CN102533780A - Basic helix-loop-helix (bHLH) transcription factor for cotton, gene for coding same, and application of bHLH transcription factor - Google Patents
Basic helix-loop-helix (bHLH) transcription factor for cotton, gene for coding same, and application of bHLH transcription factor Download PDFInfo
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Abstract
The invention discloses a basic helix-loop-helix (bHLH) transcription factor for cotton, a gene for coding the same, and application of the bHLH transcription factor. The bHLH transcription factor is named GhGL3, and is protein having an amino acid sequence shown as SEQ ID No:2 in a sequence table or protein derived from the amino acid sequence shown as SEQ ID No:2 through substitution, deletion or addition of one or more amino acids, and having the same transcription factor activity as the amino acid sequence shown as SEQ ID No:2. The gene GhGL3 for coding the bHLH transcription factor GhGL3 is one of the following nucleotide sequences: 1) a DNA sequence shown as SEQ ID No:1 in the sequence table; and 2) a DNA sequence having more than 90 percent of homology with the DNA sequence shown as the SEQ ID No:1 in the sequence table and coding the same functional protein. The gene has important significance for breeding new varieties of fiber plants, particularly new varieties of cotton.
Description
Technical field
The present invention relates to bioengineering field.More specifically, the present invention relates to a kind of bHLH transcription factor and encoding sox and the application of cotton.
Background technology
Cotton fibre is important textile raw material, and the molecular regulation mechanism of study its growth, growing will help the genetic improvement of output of cotton and quality.Cotton fibre is to grow a kind of unicellular structure that forms by the epidermic cell on the ovule outer integument that is positioned at ovary.Its four periods of formation experience: the synthesis phase and the ripening stage [1] of starting period, elongating stage (synthesizing of primary cell wall), secondary cell wall.Cotton ovule epidermic cell (except that pore epidermic cell and micropylar cell) all has the potential that is divided into cotton fibre, but has only 30% epidermic cell finally can form single celled fiber.
Though cotton fiber is to grow a kind of unicellular structure that differentiation forms by the epidermic cell of ovule outer integument, understands very few to the molecular mechanism of its formation.The development of plants biological study shows that similar cell, tissue, allelotaxis's genetic mechanism usually has high conservative property in the different plants.The formation of Arabidopis thaliana epidermal hair is at present known to be to be regulated and control by the transcription factor of some MYB classes.Transcription factor such as WD40 (AtTTG1), MYB (GL1 or WER), and bHLH albumen (GL3 or EGL3) determines the destiny [2,3,4] of Arabidopsis leaf epidermal hair cell jointly.The startup that is to say the Arabidopis thaliana epidermal hair is that (TTG1) the complex body MYB-bHLH-WD40 of product composition controls for GL1, GL3/EGL3 by three genes at least.
The early development process of cotton fiber possibly be similar to the forming process of plant mesocuticle hairs such as Arabidopis thaliana.Know that GL1 (GLABROUS1) is one of oligogene of control Arabidopis thaliana epidermal hair differentiation with WER (WEREWOLF).So; Cotton fibre is as the unicellular epidermal hair on a kind of ovary ovule outer integument; Its growth Initiated Mechanism also receives the control of MYB-bHLH-WD40 complex body regulatory mechanism probably, and more is similar to the growth startup regulation mechanism of the unicellular epidermal hair of Arabidopis thaliana.
In cotton, cloned at present the myb gene of some fibre height or specifically expressing.The R2R3MB gene that is separated to the earliest is GhMYB1-GhMYB6 [5]; The GaMYB2 R2R3MYB transcription factor of encoding is expressed in the fiber early development [6] significantly; The another one myb gene, the Gene A mMIXTA/AmMYBML1 homology [7] of GhMYB25 and control Common Snapdragon cone cell and epidermal hair differentiation; Also find to exist in the cotton AtCPC homologous gene [8] recently.Expression also is separated to many genes relevant with cotton fiber development with quantitate gene through high-throughout Microarry, comprising some cotton myb genes [7-11].In addition, in cotton, also found GhTTG gene (GhTTG1-GhTTG4) [12].Possibly also exist the bHLH that is similar in the Arabidopis thaliana and WD40 albumen and MYB protein-interacting to form startup and the growth [13] that a transcription factor complex body is controlled cotton fibre in the cotton although some investigator infers in view of the above, in cotton, also not report so far.
Summary of the invention
This research has been cloned in the cotton and Arabidopis thaliana GL3 homologous gene GhGL3, and it is an albumen that contains the bHLH structural domain.
Therefore, the purpose of this invention is to provide a kind of bHLH transcription factor and encoding sox thereof.
BHLH transcription factor name provided by the invention is called GhGL3; Be to have the protein of aminoacid sequence shown in the SEQ IDNo:2 in the sequence table, or aminoacid sequence shown in the SEQ ID No:2 is passed through one or several amino acid whose replacement, disappearance or interpolation and has identical with aminoacid sequence shown in the SEQ ID No:2 active by SEQ ID No:2 deutero-protein.
The protein that aminoacid sequence is made up of 628 amino acid shown in the SEQ ID No:2 in the sequence table.
The encoding sox GhGL3 of bHLH transcription factor GhGL3 is one of following nucleotide sequences:
1) dna sequence dna shown in the SEQ ID No:1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of SEQ ID No:1 have 90% above homology, and the identical function protein DNA sequence of encoding.
The dna sequence dna shown in the SEQ ID No:1 is by 1884 based compositions in the sequence table, and the reading frame of this gene is from 1884 bases of 5 ' the 1st base to the of end.
Utilize any carrier that can guide foreign gene in plant, to express,, can obtain transgenic cell line and transfer-gen plant that fiber number and proterties obtain changing GhGL3 gene transfered plant cell provided by the present invention.Gene of the present invention can add any enhancing promotor or inducible promoter in being building up to plant expression vector the time before its transcription initiation Nucleotide.For the ease of transgenic plant cells or plant being identified and screening, can process employed carrier, like the antibiotic marker thing that adds the alternative mark of plant or have resistance.Carry that GhGL3 expression carrier of the present invention can conventional biotechnological means imports vegetable cell through using that Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity are led etc.; By the plant transformed host both can be monocotyledons, also can be dicotyledons.Gene pairs of the present invention is cultivated the textile plant new variety, and it is significant particularly to cultivate new cotton variety.When gene of the present invention is used to change cotton fiber quantity, qualitative character, can adopt following method: 1, gene GhGL3 of the present invention is cloned in the plant conversion carrier through the RNAi method; 2, constructed plant conversion carrier is transformed renewable cotton tissue (or organ) and gene of the present invention is expressed in the tissue that transforms; The tissue (or organ) that 3, will be transformed is cultivated into plant.
More specifically, the present invention provides and the following:
1, bHLH transcription factor GhGL3; It is the protein with aminoacid sequence shown in the SEQ ID No:2, or with aminoacid sequence shown in the SEQ ID No:2 through one or several amino acid whose replacement, disappearance or interpolation and have with the identical transcription factor activity of aminoacid sequence shown in the SEQ ID No:2 by SEQ ID No:2 deutero-protein.
2, according to above 1 described transcription factor, it is characterized in that: it is the protein with aminoacid sequence shown in the SEQ ID No:2.
3, the encoding sox of bHLH transcription factor GhGL3 is one of following nucleotide sequences:
1) dna sequence dna shown in the SEQ ID No:1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of SEQ ID No:1 have 90% above homology, and the protein DNA sequence of the identical functional transcription factor of encoding.
4, according to above 3 described genes, it is characterized in that: the encoding sox of said bHLH transcription factor GhGL3 is the dna sequence dna shown in the SEQ ID No:1 in the sequence table.
5, according to above 4 described genes, it is characterized in that: the reading frame of this gene is for holding 1884 bases of the 1st base to the from 5 '.
6, contain each said expression carrier of above 3-4, preferred plant expression vector, more preferably textile plant expression vector, most preferably cotton expression vector.
7, the clone that contains above each said gene of 3-4, preferred plant clone, more preferably textile plant clone, most preferably cotton cells system.
8, the application of each said gene of above 3-4 in cultivating textile plant new variety, preferred new cotton variety.
9, a kind of method for preparing transgenic cell line or transgenic plant; Said method comprise with according to above 6 described expression vector transfered cells system or plant obtaining transgenic cell line or transgenic plant, said clone or plant optimization are plant cell or plant, more preferably textile plant clone or plant, most preferably cotton cells system or plant.
10, a kind of method for preparing transgenic plant said method comprising the steps of:
1) each described gene of above 3-4 is cloned in the plant conversion carrier through the RNAi method;
2) constructed plant conversion carrier is transformed the tissue (or organ) of renewable plant and said gene is expressed in the tissue (or organ) that transforms; With
The tissue (or organ) that 3) will be transformed is cultivated into plant,
Wherein preferred said plant is a textile plant, is more preferably cotton.
Below in conjunction with specific embodiment the present invention is described further.
Description of drawings
Fig. 1 is the expression analysis (among the figure ,-3d (my god) of GhGL3, and 0d, 3d, 8d represent respectively to bloom the same day from blooming preceding 3 days, bloom back 3 days with bloom back 8 days ovule (PA); Le representes leaf; Po representes pollen; Pe representes petal.Ovule, leaf, pollen are all from wild-type cotton Xuzhou 142 (the upland cotton kind that China cultivates is widely used in the scientific research material, is purchased from the Chinese agriculture academy of agricultural sciences).Lower floor be GhUBQ7 contrast (GhUBQ7 is a conservative gene, thus we with it as contrast, GenBank Accession No.AAZ83341.1);
Fig. 2 is GhGL3 RNAi vector construction figure;
Fig. 3 is GhGL3 RNAi transgene cotton T
0Analyze for Southern that ((M representes molecular weight marker to genomic dna (20 μ g/ swimming lane) among the figure from wild-type cotton (wt) and GhGL3 RNAi transgene cotton; WT representes the wild-type cotton; I-1 to i-10 representes that respectively the transgene cotton strain is 1 and 10).Cut with EcoRI and HindIII enzyme respectively.Probe is that (NPTII is the resistance screening gene to NPTII; Have this gene on our the used conversion carrier; Just can detect as probe with it and to carry the segmental carrier of purpose and whether change in the vegetable lamb body GenBank Accession No.ABN59488.1 over to);
(GL3i-2 among the figure representes GhGL3 RNAi transgene cotton (i-2) T1 generation to Fig. 4 for the qRT-PCR expression analysis of GhGL3 RNAi; WT representes wild-type);
Fig. 5 is phenotype analytical (the GhGL3 RNAi transgene cotton T1 representative type observation of GhGL3 RNAi transfer-gen plant.A: the sophisticated cotton fibre of wild-type, scale: 2cm; The sophisticated cotton fibre of B:GhGL3 RNAi (GL3i-2) transgene cotton, scale: 2cm; C:A schemes to amplify scale: 1cm; D:B schemes to amplify scale: 1cm);
Fig. 6 is GhGL3 dna sequence dna (SEQ ID NO:1; 1884bp, Gossypium land cotton seed (Gossypium hirsutum L.)); With
Fig. 7 is GhGL3 aminoacid sequence (SEQ ID NO:2; 628a.a., Gossypium land cotton seed (Gossypium hirsutum L.)).
Embodiment
Used vegetable material among the embodiment:
In cotton (Gossypium hirsutum L.) (wild-type cotton Xuzhou 142 (the upland cotton kind that China cultivates; Be widely used in the scientific research material; Be purchased from the Chinese agriculture academy of agricultural sciences)) florescence, the observation flowering time of listing is gathered petal, bud, flower and young bell and in ice bath, is brought back in the laboratory; Rapid the collection respectively of utensil with sterilization spent preceding 3 days ovule, florescence ovule and spent 1 to 3 day the ovule quick-frozen in liquid nitrogen in back, and-80 ℃ of preservations are subsequent use.
The expression analysis of embodiment 1, GhGL3
In order to verify whether GhGL3 is the gene of ovule or fiber specifically expressing, is material with each phase ovule, pollen and leaf, and we have carried out the RT-PCR analysis.The result shows, GhGL3 and does not express (Fig. 1) only before the florescence, express in florescence and the ovule that comes into bloom in pollen and leaf, be the gene of ovule specifically expressing.
Concrete grammar: extract the total RNA of cotton (Gossypiumhirsutum) with Plant RNeasy Kit (QIAGEN), detect the integrity of RNA through 1% agarose electrophoresis.Ss cDNA and ds cDNA's is synthetic according to SMART
TMThe method of PCR cDNA Library Construction Kit (CLONTECH), synthesizing single-stranded (ss) and double-stranded (ds) cDNA.By the synthetic Xuzhou of above method 142 cotton leaves, pollen ,-3 days ovules (PA), 0 day ovule (PA), 4 days ovules (PA), 8 days ovule (PA) sscDNA, dilute 10 times after as the template of RT-PCR.Two pairs of primers of RT-PCR are according to the cDNA sequences Design of GhGL3, and cotton GhUBQ7 is used as endogenous contrast.Primer about RT-PCR amplification GhGL3 is respectively:
GL3-SmaI：
5′-CACCCGGGGTCTACTGGAGTTCAACATCAAG-3′(Forward)
GL3-BamHI：
5 '-(Reverse) amplimer of GhUBQ7 of ACGGATCCCTCAACACTTGCTAGCAATTCTTTGC-3 ':
5′-GAAGGCATTCCACCTGACCAAC-3′(Forward)
5′-CTTGACCTTCTTCTTCTTGTGCTTG-3′(Reverse)
Utilize Pst I/BamH I and two pairs of restriction enzyme sites of Sac I/EcoR I respectively, through a pair of primer PCR amplification below using,
GL3-C01：
5′-AGTCTGCAGGAGCTCCTTGCAGAAAGTCAGAAG-3′(Forward)
GL3-C02：
5′-ATAGGATCCGAATTCTGAGAATGCTTAATGCAT-3′
(Reverse)
One section sequence of GhGL3 is made up the (structure of pPZP111-RNAi conversion carrier: utilize two restriction enzyme sites of Hind III and BamH I, 35S promoter is made up among the entering carrier pPZP111 (Invitrogen Company products) among the entering carrier pPZP111-RNAi with forward and reverse direction respectively.(NCBI, intron AJ132636) utilize two restriction enzyme sites of Pst I and Sac I to used intron, Ghsad1-intron are made up get among the carrier pPZP111, are used to make up cotton RNAi conversion carrier with cotton Ghsad1 gene in the RNAi carrier.Amplification intron primer is:
PstI-Intron：5′-ATGCTGCAGGTTAGTGTCTTTCTTTCT-3′
Sac I-Intron:5 '-GCCGAGCTCCTGTGTTCAGTAAAAATA-3 ') (Fig. 2).After order-checking is confirmed no PCR and cloned mistake fully, be transformed in the E.coli DH5 α competent cell (Invitrogen Company products), through Kan resistance screening positive colony.Identify that in intestinal bacteria connection changes the plasmid that makes up among the Agrobacterium AGL1 (Invitrogen Company products) over to after correct again, the hypocotyl and the cotyledon that used cotton 5-7 days are material, and the method that Agrobacterium is infected is carried out gene transformation.Concrete steps are with reference to (Li; X.B., X.P.Fan, X.L.Wang; Et al.The cotton ACTIN1gene is functionally expressed in fibers and participates in fiber elongation.Plant Cell, 2005.17 (3): 859-875).
The Southern analytical results of embodiment 3, GhGL3 RNAi transfer-gen plant
Paterson et al (1993) is pressed in the extraction of cotton genomic dna, genomic DNAsuitable for RFLP or PCR analysis.Plant Molecular Biology Reporter, and 1993.11 (2): the described method of 122-127 is carried out.Get 20 μ g genomic dnas, use EcoRI and HindIII (Takara product) enzyme to cut respectively.Enzyme is cut product through 0.7% agarose gel electrophoresis (30-50V; O/N) separate; On shaking table, handle running gel with ordinary method then: embathe 15min through 0.125N HCl, sex change liquid (1.5M NaCl, 0.5N NaOH) embathes 30min; Neutralizer (1.5MNaCl, 0.5M Tris-HCl pH 7.2) embathes 30min.Use 20 * SSC that southern blotting technique is arrived HybondN at last
+On the nylon membrane (Amersham).
Prehybridization, hybridization all by ordinary method carry out (Jin Dongyan. Li Mengfeng translates. molecular cloning experiment guide M. the 2nd edition. Beijing: Science Press, 1996); Hybond N
+Mannual, Amersham).
Probe mark: the Primer-a-Gene test kit with Promega company carries out, and mark spends the night.PCR Purification test kit with Qiagen company before the hybridization carries out the probe purifying.(whether NPTII is the resistance screening gene, has this gene on our conversion carrier, just can detect as probe with it to carry the segmental carrier of purpose and change in the plant materials as probe with NPTII gene (coding neomycin phosphotransferase).The GenBank Accession No.:ABN59488.1 of NPTII gene).
Wash film and carry out (Hybond N with high rigorous method
+Mannual, Amersham): 2 * SSC, 0.1%SDS, 15min; 1 * SSC, 0.1%SDS, 65 ℃, 15min; 2 * SSC, 0.1%SDS, 65 ℃, 15min.
Preservative film parcel Hybond membrane makes public to the X-ray sheet under-70 ℃.
The result is as shown in Figure 3, as can be seen from the figure, in 8 transgenic lines, has only i-2 to cut at the enzyme of EcoRI and HindIII and hybridizes to a band in the swimming lane, explains that the GhGL3 gene is to insert genome with single copy.In order further to confirm whether genetic stability of GhGL3 (i-2), we detect the copy number of T1 for GhGL3 (i-2) with Southern, and the GhGL3 gene still inserts genome with single copy as a result.Therefore we analyze for GhGL3 (i-2) transgenic line with T1.
The qRT-PCR expression analysis result of embodiment 4, GhGL3 RNAi transfer-gen plant (i-2)
In order to verify that GhGL3 is expressed in T1 and whether is suppressed for GhGL3 RNAi (i-2) transgenic plant; Gather respectively that to spend preceding 3 days ovule, florescence ovule and to spend 1 to 3 day the ovule in back be material; Extract total RNA with Plant RNeasy test kit (QIAGEN), detect the integrity of RNA through 1% agarose electrophoresis.Use TaqMan Reverse Transcription Regents test kit (Applied Biosystems) to carry out reverse transcription, resultant cDNA diluted sample is 1ng/ μ l, gets a microlitre sample and detects, and each sample is established three repetitions in experiment.PRIMEREXPRESS software (Applied Biosystems) is used in the design of gene specific primer.SYBR Green Master mix and ABI 7900 sequence detection systems (Applied Biosystems) are used in PCR reaction and detection thereof respectively.Comparative CT method (User Bulletin# 2, ABI PRISM 7700 sequence detection systems) is adopted in the processing of data.As internal reference data are carried out normalization method with 18S rRNA.Amplimer is:
GHGL3：5′-TGTCCATGGCGAGAAGGAA-3′(forward)
5′-CTGAACTGAGTGGCAATCCAAA-3′(reverse)
18SrRNA：5′-CGGCTACCACATCCAAGGAA-3′(forward)
5′-TGTCACTACCTCCCCGTGTCA-3′(reverse)
As shown in Figure 4, qRT-PCR result shows that the GhGL3 RNAi transfer-gen plant (i-2) that is expressed in of GhGL3 all is suppressed.
The phenotype analytical of embodiment 5, GhGL3 RNAi transfer-gen plant
In order to verify the effect of GhGL3 in the cotton fiber development process; We carry out phenotype to T1 for the GhGL3RNAi transgene cotton and observe (the i-2 transfer-gen plant of selecting single copy to insert); Discovery is compared with wild-type; Transfer-gen plant fiber short and sparse (Fig. 5), this explanation GhGL3 participates in the normal startup and the growth of regulating cotton.These results show that GhGL3 plays an important role in the cotton fiber development process, its mechanism of action is similar to GhMYB109.
Cotton fibre is important textile raw material, and investigators are devoted to improve its output and quality always.It has grown regulating and controlling effect in each period of cotton fiber development a large amount of gene pairss to be arranged all.The regulatory mechanism of understanding cotton fiber development has crucial meaning to improving output of cotton and quality.GhGL3 can be able to use in Gene Engineering of Cotton, finally reaches cotton genetic improvement and the purpose that improves output.
Reference
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Claims (10)
1.bHLH transcription factor GhGL3; It is the protein with aminoacid sequence shown in the SEQ ID No:2, or with aminoacid sequence shown in the SEQ ID No:2 through one or several amino acid whose replacement, disappearance or interpolation and have with the identical transcription factor activity of aminoacid sequence shown in the SEQ ID No:2 by SEQ ID No:2 deutero-protein.
2. transcription factor according to claim 1 is characterized in that: it is to have the protein of aminoacid sequence shown in the SEQ ID No:2 in the sequence table.
3.bHLH the encoding sox of transcription factor GhGL3 is one of following nucleotide sequences:
1) dna sequence dna shown in the SEQ ID No:1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of SEQ ID No:1 have 90% above homology, and the protein DNA sequence of the identical functional transcription factor of encoding.
4. gene according to claim 3 is characterized in that: the encoding sox of said bHLH transcription factor GhGL3 is the dna sequence dna shown in the SEQ ID No:1 in the sequence table.
5. gene according to claim 4 is characterized in that: the reading frame of this gene is for holding 1884 bases of the 1st base to the from 5 '.
6. contain each said expression carrier of claim 3-4, preferred plant expression vector, more preferably textile plant expression vector, most preferably cotton expression vector.
7. the clone that contains each said gene of claim 3-4, preferred plant clone, more preferably textile plant clone, most preferably cotton cells system.
8. the application of each said gene of claim 3-4 in cultivating textile plant new variety, preferred new cotton variety.
9. method for preparing transgenic cell line or transgenic plant; Said method comprises expression vector transfered cell according to claim 6 system or plant obtaining transgenic cell line or transgenic plant, and said clone or plant optimization are plant cell or plant, more preferably textile plant clone or plant, most preferably cotton cells system or plant.
10. method for preparing transgenic plant said method comprising the steps of:
1) each described gene of claim 3-4 is cloned in the plant conversion carrier through the RNAi method;
2) constructed plant conversion carrier is transformed the tissue (or organ) of renewable plant and said gene is expressed in the tissue (or organ) that transforms;
The tissue (or organ) that 3) will be transformed is cultivated into plant,
Wherein preferred said plant is a textile plant, is more preferably cotton.
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| CN102787121B (en) * | 2012-06-14 | 2014-04-30 | 浙江大学 | Method for validating transcription factor gene function |
| CN104611338A (en) * | 2015-02-28 | 2015-05-13 | 中国科学院遗传与发育生物学研究所 | GhGL3 gene promoter, vector and application thereof |
| CN104611338B (en) * | 2015-02-28 | 2018-11-16 | 中国科学院遗传与发育生物学研究所 | GhGL3 gene promoter, carrier and its application |
| CN104711266A (en) * | 2015-03-31 | 2015-06-17 | 华中师范大学 | Identification and application of cotton bHLH transcription factor gene GhFP1 and promoter thereof |
| CN104711266B (en) * | 2015-03-31 | 2018-04-27 | 华中师范大学 | The identification and application of one cotton bHLH transcription factor genes GhFP1 and its promoter |
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