CN1683530A

CN1683530A - Method for electronically cloning crop function gene using data character site information

Info

Publication number: CN1683530A
Application number: CN 200410084571
Authority: CN
Inventors: 曾华宗; 罗利军; 钟扬
Original assignee: SHANGHAI MUNICIPAL AGRICULTURAL BIOLOGICAL GENE CENTER; Fudan University
Current assignee: SHANGHAI MUNICIPAL AGRICULTURAL BIOLOGICAL GENE CENTER; Fudan University
Priority date: 2004-11-25
Filing date: 2004-11-25
Publication date: 2005-10-19
Anticipated expiration: 2024-11-25
Also published as: CN1312281C

Abstract

The present invention belongs to the field of electronic functional gene cloning technology, and is especially electronically cloning method of crop function gene with crop resistance channel reletive QTL information. The method selects interested QTL as the inquiry probe and analyzes by using the nucleotide sequence inside the QTL, and includes utilizing QTL sequence in searching dbEST library, utilizing gene predicting software in gene prediction of QTL sequence, performing homogeneous comparison between the sequence and the crop genome BAC library and other steps. Finally, PCR primer is designed based on the predicted functional gene and cDNA cloning is obtained in a RT-PCR process. The method has high reliability of the predicted result and is one new method of finding out functional gene included inside QTL. The method has no specific requirement on the crop and thus has wide applicability.

Description

Utilize the data character site information to carry out the method for electronically cloning crop function gene

Technical field

The invention belongs to functional gene electronic cloning technical field, be specifically related to a kind of method of utilizing data character site (QTL) information to carry out electronically cloning crop function gene.

Background technology

Electronic cloning is to be accompanied by the gene clone novel method that genome plan and EST (expressed sequence tag) planned development get up in recent years, cardinal principle is to utilize growing bioinformatics technique, huge arithmetic capability by robot calculator, by EST or genomic sequence assembling and splicing, utilize the method for RT-PCR (reverse transcription-polymerase chain reaction) to obtain functional gene fast, have drop into low, speed is fast, technical requirements is low and advantage such as with strong points.Along with the enforcement of rice genome plan, existing researchist utilizes the method for electronic cloning to clone the functional gene of a lot of paddy rice.The method of utilizing electronic cloning to carry out the excavation of crop functional gene at present has two kinds:

1 utilizes est database information

The electronic cloning that utilizes est database information to carry out functional gene is present the most frequently used means, and its experiment flow is seen Fig. 1.At first select interested target crop EST as the inquiry probe, search for this crop dbEST (nonredundancy EST) database, find partly overlapping EST to splice, and then be new inquiry probe to splice good EST contig (EST contig), continue search dbEST storehouse, till not having new EST to supply splicing, at last good according to splicing complete sequence design PCR primer, the method by RT-PCR obtain purpose cDNA (complementary DNA (cDNA)) and clone and carry out the sequencing checking.Except utilizing this crop EST to do the inquiry probe, can also select other species especially the nearer species total length of sibship or EST as the inquiry probe, search for the dbEST storehouse of this crop, and then being spliced into complete cDNA sequence, its main theory foundation is to have sequence conservation between the different plant species isoformgene.

2 utilize genomic information

Along with finishing of the full-length gene group sequence of increasing plant order-checking, and freely use, promoted the electronic cloning of plant function gene for the whole world.The great advantage of utilizing the genomic information data to carry out electronic cloning is exactly the restriction that the clone of gene is not subjected to crop developmental stage or special environment condition, can be with the EST of this crop that derives from any period or tissue and other crops or full length cDNA sequence as the information probe, search GenBanki (Nucleotide database), predict by artificial splicing or correspondent computer software according to the rule of intron " GU...AG " subsequently, can obtain the opening code-reading frame (ORF) of this gene complete, sequence results design PCR primer according to splicing, further take the method for RT-CR to obtain the cDNA clone of goal gene and carry out sequencing, concrete experiment flow is seen Fig. 2.

Summary of the invention

The objective of the invention is to include a large amount of functional genes in the QTL information at crop, propose a kind of have a broad applicability crop is carried out the method for functional gene electronic cloning.

The method of carrying out electronically cloning crop function gene that the present invention proposes, be to utilize the degeneration-resistant relevant QTL of crop (data character site) information to carry out the electronic cloning of functional gene, on above-mentioned two kinds of electronic cloning method bases, innovated and a kind of new electronic cloning method that forms.This method does not have specificity to species, as long as the species that are studied have relevant QTL and EST information, and the genome of this crop checks order, and all can adopt this method to carry out gene and excavate, so have extensive applicability.Its experiment flow is seen Fig. 3.Concrete steps are as follows:

Select interested QTL as the inquiry probe, utilize the nucleotide sequence of QTL inside to analyze:

1, utilizes the nucleotide sequence search dbEST storehouse of QTL inside, find all degeneration-resistant relevant est sequences with this sequence homology;

2, utilize predictive genes software (as Genscan (http://ccr-081.mit.edu/GENSCAN.html) and Fgenesh (http://www.softberry.com) software) that this QTL sequence is carried out predictive genes, find inner all genes that relate to of this sequence;

3, this sequence and this crop gene group BAC (bacterial artificial chromosome) library are carried out the homology comparison, to find the pairing crop gene group of this sequence BAC library;

4, the result of comprehensive above-mentioned three steps is positioned at predictive genes result's relative position with est sequence, because the corresponding functional gene of EST, so may be degeneration-resistant relevant gene with predicted gene that EST mates; Compare with the functional gene of predicting in the functional gene of these predictions and the crop BAC library simultaneously, with the accuracy that further predicts the outcome;

5, at last according to the functional gene design PCR primer of prediction, the method by RT-PCR obtains purpose cDNA clone and carries out the sequencing checking.

By this method, can obtain the gene of controlled target proterties among the QTL easily and efficiently, and because all corresponding corresponding degeneration-resistant est sequence of these functional genes, so the reliability that predicts the outcome is higher, be the functional gene that a kind of QTL of discovery inside is comprised, and illustrate a kind of novel method of the mutual relationship between QTL and the gene.

Description of drawings

Fig. 1 utilizes the crop est database to carry out the method flow diagram of electronic cloning, illustrates: the basic local area network connection of * is joined search tools, a kind of sequence alignment algorithm and software.

Fig. 2 utilizes crop gene group information to carry out the schema of the method for functional gene electronic cloning.

Fig. 3 utilizes the degeneration-resistant QTL information of crop to carry out the schema of the method for functional gene excavation.

Embodiment

With the paddy rice is example, and concrete operating process is:

1, select interested QTL as the inquiry probe, to Cornell paddy gene database website ( Http:// stein.cshl.org/) and Japanese paddy rice genome plan website ( Http:// rgp.dna.affrc.go.jp) obtain the Molecular Marker Information at these QTL two ends and the genome sequence between two molecule markers.

2, utilize this genome sequence to the NCBI website ( Http:// www.ncbi.nih.gov/) search dbEST storehouse, find all degeneration-resistant relevant est sequences with this sequence homology.

3, utilize predictive genes software Genscan ( Http:// ccr-081.mit.edu/GENSCAN.html) and Fgenesh ( Http:// www.softberry.com) software carries out predictive genes to this sequence, finds inner all genes that relate to of this sequence.

4, this sequence and this crop gene group BAC (bacterial artificial chromosome) library are carried out the homology comparison, to find the pairing rice genome BAC of this sequence library.

5, the result of comprehensive above-mentioned three steps is positioned at the gene correspondence position that is mated with est sequence, because the corresponding functional gene of EST, so may be degeneration-resistant relevant gene with the predicted gene of EST coupling; Compare with the functional gene of predicting in the functional gene of these predictions and the paddy rice BAC library simultaneously, with the accuracy that further predicts the outcome.

6, at last according to the functional gene design PCR primer of prediction, the method by RT-PCR obtains purpose cDNA clone and carries out experimental verification.

Utilize this method, we analyze part paddy rice QTL interval, the result shows between most QTL mark zones all and one or more degeneration-resistant relevant EST homologies, because EST represents the Partial cDNA expressed sequence of a functional gene, so determine that these QTL intervals exist one or more and degeneration-resistant relevant gene.Partial results is as follows:

On the karyomit(e) 4 between the QTL mark zone of control foundation thick (root thickness) RZ23--RM255 corresponding nucleotide sequences length be 910KB, carrying out BLAST with the dbEST database analyzes, discovery has 14 degeneration-resistant relevant EST to match, wherein EST----CA763678 is similar to the zinc finger protein (RING finger-like protein) in the Arabidopis thaliana, and zinc finger protein is and the closely-related proteinoid of plant stress-resistance.Two EST (CA759506, CA759507) and aldose reductase gene (aldose reductase-related protein) sequence homology, this gene has proved with paddy rice anti contravariance closely related.In addition, CA753947 and ethylene response factor bindin (ethylene-responsive element binding protein) homology, this ethene is considered to the regulatory factor that plays an important role in plant stress-resistance reaction, as seen in the interval RZ23-RM255 of QTL, exist at least 3 with degeneration-resistant relevant gene.

Nucleotides sequence on the karyomit(e) 3 between the interval RZ519～RZ448 of the QTL of control rice tillering number and plant height proterties and 3 paddy rice anti contravariance EST height homologies are respectively CA759165 (relevant with receptor kinase), CA765696, CA756047 (relevant with the phosphoglucomutase gene); It is the BAC genomic library of AC082645 that this sequence is positioned at genebank mark mark, is positioned at inner 94892----119035bp place, this library.The information in comprehensive this library and the result of predictive genes software, find that only there are 4 genes in this QTL inside, a gene and CA759165, two EST couplings of CA765696 are wherein arranged, be and degeneration-resistant closely-related receptor kinase gene (putativereceptor kinase) so can predict this gene; Also having a predictive genes with the CA756047 coupling in addition is phosphoglucomutase gene (phosphoglucomutase), and this gene also plays a significant role under paddy rice anti contravariance is coerced.

Claims

1, a kind of method of carrying out electronically cloning crop function gene, it is characterized in that utilizing the degeneration-resistant relevant data character site-QTL information of crop to carry out the electronic cloning of functional gene, concrete steps are as follows: select interested QTL as the inquiry probe, utilize the nucleotide sequence of QTL inside to carry out following analysis:

(1) utilizes the nucleotide sequence of QTL inside to search for the dbEST storehouse, find all degeneration-resistant relevant est sequences with this sequence homology;

(2) utilize predictive genes software that this QTL sequence is carried out predictive genes, find inner all genes that relate to of this sequence;

(3) this sequence and this crop gene group BAC library are carried out the homology comparison, to find the pairing crop gene group of this sequence BAC library;

(4) result of comprehensive above-mentioned three steps is positioned at predictive genes result's relative position with est sequence, compares with the functional gene of predicting in the functional gene of these predictions and the crop BAC library simultaneously, and is preparatory with what further predict the outcome;

(5) at last according to the functional gene design PCR primer of prediction, the method by RT-PCR obtains purpose cDNA clone and carries out the sequencing checking.