSince the use of chromosomal micro -cutting and micro -cloning technology, since obtained the Klong DNA cloning, this technology has been widely used in the construction of the plant chromosome library, t...

AI Technical Summary

Problems solved by technology

[0004](2) The ploidy of the plant genome is diverse, the chromosome structure varies greatly, and the sequence of genes is not conservative enough; the DNA content of the plant chromosome is high, the protein content is low, and most chromosomes cannot Chromosomes of elephant vertebrates show G, R or Q stripes without affecting the DNA sequence (Fukui k., M. Minezawa, Y. Kamisugi, et al. Microdissection of plant chromosome by argon-ion laser beam[J]. Theor Appl Genet., 1992, 84: 787-791); the current C-band is too damaging to the chromosomal DNA, which is not conducive to the construction of a complete DNA library; and some plant chromosomes are not only small, but also have no obvious size difference, and the bands are similar, so it is difficult to Precise identification of chromosomes and their fine structure

If each insert is 1kb, and each chromosome-specific DNA library contains more than 8×105 clones, the workload of library screening is huge