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CN113980977A - Application of cotton Gh _ A09G0075 gene in plant growth regulation - Google Patents

Application of cotton Gh _ A09G0075 gene in plant growth regulation Download PDF

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CN113980977A
CN113980977A CN202111375608.9A CN202111375608A CN113980977A CN 113980977 A CN113980977 A CN 113980977A CN 202111375608 A CN202111375608 A CN 202111375608A CN 113980977 A CN113980977 A CN 113980977A
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flowering
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gha09
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CN113980977B (en
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江静
韩栓
刘凌云
王胜楠
贾明珠
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Henan University
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Abstract

The application belongs to the technical field of cotton genetic engineering, and particularly relates to an application patent application of a cotton GH _ A09G0075 gene in plant growth regulation. The gene is located on chromosome 9 of upland cotton TM-1A genome, has 1125bp in total, and has a sequence shown in SEQ ID No. 1. The gene is located in the nucleus and mainly expressed in the leaves; the gene expression level is in positive correlation with the length of hypocotyl and the length phenotype of petiole; the gene regulates flowering factorFTRegulating the flowering-time and the expression level of the gene is positively correlated with the flowering-time, i.e., by expressionThe amount is increased to promote the flowering time to advance, and the flowering time is delayed by gene inactivation. Based on the researches, a certain gene resource foundation and a certain gene engineering technical foundation can be laid for plant phenotype regulation and new agronomic character cotton new variety cultivation.

Description

Application of cotton Gh _ A09G0075 gene in plant growth regulation
Technical Field
The application belongs to the technical field of cotton genetic engineering, and particularly relates to an application patent application of a cotton GH _ A09G0075 gene in plant growth regulation.
Background
Light has many effects on plant growth and development, and among them, phytochrome (phytochrome) is the earliest and most well studied protein as the receptor of red light and far-red light in plants. Based on the spectral difference, the photosensitizing pigments are correspondingly classified into two types, red light absorption type and far-red light absorption type. Further research shows that the 15 th subfamily of the transcription factor bHLH family, which is the phytochrome interacting factors, has important influence on the function of the phytochrome through the interaction with far-red light absorption type phytochrome.
So far, in Arabidopsis thaliana (Arabidopsis thaliana) 7 members of the PIFs family were found (PIF 1, PIF3-PIF 8), rice (Oryza sativa) 6 members of the group (OsPIL 11-OsPIL 16), maize (corn: (A))Zea mays) There were 7 members (ZmPIF 1-ZmPIF 7). Partial studies with arabidopsis PIF4 have shown that this transcription factor is associated with the modulation of a plant's partial growth phenotype, which also suggests that the function of the phytochrome-acting factor PIF is likely to be diverse, rather than unique.
Based on the studies of PIF in arabidopsis thaliana and other plants, some researchers have performed cloning studies of related PIF genes in cotton. However, due to the diversity of functions of transcription factors and the complexity of biological research, the deep research on different PIF genes in cotton has extremely important scientific and technological significance for related gene function analysis and new variety breeding.
Disclosure of Invention
The application aims to provide cottonGH_A09G0075The gene lays a certain technical foundation for regulating and controlling the relevant characteristics of plant growth from the gene level.
The technical solution adopted in the present application is detailed as follows.
CottonGH_A09G0075The gene is located on chromosome 9 of upland cotton TM-1A genome, has 1125bp in total, has a sequence shown as SEQ ID No.1, and specifically comprises:
ATGAACCACTGTAGTTCAGATTGGAATTTTGATTCTGATCTTCCAATCTCCAATCAAAAAAAACTCTTGGGTCAAGATAATGAACTAGTAGAGCTTTTATGGCAGAATGGGAAGGTAGTTTTGCAAAGCCAAACTCATAAGAAACAAGATGATGAAACAGTCTCGTGGATCCAAAACCCTTTTGAGGAATCATTTGAGAAAGAGATGTTTTCCAACTTTTTCTCTGAATTTCCAGCTTATGATCCCATGGATCACCAACATGAACAACAAGAGGATGATAAAAGTTTAAAACATAAGAACAATTTGATGCAAAGAGAAGTTAATGAACTTTCAGGAATGGCAATTGGGTCAAGCCATTGTGGAAGCAATCAAGTTCGAAATGATGGAGATCTTAGCAGAGGATCAAGCAATGGAATTGGAACTACATCCACTGGTTTATCTGTAGGGACATCTAAAGATGATGAAGATGATTATAATGGTGGCCAAGTTGAAAATGAGAAAGGAACTTCAGTTGGAGTCACTAGTAGCCAGAAAAGGAAGAACGGAGATGGTAGGGAAGATTATGAGTGTCAAAGTGAGTTTGCTGAAGATAAGCCAGTCCGACGATCTGGGTCTTATCGTCGCCGGAGTCGAGCTGCCGAAGTTCATAATTTATCGGAGAGGAGAAGAAGGGATAGGATCAATGAGAAGATGAGAGCATTACAAGAGCTCATCCCTCACTGCAATAAGACAGATAAAGCATCTATGCTAGATGAGGCCATTGAATATTTGAAGTCACTTCAATCACAACTTCAGGTCATGTGGATGGGAAATGGGATGGCCCCAATGATGTTCCCTGGAATCCAACATTATATGTCCCCCATGGCAATGGGAACAGCTCCACCTACAATGCCTTCAATTCAAAACCAACGTTTTATTCAAAATCCCACTTTTTCTGAACAATATGCACGTTTTTTAGGCTTTCAACATATGCAAACAGCTTCTCAGCCAATTAATATGTTTGGTTATGGCTCCCAAACTACACCTCAAAGTCCCACGGTATTAGGCTTTAGCAATGGCAGTAACCAACTCAATGGAGGAATAACTGCTGCCAATAACACTTCTCTAAGTGGCAAGATAGGATGA。
cottonGH_A09G0075The GH _ A09G0075 protein encoded by the gene has an amino acid sequence (384 amino acids) shown as SEQ ID No.2, and specifically comprises the following steps:
MSFKASISLAMNHCSSDWNFDSDLPISNQKKLLGQDNELVELLWQNGKVVLQSQTHKKQDDETVSWIQNPFEESFEKEMFSNFFSEFPAYDPMDHQHEQQEDDKSLKHKNNLMQREVNELSGMAIGSSHCGSNQVRNDGDLSRGSSNGIGTTSTGLSVGTSKDDEDDYNGGQVENEKGTSVGVTSSQKRKNGDGREDYECQSEFAEDKPVRRSGSYRRRSRAAEVHNLSERRRRDRINEKMRALQELIPHCNKTDKASMLDEAIEYLKSLQSQLQVMWMGNGMAPMMFPGIQHYMSPMAMGTAPPTMPSIQNQRFIQNPTFSEQYARFLGFQHMQTASQPINMFGYGSQTTPQSPTVLGFSNGSNQLNGGITAANNTSLSGKID。
cottonGH_A09G0075The gene is applied to plant growth regulation, and the cotton GH _ A09G0075 is positioned in a cell nucleus and mainly expressed in leaves; the gene expression level is in positive correlation with the length of hypocotyl and the length phenotype of petiole; the gene regulates flowering factorFTIs regulated by the level of gene transcriptionThe flowering time is shortened, and the expression level of the gene is in positive correlation with the flowering time, namely, the flowering time is promoted to be advanced by increasing the expression level, and the flowering time is delayed by inactivating the gene.
Method for breeding new transgenic arabidopsis variety transformed with cottonGH_A09G0075Gene by over-expression of cottonGH_A09G0075The gene and the arabidopsis thaliana new variety have the characteristics of a new variety with elongated hypocotyl length, elongated petiole length and advanced flowering time.
Based on the research of the Arabidopsis PIF and the research of the cotton PIF gene in the prior art, different cotton varieties are researchedG.hirsutum(upland cotton),G.raimondii(Ramond cotton) andG.arboreumthe GhPIF family gene in tree cotton and Asian cotton is compared and screened, and the inventor selects cottonGH_A09G0075The gene is used as a research object, and the function of the gene is preliminarily analyzed and researched.
Based on the comparative analysis of related genetic relationship, the inventor believes that the cotton isGH_A09G0075The gene has closer genetic relationship with Arabidopsis PIF4 in evolution, and has obvious promotion effect on stem growth, petiole elongation, flowering time and the like in the aspect of plant growth and development. Based on the researches, a certain gene resource foundation and a certain gene engineering technical foundation can be laid for plant phenotype regulation and new agronomic character cotton new variety cultivation.
Drawings
FIG. 1 shows the homology alignment result of GH _ A09G0075 amino acid sequence;
FIG. 2 shows the genetic relationship analysis results of Arabidopsis PIF4 and upland cotton GH _ A09G0075 genes;
FIG. 3 shows the expression localization results of GH _ A09G0075 cells;
FIG. 4 shows the detection results of the expression levels of GH _ A09G0075 in different cotton varieties and different tissues (roots, stems, true leaves and cotyledons); wherein A is inG.arboreum(Cotton gossypii, Asian cotton) and B is inG.raimondii(Redmond cotton) assay conditions, C is inG.hirsutum(upland cotton) test cases;
FIG. 5 shows the tissue expression detection of GH _ A09G0075 in Arabidopsis; i to VI are different developmental stages and different tissue detection results, wherein: i, seedlings of 2 days old; II, 7-day-old seedlings; III, seedlings of 2 weeks old; IV, stem; v, inflorescence; VI, silique;
FIG. 6 shows the effect of GH _ A09G0075 on growth regulation of Arabidopsis after anaplerosis; wherein: a, detecting the expression level of GhPIF4 in different experimental materials (RT-PCR); b is the visual comparison of the quantity of leaves of each material when bolting and flowering; c is the statistical result of the number of leaves of each material when bolting and flowering; d, visually comparing the flowering phenotypes of the materials at the same time point; e, counting and comparing the ratio of flowering plants in the growth process of each material;
FIG. 7 is the effect of GH _ A09G0075 on elongation growth of hypocotyls of Arabidopsis; wherein: a is the visual comparison of the length of the hypocotyl of each material; b is the counting result of the length of the hypocotyl of each material;
FIG. 8 is a graph showing the effect of GhA09 on the elongation growth of the hypocotyl of Arabidopsis thaliana under dark culture conditions; wherein: a is the visual comparison of the length of the hypocotyl of each material; b is the counting result of the length of the hypocotyl of each material;
FIG. 9 is the effect of GH _ A09G0075 on the elongation growth of Arabidopsis petioles; wherein: a is the visual comparison of the lengths of the petioles of the materials; b is the result of the leaf stalk length statistics of each material;
FIG. 10 is overexpressionGH_A09G0075The GhA09 gene in the strain has influence on the growth phenotype of arabidopsis; wherein: a, detecting the expression level of GhA09 genes in each experimental material (RT-PCR); b is the visual comparison of the quantity of leaves of each material when bolting and flowering; c is the statistical result of the number of leaves of each material when bolting and flowering; d, visually comparing the flowering phenotype of each material; e, comparing the ratio of flowering plants in each growth period; f in each materialFTAnd (4) carrying out statistical comparison on the gene expression level.
Detailed Description
The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description will be given of some experimental background cases in the following embodiments.
Biological material:
the cotton varieties mainly involved areG.hirsutum(including TM-1 and JI 11), G.raimondii and G.aThe varieties are common cotton research varieties in the prior art and can be obtained from public channels;
mutants involved in Arabidopsis thalianapif4-2From the american arabidopsis seed resource center;
the over-expression plasmid vector pCAMBIA1300, the plasmid pCMBIA-1391-GUS and the plasmid pHBT-GFP are all common plasmids in the genetic engineering technology and can be obtained from public channels;
coli DH5 α competent cells, purchased from whole gold organisms;
agrobacterium GV3101 and GV3101 (p 19) competent cells, purchased from Bomeyer organisms;
experimental reagents and instrumentation:
related restriction enzymes are all products of Takara company;
plant RNA extraction kit, product of LifeFeng company;
AceQ qPCR SYBR®green Master Mix, 2 XTaq Plus Master Mix II, one-step cloning kit, all Vazyme company products;
RNA reverse transcriptase, KOD ONE Hi-Fi enzyme, product of Toyobo company;
a plasmid extraction kit, an agarose gel recovery kit, a DNA purification kit, products of Omega company;
enhanced RIPA lysate, a bosch de bio products;
MCX column, wawter corporation, usa;
DNA Marker (2K), product of all-gold Biotechnology Ltd.
Example 1
In the early research process aiming at the arabidopsis PIF4, the arabidopsis PIF4 has wide influence on the processes of light modulation and control morphogenesis, high temperature, low temperature, drought stress response, biological clock, flowering and the like, and in view of the functional diversity of the transcription factors, the inventor considers that the PIF related genes in cotton need to be further researched based on the analysis requirement of cotton genome, so that a certain technical basis is laid for cotton cultivation.
In the research process, the inventor firstly combines the amino acid sequence of Arabidopsis AtPIF4 with the upland cotton in cotton (G.hirsutum) Raymond cotton (G.raimondii ) And Asian cotton (G.arboreum) The unknown homologous protein sequences in (1) are preliminarily aligned, and the results are shown in FIG. 1. After analysis, it can be seen that:
arabidopsis AtPIF4 amino acid sequence and Gh _ A09G0075 (short for) in upland cottonGhA09) The homology is high, highly homologous amino acid sequences also exist in other two cottons, and the sequences of the key functional domain bHLH as a transcription factor are completely identical, so that it can be preliminarily 'guessed' that the Gh _ A09 may have similar functions to those of Arabidopsis PIF and may have certain influence on the growth and development regulation of cotton.
Based on the related genome sequence information, the inventors further carried out comparative analysis on the evolution relationship of the arabidopsis AtPIF4 gene and the upland cotton GH _ A09. As shown in fig. 2, the analysis shows that the AtPIF4 gene has a close evolutionary relationship with gossypium hirsutum GH _ a09, which further provides a reference for "guessing" the specific function of GH _ a09 by the function of AtPIF 4.
Based on the comparison result, further research and clarification of cottonGhA09The gene function is obtained by further cloningGhA09The specific process of the gene is described below.
(I) extracting the genome of upland cotton TM-1
Extracting RNA of upland cotton TM-1 by referring to the specification of a plant RNA extraction kit and the prior art, and performing reverse transcription to obtain cDNA for later use;
meanwhile, referring to AtPIF4 gene and the existing cotton genome sequencing result, the primer pair for PCR amplification is designed as follows:
35S::Gh A09-GFP-F：5’-GAGCTCGGTACCCGGGGATCCATGAACCACTGTAGTTCAGATTGGAA-3’，
35S::Gh A09-GFP-R：5’-CATGTCGACTCTAGAGGATCCTCCTATCTTGCCACTTAGAGAAGTGT-3’；
(II) PCR amplification
Carrying out PCR amplification by using the cDNA prepared in the step (I) as a template and using a designed primer pair; during PCR amplification, a 50 mu L amplification system is designed as follows:
2×KOD OneTM PCR Master Mix，25 μL；
Forward Primer（10 μM），1.5 μL；
Reverse Primer（10 μM），1.5 μL；
cDNA template, 2.0 μ L;
Nuclease-free water，20 μL；
the PCR amplification procedure was as follows: heating at 98 deg.C for 3 min; 98 ℃, 30s, 60 ℃, 30s, 72 ℃, 90s, 35 cycles; 72 ℃ for 5 min; the amplification product was stored at 4 ℃ until use.
And (3) carrying out electrophoresis detection on the PCR amplification product, further recovering and purifying by referring to a related kit, and carrying out sequencing identification. The specific sequencing result of the Gh _ A09G0075 is shown as SEQ ID No.1 is shown.
(III)GhA09Gene mapping
Based on the above sequencing results, in order to further clarify the action characteristics of the Gh _ a09G0075 gene at the cell level, the inventors examined the localization of the gene in the cell, and the specific situation is briefly described as follows.
Firstly, referring to the conventional construction method of recombinant plasmid vector, the plasmid pHBT-GFP is taken as the vector to be recombined and integratedGhA09A gene; note that, in the recombination process, PCR amplification was performedGhA09When the gene is used, the primer sequence is designed as follows:
pHBT::GhA09-F：5’-GGTACCGCGGGCCCGGGATCCATGAACCACTGTAGTTCAGATTGGAA-3’，
pHBT::Gh A09-R： 5’-GCCCTTGCTCACCATGGATCCTCCTATCTTGCCACTTAGAGAAGTGT-3’。
subsequently, arabidopsis thaliana protoplasmic mesophyll cells are used as transformation objects, the recombinant plasmid vectors are transformed, and the transformed cells are observed under a laser confocal microscope after being incubated overnight.
During the experiment, to transformpHBT::GFPPlasmid and recombinant vector containing nuclear markerpHBT::NLSThe transformed cell group (prepared conventionally, referred to the prior art) was used as a control.
The results are shown in FIG. 3. The observation can find that:pHBT::GhA09the position where the carrier emits fluorescence can be determined by the cellThe position markers are overlapped, which shows thatGhA09The gene is mainly expressed in the nuclear part, and meets the basic characteristics of the gene as a transcription factor.
(IV) tissue expression patterns
To further determine the expression characteristics of the Gh _ A09 gene in plant tissue level and to determine the expression difference between the gene and its homologous gene, the inventors designedG. raimondiiAndG. arboreumneutralization ofGhA09Genes with the highest homologyGorai.006G008800AndGa09G0085as a control, the three genes were detected by fluorescent quantitative PCR for 14 days of growthG. raimondii、G. arboreumAndG. hirsutumexpression levels in various tissues of cotton. The specific experimental conditions are briefly described below.
Firstly, extracting the genome of each sample by referring to the existing fluorescent quantitative PCR technology;
then, designing a primer for detection, and carrying out fluorescent quantitative PCR amplification, wherein during PCR detection, the related primer is designed as follows:
to is directed atGa09G0085Then, UBQ7 is used as an internal reference gene, and a primer pair is designed as follows:
qGaUBQ7- F：5’-TCGAGTCTTCGGACACCATTG-3’，
qGaUBQ7-R：5’-TTGTTGGTCAGGTGGAATGC-3’；
qGaPIF4-F：5’-AACAGCTCCACCTACAATGC-3’，
qGaPIF4-R：5’-TTGGCTGAGAAGCTGTTTGC-3’；
to is directed atGorai.006G008800Then, UBQ7 is used as an internal reference gene, and a primer pair is designed as follows:
qGoraiUBQ7-F：5’-TGTGGGCTCACTTACGTTTAC-3’，
qGoraiUBQ7-R：5’-TTTTTGCAAGACACTAACCGC-3’；
qGoraiPIF4-F：5’-TGTGAGTTTGCTGAAGTTAAGC-3’，
qGoraiPIF4-R：5’-AAATTATGAACTTCGGCAGCTC-3’；
to is directed atGhA09When the gene is used, UBQ7 is used as an internal reference gene, and a primer pair is designed as follows:
qGhUBQ7-F：5’-GAAGGCATTCCACCTGACCAAC-3’，
qGhUBQ7-R：5’-CTTGACCTTCTTCTTCTTGTGCTTG-3’；
qGhA09-F：5’-TCTCGTGGATCCAAAACCCT-3’，
qGhA09-R：5’-GCTTCCACAATGGCTTGACC-3’。
the specific quantitative results are shown in FIG. 4. It can be seen that the expression characteristics of the related homologous genes in different cotton materials have certain similarity, but the expression amounts in different materials also have certain difference. Then is turned onGhA09In the light of the above-mentioned reasons,GhA09the expression was low in roots and the expression was highest in leaves.
Example 2
Based on the preliminary study of example 1, to further clarifyGhA09The expression characteristics of the gene take a model plant Arabidopsis thaliana as a transformation object, and the inventor makes clear the specific tissue and organ conditions of the gene on plant growth by constructing a related recombinant expression vector and transforming the related recombinant expression vector into Arabidopsis thaliana materials with different phenotypes. The specific experimental conditions are briefly described below.
First, referring to the prior art and example 1, plasmid pCMBIA-1391-GUS was used as a vector to integrate and recombineGhA09A gene;
subsequently, the above preparation is integrated withGhA09Transforming agrobacterium with recombinant plasmid vector of gene and further preparing dip dyeing liquid for transfection;
then, WT and WT at flowering stage were selected separatelypif4-2The arabidopsis thaliana material is subjected to dip-dyeing transformation by using a method of dip-dyeing arabidopsis thaliana inflorescences by using agrobacterium;
and finally, performing resistance screening and identification on a hygromycin screening culture medium to finally obtain a transgenic strain with correct transformation.
Using GUS histochemical staining method for transformationGhA09Transgenic Arabidopsis of genesGhA09The expression time and position of the gene are detected. The results are shown in FIG. 5.
As can be seen from the visual results of GUS staining,GhA09is obviously existedThe expression level of (2) was very low, with little expression in the stem, little expression in the flower and pod, and highest in the leaf.
Example 3
To further clarify the results of example 2GhA09The actual function of the gene is complemented by the inventor by adopting the gene function complementation experimentGhA09Further verification of gene function was performed, and the detailed experimental conditions and results are summarized below.
First, referring to the prior art and the above examples, the overexpression plasmid vector pCAMBIA1300 is used as the vector for recombinant integrationGhA09A gene;
subsequently, the above preparation is integrated withGhA09Transforming agrobacterium with recombinant plasmid vector of gene and further preparing dip dyeing liquid for transfection;
then, WT and WT at flowering stage were selected separatelypif4-2The arabidopsis thaliana material is subjected to dip-dyeing transformation by using a method of dip-dyeing arabidopsis thaliana inflorescences by using agrobacterium;
and finally, performing resistance screening and identification on a hygromycin screening culture medium to finally obtain a transgenic strain with correct transformation.
Make-up for correct conversionAtpif4-2And observing the phenotype of the material. Specific phenotypic results are shown in figure 6.
Analysis can see that, in terms of the visual phenotype at the flowering time point, under normal growth conditions,Atpif4-2the flowering time is delayed but inGhA09Gene transfer into anaplerotic Arabidopsis thalianaAtpif4-2After that, can recoverAtpif4The phenotype of delayed flowering time, particularly the statistics of bolting days show that the flowering time of WT is close to that of 1# and 4# anaplerotic material, the flowering starts in about 27 days,Atpif4the flowering time is delayed for 2 days; the statistical results of the number of synchronized rosette leaves showed that the ratio of WT to 1# and 4# anaplerotic materialAtpif42 pieces less. Based on the above results, it was confirmed that,GhA09the gene has direct influence on the flowering time of arabidopsis thaliana and has the function and application effect of promoting early flowering.
Example 4
To further clarifyGhA09Amount of Gene expression in plantsInfluence of the phenotype, the inventors onGhA09The gene expression level is further over-expressed, and the influence on plant growth and development under the condition of over-expression of the gene is evaluated. The specific experimental conditions are briefly described below.
First, the compound constructed in example 3 containsGhA09Transforming agrobacterium with recombinant plasmid vector of gene and further preparing dip dyeing liquid for transfection;
then, selecting a WT arabidopsis material in a flowering period, and carrying out dip-dyeing transformation by using a method of carrying out dip-dyeing on arabidopsis inflorescences by using agrobacterium;
then, resistance screening and identification are carried out on a hygromycin screening culture medium, and finally correct transformation is obtainedGhA09-OE transgenic homozygous overexpression lines.
Finally, willGhA09-OE homozygous transgenic plants and untransformed WT plants (control group) were cultivated and evaluated in combination with the growth of transgenic material in example 3GhA09Influence on the development of plants at different growth stages under the condition of gene overexpression.
(1) Effect on hypocotyl elongation growth:
detecting and counting WT growing on MS culture medium for 7 days at 22 deg.C,Atpif4、GhA09Anaplerosis and overexpression of the hypocotyl length of the material.
The results are shown in figure 7 of the drawings,Atpif4the length of the hypocotyl of the mutant is about 1.3mm,GhA09the length of the hypocotyl of the anaplerosis material is similar to that of WT and is about 2.3mm,GhA09the length of the hypocotyl of the overexpression material is greater than WT and is about 3.7 mm. This result indicates that:GhA09the gene has positive regulation on the growth length phenotype of the Arabidopsis hypocotyl, can obviously shorten the growth length of the hypocotyl after mutation inactivation, but can obviously prolong the growth length of the hypocotyl after overexpression.
(2) Effect of GH _ a09 on growth of hypocotyl elongation in arabidopsis under dark conditions:
to clarifyGhA09Whether the influence of the gene on the growth of the hypocotyl is related to the illumination condition or not, the WT is determined,Atpif4、GhA09Anaplerotic and overexpression material at 22 deg.CAfter germination and dark culture for 4 days in the field, the growth length of hypocotyls of etiolated seedlings is observed and counted, and the result is shown in FIG. 8.
As can be seen from the analysis,Atpif4the hypocotyl growth length of the material is 0.57cm and is significantly shorter than that of the WT, the hypocotyl length of the GhA09 anaplement material is consistent with that of the WT and is about 1.1cm, and the hypocotyl length of the GhA09-OE material is significantly longer than that of the WT and is about 1.6 cm. This result indicates that: the growth length phenotype of the Arabidopsis hypocotyls is not only influenced by the lighting conditions, but also byGhA09Regulation of expression of the gene; that is, under dark conditions without light,GhA09the gene still has about positive regulation on the growth length phenotype of Arabidopsis hypocotyl.
(3) Influence on growth of petiole elongation
For WT growing normally for 23 days,Atpif4、GhA09The lengths of the 1 st and 2 nd true petioles were counted for anaplerosis (example 3) and overexpression material of this example.
As a result, as shown in FIG. 9, the average length of the WT petioles was 6.1mm,pif4the average length of the mutant petioles is 3.4cm,GhA09transformation ofAtpif4The average petiole length of the anaplerotic material is 5.7cm, and the petiole length of the overexpression material is 7.5 mm. Namely:Atpif4the results that the petioles are shorter than the WT, the length of the leaf petiole of the anaplerotic material is consistent with that of the WT, and the length of the leaf petiole of the over-expression material is significantly longer than that of the WT (A, B) show that:GhA09based on the fact that the growth length phenotype of the Arabidopsis petiole is about forward regulation, the length of the petiole can be obviously shortened after mutation inactivation, but the growth length of the petiole can be obviously prolonged after overexpression.
(4) Influence of flowering phase
During the flowering phase, grown for 15 days under long-day conditions (light/dark = 16/9)GhPIF4And counting the bolting days, the number of rosette leaves and the expression condition of the FT gene (Flowering locus T) of the over-expression transgenic line. The results are shown in FIG. 10.
As can be seen from the analysis,GhA09-flowering time of OE overexpression transgenic plants was about 3 days earlier than WT. Further, the method comprisesAtFTThe detection result of gene expression level shows that the over-expression gene materialAtFTThe expression level of the gene expression is obviously higher than that of WT. That is to say that the first and second electrodes,GhA09the cotton gene plays a role in regulating the flowering time point of arabidopsis thaliana by regulating the expression of an arabidopsis thaliana flowering factor FT.
SEQUENCE LISTING
<110> university of Henan
Application of <120> cotton Gh _ A09G0075 gene in plant growth regulation
<130> none
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1124
<212> DNA
<213> Gossypium hirsutum
<400> 1
tgaaccactg tagttcagat tggaattttg attctgatct tccaatctcc aatcaaaaaa 60
aactcttggg tcaagataat gaactagtag agcttttatg gcagaatggg aaggtagttt 120
tgcaaagcca aactcataag aaacaagatg atgaaacagt ctcgtggatc caaaaccctt 180
ttgaggaatc atttgagaaa gagatgtttt ccaacttttt ctctgaattt ccagcttatg 240
atcccatgga tcaccaacat gaacaacaag aggatgataa aagtttaaaa cataagaaca 300
atttgatgca aagagaagtt aatgaacttt caggaatggc aattgggtca agccattgtg 360
gaagcaatca agttcgaaat gatggagatc ttagcagagg atcaagcaat ggaattggaa 420
ctacatccac tggtttatct gtagggacat ctaaagatga tgaagatgat tataatggtg 480
gccaagttga aaatgagaaa ggaacttcag ttggagtcac tagtagccag aaaaggaaga 540
acggagatgg tagggaagat tatgagtgtc aaagtgagtt tgctgaagat aagccagtcc 600
gacgatctgg gtcttatcgt cgccggagtc gagctgccga agttcataat ttatcggaga 660
ggagaagaag ggataggatc aatgagaaga tgagagcatt acaagagctc atccctcact 720
gcaataagac agataaagca tctatgctag atgaggccat tgaatatttg aagtcacttc 780
aatcacaact tcaggtcatg tggatgggaa atgggatggc cccaatgatg ttccctggaa 840
tccaacatta tatgtccccc atggcaatgg gaacagctcc acctacaatg ccttcaattc 900
aaaaccaacg ttttattcaa aatcccactt tttctgaaca atatgcacgt tttttaggct 960
ttcaacatat gcaaacagct tctcagccaa ttaatatgtt tggttatggc tcccaaacta 1020
cacctcaaag tcccacggta ttaggcttta gcaatggcag taaccaactc aatggaggaa 1080
taactgctgc caataacact tctctaagtg gcaagatagg atga 1124
<210> 2
<211> 384
<212> PRT
<213> Gossypium hirsutum
<400> 2
Met Ser Phe Lys Ala Ser Ile Ser Leu Ala Met Asn His Cys Ser Ser
1 5 10 15
Asp Trp Asn Phe Asp Ser Asp Leu Pro Ile Ser Asn Gln Lys Lys Leu
20 25 30
Leu Gly Gln Asp Asn Glu Leu Val Glu Leu Leu Trp Gln Asn Gly Lys
35 40 45
Val Val Leu Gln Ser Gln Thr His Lys Lys Gln Asp Asp Glu Thr Val
50 55 60
Ser Trp Ile Gln Asn Pro Phe Glu Glu Ser Phe Glu Lys Glu Met Phe
65 70 75 80
Ser Asn Phe Phe Ser Glu Phe Pro Ala Tyr Asp Pro Met Asp His Gln
85 90 95
His Glu Gln Gln Glu Asp Asp Lys Ser Leu Lys His Lys Asn Asn Leu
100 105 110
Met Gln Arg Glu Val Asn Glu Leu Ser Gly Met Ala Ile Gly Ser Ser
115 120 125
His Cys Gly Ser Asn Gln Val Arg Asn Asp Gly Asp Leu Ser Arg Gly
130 135 140
Ser Ser Asn Gly Ile Gly Thr Thr Ser Thr Gly Leu Ser Val Gly Thr
145 150 155 160
Ser Lys Asp Asp Glu Asp Asp Tyr Asn Gly Gly Gln Val Glu Asn Glu
165 170 175
Lys Gly Thr Ser Val Gly Val Thr Ser Ser Gln Lys Arg Lys Asn Gly
180 185 190
Asp Gly Arg Glu Asp Tyr Glu Cys Gln Ser Glu Phe Ala Glu Asp Lys
195 200 205
Pro Val Arg Arg Ser Gly Ser Tyr Arg Arg Arg Ser Arg Ala Ala Glu
210 215 220
Val His Asn Leu Ser Glu Arg Arg Arg Arg Asp Arg Ile Asn Glu Lys
225 230 235 240
Met Arg Ala Leu Gln Glu Leu Ile Pro His Cys Asn Lys Thr Asp Lys
245 250 255
Ala Ser Met Leu Asp Glu Ala Ile Glu Tyr Leu Lys Ser Leu Gln Ser
260 265 270
Gln Leu Gln Val Met Trp Met Gly Asn Gly Met Ala Pro Met Met Phe
275 280 285
Pro Gly Ile Gln His Tyr Met Ser Pro Met Ala Met Gly Thr Ala Pro
290 295 300
Pro Thr Met Pro Ser Ile Gln Asn Gln Arg Phe Ile Gln Asn Pro Thr
305 310 315 320
Phe Ser Glu Gln Tyr Ala Arg Phe Leu Gly Phe Gln His Met Gln Thr
325 330 335
Ala Ser Gln Pro Ile Asn Met Phe Gly Tyr Gly Ser Gln Thr Thr Pro
340 345 350
Gln Ser Pro Thr Val Leu Gly Phe Ser Asn Gly Ser Asn Gln Leu Asn
355 360 365
Gly Gly Ile Thr Ala Ala Asn Asn Thr Ser Leu Ser Gly Lys Ile Asp
370 375 380

Claims (4)

1. CottonGH_A09G0075The gene is characterized in that the gene is located on chromosome 9 of a genome of upland cotton TM-1A, the gene has 1125bp in total, and the sequence is shown as SEQ ID No. 1.
2. The cotton of claim 1GH_A09G0075The GH _ A09G0075 protein encoded by the gene is characterized by being a bHLH type transcription factor, and the amino acid sequence of the protein is shown as SEQ ID No. 2.
3. The cotton of claim 1GH_A09G0075The gene is applied to plant growth regulation, and is characterized in that the cotton GH _ A09G0075 gene is positioned in a cell nucleus and mainly expressed in leaves; the gene expression level is in positive correlation with the length of hypocotyl and the length phenotype of petiole; the gene regulates flowering factorFTOf (2) aThe flowering time is regulated by the transcription level, and the expression level of the gene is in positive correlation with the flowering time, namely, the flowering time is promoted to be advanced by increasing the expression level, and the flowering time is delayed by inactivating the gene.
4. Use of the cotton of claim 1GH_A09G0075A method for breeding a new transgenic Arabidopsis variety with a gene, characterized in that the new transgenic Arabidopsis variety is transformed with cottonGH_A09G0075Gene by over-expression of cottonGH_A09G0075The gene and the arabidopsis thaliana new variety have the characteristics of a new variety with elongated hypocotyl length, elongated petiole length and advanced flowering time.
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