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Construction method and application of Pichia stipitis large-fragment DNA (deoxyribonucleic acid) genome library

A Pichia stipitis and genome library technology, applied in the field of bioengineering, can solve problems such as unknown gene sequences

Inactive Publication Date: 2013-05-08
JIANGNAN UNIV
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Problems solved by technology

However, the research on the gene level of Pichia stipitis is still in its infancy, and there are few literatures that can be retrieved. Although its genome sequencing has been preliminarily completed, many key functional target gene sequences (such as cellobiase-related genes) are still unknown. unknown

Method used

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  • Construction method and application of Pichia stipitis large-fragment DNA (deoxyribonucleic acid) genome library
  • Construction method and application of Pichia stipitis large-fragment DNA (deoxyribonucleic acid) genome library
  • Construction method and application of Pichia stipitis large-fragment DNA (deoxyribonucleic acid) genome library

Examples

Experimental program
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Embodiment 1

[0029] Example 1 Construction of Pichia stipitis CBS 5773 Large Fragment DNA Genomic Library

[0030] 1.1 Extraction of Pichia stipitis genomic DNA

[0031] Take Pichia stipitis CBS 5773 and inoculate it in YEPD liquid medium, shake and cultivate overnight at 30°C, the shaker speed is 200r / min; take 2mL culture into a 5mL centrifuge tube, centrifuge at 6000r / min for 5min, discard Collect the supernatant, add 0.5mL Yeast Chromosome Extraction Reagent Ⅰ (0.1mol / L Na2EDTA-0.9mol / L Sorbitol, pH7.5), resuspend the bacteria; add 30μL 5mg / mL snail Enzyme, treat the bacterial cells at 37°C for 5 hours (at this time, most cells form protoplasts), centrifuge at 6000r / min for 5 minutes, discard the supernatant, and collect the bacterial cells; add 0.5mL yeast chromosome extraction reagent II (20mmol / L Na2EDTA-50mmol / L Tris-HCl, pH7.4), resuspend the cells, add 50μL of SDS with a mass volume concentration of 10%, and incubate at 65°C for 30min to lyse the cells; add 200μL of 5mol / L potas...

Embodiment 2

[0041] Example 2 Quality Evaluation of Pichia stipitis Large Fragment DNA Genomic Library

[0042] According to the number of positive clones obtained in the genomic library and the size of the average inserted fragment, according to the formula: library capacity = number of positive clones in the library × average size of inserted fragments / size of the biological genome, calculate the size of the genomic library The size of the storage capacity.

[0043] Among them, it is known that the genome size of Pichia stipitis CBS 5773 is 15441179bp, the number of positive clones in the library is 3000, and the average size of the inserted fragment is 5000bp. The calculated library capacity of the genome library is: library capacity = 3000×5000 / 15441179=97%, that is, the total length of the inserts contained in the library is 0.97 times the total length of the Pichiastipitis CBS 5773 genome, that is, any DNA sequence of Pichia stipitis was screened from the library The exact probabi...

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Abstract

The invention provides a construction method of a Pichia stipitis large-fragment DNA (deoxyribonucleic acid) genome library, which comprises the following steps: (1) extracting genome DNA of Pichia stipitis of which the collection number is ATCC NO:58376; (2) constructing a recombinant vector; (3) screening a positive clone; and (4) performing enzyme cutting identification on the positive clone, and saving a library. The invention successfully constructs the Pichia stipitis large-fragment DNA genome library for the first time; the storage capacity of the library is high (the cloning probability is 97%), and the coverage rate is high (N=1.08); and a gene related to cellobiase can be successfully screened by using the genome library, thereby laying a foundation for the screening of an unknown functional target gene, the functional research on the related gene and the further reconstruction on the gene level so as to obtain an ideal industrially-produced strain.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for constructing a large-segment DNA genome library of Pichia stipitis and an application of the genome library in screening unknown functional genes. Background technique [0002] With the development of molecular biology and bioengineering technology, the discovery of new genes and the study of the functions of new genes have become the hotspots of functional genomics research, and the construction of large fragment DNA genome libraries is the basic method of functional genomics research. Genome library refers to a recombinant DNA clone population containing random fragments of all genes of a certain organism, which can truly reflect all the information of the genome of this species. Genomic libraries are mainly used to preserve the genetic information of species and can be used to screen unknown genes of interest with specific functions. [0003] I...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12N15/31C12N15/81C12N15/10
Inventor 张梁石贵阳马经纬薛卫鄢贵龙
Owner JIANGNAN UNIV
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