CN117821473A - Main gene GhFBA1 of cotton branch included angle and application thereof in regulation and control of cotton plant type - Google Patents
Main gene GhFBA1 of cotton branch included angle and application thereof in regulation and control of cotton plant type Download PDFInfo
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Abstract
The invention discloses a cotton fruit branch included angle major gene GhFBA1 and application thereof in regulation and control of cotton plant types. Experiments prove that the knockout or overexpression of the GhFBA1 gene in cotton can obviously reduce or increase the growth angle of the fruit branches of the cotton, so that the cotton plant type is changed, and the gene or the protein encoded by the gene plays an important role in controlling the cotton plant type. Namely, the GhFBA1 can be used as a molecular breeding tool to improve cotton germplasm, and directionally reduce the included angle of cotton fruit branches so as to create a cotton breeding material with compact plant type and small included angle of the fruit branches, thereby having important practical application significance for cotton breeding.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a cotton fruit branch included angle major gene GhFBA1 and application thereof in regulation and control of cotton plant types.
Background
Cotton is an important economic crop, not only provides an important natural fiber raw material for textile industry, but also has important application in the fields of national defense, medicine, automobile industry and the like, and has an irreplaceable position in national economic development. Xinjiang is the largest cotton production base in China, the cotton picking area of the whole area is rapidly increased, and the cotton picking rate of the main production area in northern Xinjiang is over 90 percent.
For many years, "close planting dwarfing, early cultivation promotion and agricultural machinery and agriculture integration" are always important technical approaches for high yield and high efficiency in cotton-producing areas in Xinjiang. The method is one of important ways for promoting early ripening and high yield of Xinjiang cotton by cultivating high-yield groups with high density, even distribution and uniformity and fully utilizing light energy in the middle period before cotton growth. On the other hand, the implementation of cotton mechanical harvesting becomes a necessary way for sustainable development of cotton planting industry. The breeding of new cotton varieties suitable for mechanical picking needs to be based on the consideration of yield, quality, precocity, resistance and the like, and the breeding difficulty is relatively high.
The included angle of fruit branches is an important plant type character of cotton. The method is an important factor affecting the illumination intensity and Cheng Ling rate in the population, and the crown layer with smaller angle of the upper fruit branches can enable the light-receiving part of the upper cotton to be converged, so that the light reaches the middle lower part of the cotton crown layer, thereby increasing the light energy utilization rate and improving the cotton yield. Meanwhile, the included angle of the fruit branches is also one of important characters required by mechanical cotton picking breeding. Therefore, identification of the cotton branch angle-related regulatory genes is very important for directional improvement of cotton plant types.
Disclosure of Invention
The invention aims to provide application of a cotton gene GhFBA1 in regulation and control of cotton plant types, and solves the problems of the prior art in cultivating compact plant types and cotton varieties suitable for mechanical harvesting characteristics.
The invention aims at providing an application of a GhFBA1 gene in regulating and controlling cotton plant type characters and/or cotton breeding, wherein the coding region sequence of the GhFBA1 gene is shown as SEQ ID NO. 2.
Further, the genomic nucleotide sequence of the GhFBA1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO.3.
Further, the plant refers to cotton, and the plant type trait includes fruit branch growth angle.
The second object of the invention is to provide a method for regulating cotton plant type, which regulates cotton plant type by over-expressing or knocking down GhFBA1 gene, wherein the nucleotide sequence of the GhFBA1 gene is shown as SE Q ID NO. 1.
Further, the knocking down is to knock out the GhFBA1 gene in the cotton by using a CRISPR/Cas9 mediated gene editing technology or an RNAi technology, so that the cotton shows a phenotype that the growth angle of fruit branches is remarkably reduced.
Further, the method comprises: and constructing a CRISPR/Cas9 gene editing vector or RNAi vector of the GhFBA1 gene, and converting the vector or RNAi vector into a cotton plant to obtain a cotton plant with stable inheritance of GhFBA1 gene deletion or reduced gene expression, wherein the cotton plant shows a phenotype of remarkably reduced fruit branch growth angle.
Further, sequences shown as SEQ ID NO.8 and SEQ ID NO.9 in the GhFBA1 gene are selected as targets for constructing a CRISPR/Cas9 gene editing vector or an RNAi vector.
The invention also aims to provide a cotton variety cultivation method with compact plant type and small branch included angle, which comprises the following steps: and constructing a CRISPR/Cas9 gene editing vector or RNAi vector of the GhFBA1 gene, and transforming the vector or RNAi vector into a cotton plant to obtain a cotton plant with stable inheritance of GhFBA1 gene deletion or reduced gene expression, wherein the cotton plant shows a phenotype of remarkably reduced fruit branch growth angle.
Further, the nucleotide sequence of the GhFBA1 gene is shown as SEQ ID NO. 1.
Further, sequences shown as SEQ ID NO.8 and SEQ ID NO.9 in the GhFBA1 gene are selected as targets for constructing a CRISPR/Cas9 gene editing vector or an RNAi vector.
The beneficial effects are that:
the invention identifies the main gene GhFBA1 for controlling the angle of the cotton fruit branches by a map cloning technology, and further proves that the knockout or over-expression of the GhFBA1 gene in cotton can obviously reduce or increase the growth angle of the cotton fruit branches, thereby changing the cotton plant type, and indicating that the gene or the protein encoded by the gene plays an important role in controlling the cotton plant type. The gene has important value for theoretical researches such as molecular mechanism formed by analyzing the included angle of cotton branches. The invention can directionally reduce the included angle of cotton branches to create cotton breeding materials with compact plant types and small included angle of branches, and has important practical application significance for cotton breeding.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a map-based clone of GhFBA1. A and B: the jaw cotton No. 22 (left) and EH21 (right) fruit branch included angle phenotype comparison; c, introducing fragment of yellow brown cotton of EH 21; d: the phenotype of jaw cotton No. 22 and EH21 branch angle in 4 independent environments. E, F: fine positioning of qFBA-A 11.
FIG. 2 shows the effect of GhFBA1 gene knockout on cotton fruit branch angle. A and B: the fruit branch included angle phenotype of the GhFBA1 gene knockout strain; c: sequencing results of the GhFBA1 gene knockout target.
FIG. 3 shows the effect of GhFBA1 gene overexpression on cotton fruit branch angle.
FIG. 4 is F 2 Group cotton fruit branch included angle phenotype frequency distributionAnd (5) a histogram.
Detailed Description
The following examples are only for more clearly illustrating the technical aspects of the present invention, and thus are merely examples, which should not be construed as limiting the scope of the present invention. It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified. Those of skill in the art, without any particular mention of the techniques or conditions, may follow the techniques or conditions described in the literature in this field or follow the product specifications.
Example 1 localization and map cloning of GhFBA1
(1) Initial positioning of GhFBA1
Taking tetraploid wild cotton seed yellow brown cotton as donor parent, loose upland cotton variety jaw cotton 22 as acceptor parent, first hybridizing jaw cotton 22 as female parent with yellow brown cotton to obtain F 1 And (3) carrying out continuous backcross for 4 generations by taking jaw cotton No. 22 as a recurrent male parent, and then carrying out continuous selfing for 5 generations, thus finally obtaining the yellow brown cotton introgression line population containing 264 lines. The 264 yellow brown cotton introgression lines are subjected to introgression fragment identification by utilizing genome resequencing, 1662 yellow brown cotton chromosome introduced fragments are identified in total, and the yellow brown cotton genome is divided into 2341 recombination modules.
The fruit branch included angle characters of the yellow brown cotton introgression line group in 4 independent environments (the Wuhan in 2017, the Wuhan in 2018, the stone river in 2017 and the stone river in 2018) are examined, and by combining the genotypes of the introgression line group, the fruit branch included angle main effective site qFBA-A11 with stable environment is detected, and the fruit branch included angle can be reduced by 30% due to the allelic variation of the yellow brown cotton.
(2) Fine localization and map cloning of GhFBA1
Crossing and selfing an introgression line EH21 (with a fruit branch included angle of about 45 DEG) with a corresponding introduction fragment and recurrent parent jaw cotton No. 22 (with a fruit branch included angle of about 63 DEG) to obtain F 2 And (3) carrying out investigation and measurement on the fruit branch included angle of each individual plant in the mature period by the generation separation population (1364 individual plants). The results show that F 2 The fruit branch included angle of individual plants in the population is between 30 ° and 80 °, and shows a bimodal distribution (fig. 4), wherein 334 plants with small included angles (less than 53 °) and 943 plants with large included angles (greater than 57 °) are present, the ratio being in accordance with a separation ratio (χ) of 3:1 2 ＝0.91，P>0.05 (fig. 1). Indicating that the fruit branch included angle character of EH21 is controlled by a pair of dominant genes.
In the candidate region of qFBA-A11, 40 InDel markers (marker names GhA-1-GhA-40) were developed in the range of 9.57Mb to 10.84Mb corresponding to the chromosome of reference genome A11 using the resequenced data of EH21, and the pairs F were labeled with these markers 2 Genotyping 1623 individuals of the population to obtain 68 different heterozygous recombinant individuals, and further carrying out F derived from the recombinant individuals 2:3 The pedigrees were characterized for the angle-of-fruit phenotype, and the candidate gene for qFBA-A11 was further located between the GhA-34 and GhA-40 markers (FIG. 1), corresponding to the A11 chromosome 9.95Mb to 10.12Mb. Meanwhile, the markers GhA-34 and GhA-40 can be used as closely linked molecular markers of the collateral included angle genes and used for molecular marker assisted selective breeding. GhA11-34 and GhA11-40 marker sequences:
GhA11-34F：5’ATGCCTTTCTTTGTCTCTATGGT 3’(SEQ ID NO.4)
GhA11-34R：5’TGATGAAAGAATGGAGACATACAC 3’(SEQ ID NO.5)
GhA11-40F：5’GAAGCAATAAAAGCATAGGAAAG 3’(SEQ ID NO.6)
GhA11-40R：5’AAAATCAATGCTGGCACTATGTA 3’(SEQ ID NO.7)
the mutation of the candidate gene was analyzed in the interval, and it was found that EH21 had only one transposon insertion in the candidate interval ghir_a11g010710 gene, resulting in deletion of the ghir_a11g010710 gene in yellow brown cotton and its corresponding introduced line, designated GhFBA1. The corresponding genome sequence of GhFBA1 is SEQ ID NO.1, the corresponding CDS sequence is SEQ ID NO.2, and the corresponding protein sequence is SEQ ID NO.3.
Example 2 knockout experiments of GhFBA1 in cotton
(1) Vector construction
According to the genomic sequence (SEQ ID NO. 1) and CDS sequence (SEQ ID NO. 2) of GhFBA1, two CRISPR/Cas9 targets are designed at the 1 st exon, and the sequences are as follows: TTGCAATTGACTC CAATACA (SEQ ID NO. 8) and GCACTGGTTGACATGCTGGA (SEQ ID NO. 9). Designing a corresponding primer according to a target sequence for constructing a CRISPR vector of the GhFBA1 gene, and specifically:
cFBA1-1as：5’TGTATTGGAGTCAATTGCAAtgcaccagccgggaat3’(SEQ ID NO.10)；
cFBA1-1s：5’TTGCAATTGACTCCAATACAgttttagagctagaaata 3’(SEQ ID NO.11)；
cFBA1-2as：5’TCCAGCATGTCAACCAGTGCtgcaccagccgggaat3’(SEQ ID NO.12)；
inf cFBA1-2as：5’ttctagctctaaaacTCCAGCATGTCAACCAGTGC3’(SEQ ID NO.13)。
first, a GhFBA1-gRNA sequence (SEQ ID NO. 14) driven by the GhU6.7 promoter was obtained by the method of overlay PCR (see Zhang, jun et al, "High efficien T multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system," Plant Biotechnology Journal 16.1.1 (2018): 137-150.), and ligated to pRGEB32-GhU6.7 vector using T4 DNA ligase (see Zhang, jun et al, "High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CR ISPR/Cas9 system," Plant Biotechnology Journal 16.1.1 (2018): 137-150.), to obtain a knock-out vector of the Gh FBA1 gene.
(2) Agrobacterium-mediated genetic transformation of cotton
A. Culturing aseptic seedlings: selecting seeds with plump seeds and normal development (the variety is Jin 668), peeling cotton seed coats, soaking in 2% sodium hypochlorite solution for 10min, washing with sterile water for more than 3 times, inoculating to a sterile seedling culture medium, inserting roots into the culture medium for seedling support after the seeds are exposed, sealing, and culturing in a dark incubator at 28 ℃ for 5-6d.
B. Cutting the hypocotyl into small sections, infecting with activated agrobacterium containing GhFBA1 knockout vector, discarding bacterial liquid, and blow-drying; spreading the hypocotyl on a co-culture medium (MS culture medium+2, 4-D0.1 mg/L+KT0.1 mg/L+30g/L glucose+2.6 g/L Phytagel, pH: 5.85-5.95) with filter paper, and culturing in a constant temperature incubator at 20 ℃ for 36-48 h;
C. obtaining regenerated material: the infected hypocotyl was inoculated into DK callus induction medium (MS medium +2,4-D0.1mg/L +KT0.1 mg/L +30g/L glucose +2.6g/L Phytagel +kanamycin 50mg/L +cephalosporin 400mg/L, pH: 5.85-5.95.). Culturing at 28 deg.c in light culture room (16 hr + 8 hr), and subculturing with fresh callus inducing medium once every 20-30 d.
D. When the callus grows into rice grains, the rice grains are transferred into a differentiation culture medium (MS culture medium, IBA 0.5mg/L, KT 0.1mg/L, glutamine 1.0g/L, asparagine 0.5g/L, glucose 30g/L, and Phytagel 2.6g/L, pH is 6.1-6.2) to be further differentiated into embryoid bodies.
E. The differentiated plantlets are transferred to rooting culture medium (1/2 MS culture medium+15 g/L glucose+2.6 g/L Phytagel, pH: 6.1-6.2) until plantlets with good rooting are grown.
(3) Identification and phenotype of GhFBA1 knockout offspring
The DNA of leaves of T0 generation and T1 generation transgenic positive plants are extracted from the obtained 15 transgenic positive cotton lines, the gene sequence of GhFBA1 is amplified by PCR of a designed primer (SEQ ID NO.15: F:5'-GAAAAGACTTGTGCT TTATGTGCAG-3'; SEQ ID NO.16: R: 5'-AGGATCGAAACCGAAGGTGC-3'), and the homozygous mutant strains GhFBA1_KO-1 to KO-9 with the GhFBA1 knocked out are screened by sanger sequencing to carry out field phenotype identification. After cotton harvesting, the fruit branch angle, leaf branch angle and leaf angle of the above GhFBA1 gene knockout lines GhFBA1_KO-1 to KO-9 and wild type Jin668 plants were measured and counted, and the results are shown in FIG. 2.
As can be seen from fig. 2, the branch angle of the strain with the GhFBA1 gene knocked out was significantly lower than that of the wild-type control plant. Further field investigation of the main agronomic trait phenotypes of 5 high-generation knockout lines showed that the investigated 3 yield traits (effective bell number, single bell weight, coat division) and 5 fiber quality traits were not significantly different from the transgenic recipients, indicating that gene knockout of GhFBA1 could directionally reduce cotton fruit branch angle.
TABLE 1 major agronomic trait phenotypes of GhFBA1 knockout plants
Example 3 overexpression experiments of GhFBA1 in Cotton
(1) Construction of the overexpression vector
A pair of primers was designed based on the coding sequence of GhFBA1. The primer is used for amplifying the GhF BA1 sequence in the jaw cotton No. 22, the product agarose gel electrophoresis detection is carried out, the recovered target product is connected with the over-expression vector pK2GW7, and the recombinant plasmid with correct sequencing is named as pK2GW7-FBA1. The recombinant plasmid was transformed into Agrobacterium competent cells for transformation of the cotton transgenic receptor Jin668.
(2) Genetic transformation
The genetic transformation method is the same as in example 2.
(3) Identification and phenotype of GhFBA1 over-expression offspring
The obtained 8 transgenic positive cotton lines which are transformed independently are subjected to DNA extraction of leaves of T0 generation and T1 generation transgenic positive plants, primers (SEQ ID NO.17:5'-TCCCACTATCCTTCGCA AGAC-3'; SEQ ID NO.18: 5'-TCAAACCTCAGCAAGATCTGGCGAT-3') are designed to carry out PCR amplification on 35S promoter and GhFBA1 gene sequences, and homozygous mutant strains GhFBA1_OE-1 to 5 containing GhFBA1 overexpression elements are screened by Sanger sequencing to carry out field phenotype identification. After cotton harvesting, the angle between the branches of GhFBA1_OE-1 to 5 and wild type Jin668 plants was measured and counted and the results are shown in FIG. 3. As can be seen from fig. 3, the branch angle of the strain overexpressing the GhFBA1 gene was significantly greater than that of the wild-type control plant.
The above detailed description describes in detail the practice of the invention, but the invention is not limited to the specific details of the above embodiments. Many simple modifications and variations of the technical solution of the present invention are possible within the scope of the claims and technical idea of the present invention, which simple modifications are all within the scope of the present invention.
Claims (10)
- The application of the GhFBA1 gene in regulating cotton plant type characters and/or cotton breeding is characterized in that the coding region sequence of the GhFBA1 gene is shown as SEQ ID NO. 2.
- 2. The use according to claim 1, wherein the nucleotide sequence of the GhFBA1 gene is shown in SEQ ID No.1 and the amino acid sequence of the encoded protein is shown in SEQ ID No.3.
- 3. The use according to claim 1 or 2, wherein the plant is cotton and the plant type trait comprises fruit branch growth angle.
- 4. A method for regulating cotton plant type is characterized in that the cotton plant type is regulated by over-expressing or knocking down GhFBA1 gene, and the nucleotide sequence of the GhFBA1 gene is shown as SEQ ID NO. 1.
- 5. The method of claim 3, wherein the knockdown is a knockout of the GhFBA1 gene in cotton using CRISPR/Cas9 mediated gene editing techniques or RNAi techniques.
- 6. The method according to any one of claims 4 or 5, wherein the method comprises: and constructing a CRISPR/Cas9 gene editing vector or RNAi vector of the GhFBA1 gene, and transforming the vector into a cotton plant to obtain a cotton plant with stable inheritance of GhFBA1 gene deletion or reduced gene expression.
- 7. The method of claim 6, wherein the sequence shown as SEQ ID No.8 and SEQ ID No.9 in the GhFBA1 gene is selected as a target point for constructing a CRISPR/Cas9 gene editing vector or an RNAi vector.
- 8. A cotton variety cultivation method with compact plant type and small branch included angle is characterized by comprising the following steps: and constructing a CRISPR/Cas9 gene editing vector or RNAi vector of the GhFBA1 gene, and transforming a cotton plant to obtain a cotton plant with stable inheritance of GhFBA1 gene deletion or reduced gene expression.
- 9. The method of claim 8, wherein the nucleotide sequence of the GhFBA1 gene is set forth in SEQ ID No. 1.
- 10. The method according to any one of claims 8-9, wherein the sequence shown as SEQ ID No.8 and SEQ ID No.9 in the GhFBA1 gene is selected as target point for the construction of CRISPR/Cas9 gene editing vector or RNAi vector.
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