CN105018520B - The plant expression vector and application thereof of 4 pairs of cotton DELLA protein gene GhGAIs expression of regulation and control - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and in particular relates to a plant expression vector for regulating and controlling the expression of four pairs of cotton DELLA genes GhGAIs and an application thereof. The technical problem to be solved by the invention is to provide a new option for increasing cotton yield. The technical scheme of the present invention is to simultaneously regulate the expression of four pairs of cotton DELLA genes GhGAIs in cotton fiber by using RNAi technology. The present invention also provides host cells containing the vector. After the target gene is regulated by the carrier of the present invention, the improved cotton's lint score and lint index are obviously improved, and the cotton yield potential is obviously increased. The method of the invention is simple and easy to implement, has remarkable effect and has the application prospect of generating huge economic benefits.

Description

A plant expression vector for regulating the expression of 4 pairs of cotton DELLA protein genes GhGAIs and its use

technical field

The invention belongs to the technical field of plant genetic engineering, and in particular relates to a plant expression vector for regulating the expression of four pairs of cotton DELLA protein genes GhGAIs and an application thereof.

Background technique

Cotton is the most important natural fiber crop. my country is the largest cotton-producing country and textile exporter in the world. Cotton plays a pivotal role in my country's national economy.

Cotton fiber is a single-celled fiber developed from the epidermal cells of the outer integument of cotton through four stages of initiation, elongation, secondary wall synthesis and maturation. Plant hormones play a very important role in the regulation of cotton fiber development. Up-regulation of gibberellin (GA), auxin (IAA) and brassinosteroid (BR) synthase genes in cotton fibers and ovules can increase the levels of related hormones in ovules and fibers, which can promote the initiation or growth of cotton fibers , directional improvement of cotton fiber yield and quality traits, indicating that plant hormones play a very important role in regulating the development of cotton fibers. There are few studies on targeting phytohormone signal transduction elements to regulate phytohormone signal transduction in fiber development.

DELLA protein is a key negative regulator in the GA signaling pathway. GA induces the ubiquitination and degradation of DELLA protein, releases the inhibition of DELLA protein, and promotes the expression of GA-responsive genes. In addition, a variety of hormones and environmental signals can directly or indirectly regulate the degradation of DELLA proteins, thereby affecting the growth and development of plants and their responses to the environment. So far, there has been no report on improving cotton fiber by regulating cotton DELLA gene. According to the results of cotton genome sequencing, the DELLA protein in cotton is encoded by a gene family, and a single cotton genome contains 4 coding genes, while the allotetraploid upland cotton contains 4 pairs of coding genes, named GhGAI1A and GhGAI1D, respectively. GhGAI2A and GhGAI2D, GhGAI3A and GhGAI3D, GhGAI4A and GhGAI4D (Table 1). Each pair of genes is orthologous genes on different subgenomes (A and D subgenomes), with the same nucleotide sequence>95%. Previous studies have analyzed the functions of some cotton DELLA protein genes by means of cloning and heterologous expression, but there is no report on the function of cotton DELLA protein genes in cotton fiber development, and there is no report on the improvement of cotton fiber yield and quality by regulating DELLA protein genes. reports.

Table 1 Cotton DELLA protein gene and its coding sequence in the sequenced genome

Gene upland cotton Raymond Cotton Asian cotton GhGAI1A Gh_A07G0717 Cotton_A_30811 GhGAI1D Gh_D07G0779 Gorai.001G089400 GhGAI2A Gh_A01G1242 Cotton_A_41295 GhGAI2D Gh_D01G1446 Gorai.002G177000 GhGAI3A Gh_A06G0504 Cotton_A_20431 GhGAI3D Gh_D06G0560 Gorai.010G067000 GhGAI4A Gh_A05G0135 Cotton_A_11125 GhGAI4D Gh_D05G0197 Gorai.009G021700

Contents of the invention

The technical problem to be solved by the invention is to provide a new option for increasing cotton yield.

The technical scheme of the present invention is to regulate the plant expression of four pairs of cotton DELLA genes GhGAIs and carry GhGAI3D, GhGAI4A and GhGAI4D.

Specifically, the vector contains RNAi elements targeting the four pairs of genes in series, and the RNAi elements have the nucleotide sequence shown in SEQ ID No.5.

Specifically, the promoter regulating the RNAi element is a specific promoter for the synthesis phase of the secondary wall of fibers.

Preferably, the promoter is the cotton FbLate-2 gene promoter (pFbl2), the nucleotide sequence of which is shown in SEQ ID No.6.

Specifically, the plant expression vector has the nucleotide sequence shown in SEQ ID No.7.

The present invention also provides host cells containing the vector.

Specifically, the host cell is Agrobacterium tumefaciens.

The present invention also provides the substance for increasing cotton fiber yield, the main active ingredient of which is the plant expression vector, or the host cell containing the plant expression vector.

The invention also provides the use of the carrier in increasing the cotton fiber yield.

The present invention also provides the use of the host cell in improving cotton fiber yield.

Beneficial effects of the present invention: the present invention proves that the DELLA protein plays an important role in the growth and development of cotton fibers through research and analysis. And by comprehensively down-regulating the expression of 8 DELLA genes GhGAIs during the synthesis of cotton fiber secondary wall, the transgenic cotton with significantly improved coat score (the percentage of cotton fiber in seed weight) and coat index (the weight of fiber on a hundred cotton seeds) was obtained . The test result proves that after the target gene is regulated by the carrier of the present invention, the improved cotton's lint count and lint index are obviously improved, and the fiber output is obviously increased. The method of the invention is simple and easy to implement, has remarkable effect and has the potential of generating huge economic benefits.

Description of drawings

Figure 1: Gene structure diagram of the T-DNA region of the expression vector of GAIsRNAi

CaMV35S-P, cauliflower mosaic virus 35S promoter; CaMV35S-T, cauliflower mosaic virus 35S terminator; D1-D4, coding sequences of DELLA and VHYNP domains of cotton GAI1-4 genes, two inverted repeats of D1 -D4 tandem sequence and spacer sequence constitute the GAIsRNAi element; GUS: NPTII, β-glucosidase and neomycin phosphotransferase fusion gene; LB, T-DNA left border; Nos-T, Agrobacterium opine synthase Gene terminator; pFbl2, cotton FbLate-2 gene promoter; RB, T-DNA right border.

Figure 2: Construction process of p5-pFbl2-GAIRNAi expression vector

See embodiment 2 for the specific experimental method. CaMV35S-P, cauliflower mosaic virus 35S promoter; CaMV35S-T, cauliflower mosaic virus 35S terminator; GAI-DELLA, tandem sequence of 4 cotton GAI fragments; GUS: NPTII, β-glucosidase and neomycet Phosphotransferase fusion gene; LB, T-DNA left border; Nos-T, Agrobacterium opine synthase gene terminator; pFbl2, cotton FbLate-2 gene promoter; RB, T-DNA right border; REP origin , the origin of plasmid replication. Relevant enzyme cutting sites and positions are marked on each vector.

Figure 3: Expression levels of 4 pairs of cotton DELLA protein genes (GhGAI1A and GhGAI1D, GhGAI2A and GhGAI2D, GhGAI3A and GhGAI3D, GhGAI4A and GhGAI4D) in fibers of GAIsRNAi transgenic cotton 18 days after flowering

WT stands for Non-GMO cotton. FGi is GAIsRNAi transgenic cotton, and the numbers behind it show different transformants and homozygous lines.

Detailed ways

Routine experimental operations used in the following examples:

1. Extraction of DNA

Genomic DNA was extracted using Plant Genomic DNA Rapid Extraction Kit (Aidlab). See the instructions for detailed steps.

2. Extraction of RNA

RNA was extracted using the EASYspin Plant RNA Rapid Extraction Kit (Aidlab). See the instructions for detailed steps.

3. PCR amplification of DNA fragments

The amplification system is as follows: 10×Ex PCR buffer (Mg ²⁺ free) 2.5 μL, 2.5 mmol/L dNTPs 2 μL, 25 mmol/L MgCl _{2 2} μL, primer 1 (5 μmol/L) 1 μL, primer 2 (5 μmol/L) 1 μL , Ex Taq DNA polymerase 1U, genomic DNA about 60ng, add ddH ₂ O to 25μL.

The amplification program was: 94°C, 5min; 94°C, 30sec, 56°C, 30sec, 72°C, 1.5min, 35 cycles; 72°C extension, 10min.

4. Recovery, ligation and cloning of DNA fragments

DNA fragments were recovered using the BioFlux Gel Recovery Kit. DNA fragment ligation was performed using T4 DNA ligase. The recovered fragments were connected with the pUCm-T (Shanghai Sangong) vector to establish the following ligation system: 1 μL of 10×T4 DNA ligation buffer, 1 μL of vector DNA fragments, 1 μL of exogenous ligation product DNA fragments, 1 μL of T4 DNA ligase, and double-distilled water Make up the volume to 10 μL. The molar ratio of vector DNA fragments to exogenous ligation product DNA fragments was 1:3, and ligated at 16°C for 12 hours. Then the ligation product was transformed into Escherichia coli DH5α. The obtained resistant clones were cultured overnight in liquid, and the plasmids were extracted with BioFlux plasmid extraction kit, and sequenced in Invitrogen after verification by enzyme digestion.

5. GUS histochemical staining

Since the expression vector used in the laboratory has a GUS reporter gene, GUS histochemical staining is generally used to detect and track the transgene. Specific method: Take a small amount of transgenic cotton leaves (with wounds) and place them in a 96-well plate, add GUS staining solution [0.1mol/LK ₃ Fe(CN) ₆ , 0.1mol/LK ₄ Fe(CN) ₆ , 0.01mol/L Na ₂ EDTA, 500mg/L X-Gluc, 1% Triton X-100 (v/v), 0.14mol/L sodium phosphate buffer (pH7.0)], placed at 37°C for about 2 hours, fully stained Then decolorize with 75% ethanol. The plants whose leaves can be stained with specific blue by GUS staining solution are positive for the transgene.

Example 1 Regulation of the acquisition of the RNAi element GAIsRNAi of cotton DELLA gene

DELLA protein belongs to a large GARS protein family. In addition to the GARS domain shared by the family at the C-terminus, there are about 100 amino acid DELLA and VHYNP domains unique to DELLA proteins at the N-terminus. As mentioned above, upland cotton contains 4 pairs of orthologous genes of DELLA protein, and the nucleotide sequence similarity of the paired symbiotic homologous genes is very high (identical nucleotides > 95%), and the nucleotide sequence similarity of the non-orthologous genes is very high. Nucleotide sequence similarity is low. In order to comprehensively regulate the expression of DELLA protein genes in cotton, we selected D genome sequences (SEQ ID No. 1-4) encoding DELLA and VHYNP domains of 4 DELLA proteins based on 4 pairs of orthologous DELLA protein genes. The four sequences were concatenated by the PCR method, and finally amplified into an inverted repeat sequence containing a spacer sequence, that is, the GAIsRNAi element (SEQ ID No. No. 5). After the sequence is transcribed in cotton, it can form a double-stranded RNA sequence targeting 4 pairs of DELLA protein genes, thereby down-regulating the expression levels of these genes through the RNAi mechanism. At the same time, the coding sequences of DELLA and VHYNP domains are unique to the DELLA protein gene, and the expression of GAIsRNAi elements will not affect the expression and function of other GRAS proteins.

First, the DELLA and VHYNP domain coding sequences of GAI1-4 were amplified using the upland cotton genomic DNA as a template, and the primers were GAI1i-F/R, GAI2i-F/R, GAI3i-F/R and GAI4i-F/R, respectively. (Table 2), the method is the same as the DNA fragment amplification of the above-mentioned conventional test operation. The amplified GAI1-4 fragments were further recovered by the above DNA fragment recovery method.

Table 2 GAIsRNAi element amplification primer sequence

Primer Base sequence (5'--3') use GAI1i-F GACGAGTTATTAGCTGTTTTGG GAI1 fragment amplification upstream primer GAI1i-R AAGCTCATCGTTGAACTCGATCAACAAATTTTG GAI1 fragment amplification downstream primers GAI2i-F CGAGTTCAACGATGAGCTTTTGGCGGTTTTG GAI2 fragment amplification upstream primer GAI2i-R TAGTCCATCGTTAAGTTCAGAGAGCATGC GAI2 fragment amplification downstream primers GAI3i-F CTGAACTTAACGATGGACTACTCGCCGGT GAI3 fragment amplification upstream primer GAI3i-R GAAAACCATCCTCAGCGAACTCAGTTAGC GAI3 fragment amplification downstream primers GAI4i-F TTCGCTGAGGATGGTTTTTCTAGCCGGAG GAI4 fragment amplification upstream primer GAI4i-R CTCCTACTTGTCCCCTCCGTTAACTGATTATTCAAACT GAI4 fragment amplification downstream primers

Second, the recovered four GAI fragments were concatenated using the asymmetric overlap PCR method. Connect GAI1 and GAI2, GAI3 and GAI4 in series respectively. Construct 4 amplification systems as follows:

(1) The recovered GAI1 fragment is about 5 ng, 2 μL primer GAI1i-F (5 μmol/L), 1 μL primer GAI1i-R (1 μmol/L);

(2) The recovered GAI2 fragment is about 5 ng, 1 μL primer GAI2i-F (1 μmol/L), 2 μL primer GAI2i-R (5 μmol/L);

(3) The recovered GAI3 fragment is about 5 ng, 2 μL primer GAI3i-F (5 μmol/L), 1 μL primer GAI3i-R (1 μmol/L);

(4) The recovered GAI4 fragment is about 5 ng, 1 μL of primer GAI4i-F (1 μmol/L), and 2 μL of primer GAI4i-R (5 μmol/L).

The remaining components of the amplification system are the same as the conventional amplification system. The amplification program was: 94°C, 5min; 94°C, 30sec, 56°C, 30sec, 72°C, 30sec, 30 cycles; 72°C extension for 3min. Mix system 1 and system 2, system 3 and system 4 respectively, 56°C, 1min, 72°C, 3min. After the amplified product was subjected to agarose electrophoresis, the target bands GAI1+GAI2 and GAI3+GAI4 were recovered respectively.

Further use GAI1+GAI2 and GAI3+GAI4 as templates to complete the concatenation of four GAI fragments. Two amplification systems were constructed as follows:

(5) About 5 ng of recovered GAI1+GAI2 fragment, 2 μL primer GAI1i-F (5 μmol/L), 1 μL primer GAI3i-R (1 μmol/L);

(6) The recovered GAI3+GAI4 fragment is about 5 ng, 1 μL of primer GAI3i-F (1 μmol/L), and 2 μL of primer GAI4i-R (5 μmol/L).

The remaining components of the amplification system are the same as the conventional amplification system. The amplification program was: 94°C, 5min; 94°C, 30sec, 56°C, 30sec, 72°C, 30sec, 30 cycles; 72°C extension for 3min. Mix System 5 and System 6, 56 °C, 1 min, 72 °C, 3 min. After the amplified product was subjected to agarose electrophoresis, the target band GAI1+GAI2+GAI3+GAI4 was recovered.

When designing the primers, the amplified fragment of the GAI4 primer contained the coding sequence of the DELLA and VHYNP domains of GhGAI4 and the adjacent sequences of the domains. The adjacent sequence of this domain is equivalent to the essential component of RNAi element-the spacer sequence, which is conducive to the formation of double-stranded RNA by two inverted repeats.

Finally, method ¹ of directly amplifying hairpin RNA was used to amplify the fragment GAI1+GAI2+GAI3+GAI4 as a template to obtain the inverted repeat sequence of 4 GAI fragments containing spacers. The reaction system contains about 5 ng of recovered GAI1+GAI2+GAI3+GAI4, 2 μL primer GAI1i-F (5 μmol/L), 1 μL primer GAI4i-R (1 μmol/L), and the rest of the components are the same as the conventional amplification system. The amplification program was: 94°C, 5min; 94°C, 30sec, 56°C, 30sec, 72°C, 1min, 35 cycles; 72°C extension for 3min. After the amplified product was subjected to agarose electrophoresis, the target band of about 1800 bp was recovered, recovered, cloned and sequenced to verify by conventional methods, and finally the GAIsRNAi element was obtained (Figure 1).

GAIsRNAi elements sequentially include GAI1+GAI2+GAI3+GAI4 (including DELLA and VHYNP domain coding sequences and adjacent sequences of domains) + GAI4 inverted repeats + GAI3 inverted repeats + GAI2 inverted repeats + GAI1 inverted repeats sequence. The sequence of the GAIsRNAi element is as follows, where the spacer is underlined:

agcctcctacttgtccctccgagttcatcatggatcctgaaaccaatcagacggtggtaagcgacgcatgga ccactgccgaacctcatatgccgcaggtgcaccagaatatttcttaccagcaacaaagtttgaataatcagttaacg gttgataatcagttaacggttgtaacagcaatggaggaagattccggtatacggttggttcatatgttgatgacgtg tgcggagtgcgttcaacgtggagacttctcattggct

Example 2 Construction of GAIsRNAi expression vector specific to the synthesis phase of the fiber secondary wall

The procedure for constructing the GAIsRNAi element into the plant expression vector p5 is shown in FIG. 2 . p5 is a binary plant expression vector transformed from the traditional plant expression vector pBI121. Its T-DNA segment (the region between RB and LB, Figure 2) was replaced with a constitutive CaMV35S promoter (CaMV35S-P) controlling the fusion gene expression cassette of reporter gene GUS and marker gene NptII, and another gene expression cassette composed of CaMV35S- P-controlled expression cassette.

Use the primers (pFbl2F, 5'-aagctTGCAGACTTAGGATTGGATG-3' and pFbl2R, 5'-ggatccGGTTAACCGAAATACAAAGCA-3') to amplify and clone the promoter pFbl2 from the cotton genome according to the aforementioned routine method, and add HindⅢ and BamHI at both ends of the promoter location. Cut the pFbl2 promoter from the cloning vector with HindⅢ and BamHI (HindⅢ is partial digestion), and connect it to the p5 vector cut with HindⅢ and BamHI to construct the p5-pFbl2 vector. Further use BamHI and KpnI to excise the GAIsRNAi element from the cloning vector (BamHI is partially digested), and insert it into the corresponding site of the p5-pFbl2 vector to obtain the final expression vector p5-pFbl2-GAIsRNAi. All restriction endonucleases were purchased from Roche Company and operated according to the instruction manual. The recovery, ligation and Escherichia coli transformation of DNA fragments were carried out according to the aforementioned conventional operation methods. Referring to the Bio-RAD MicroPulser user manual, the above vector was introduced into Agrobacterium LBA4404 by electric shock transformation.

The genetic transformation of embodiment 3 cotton

The genetic transformation of cotton with the above expression vector was carried out by the method mediated by Agrobacterium tumefaciens, and the formulation of the medium used is shown in Table 3. The specific method is as follows: shell the plump cotton seeds of wild-type upland cotton Ji Mian No. 14, place a small amount (about 20 to 40 seeds) of the shelled seeds in a sterilized 100mL triangular flask, and first use 75% alcohol Pre-wash the seeds for 1 minute, pour out the alcohol and then add 0.1% HgCl ₂ to sterilize for about 12 minutes (constantly shake the Erlenmeyer flask for sterilization), pour out the mercuric chloride, add sterile water to rinse thoroughly, rinse about 10 times, and finally A proper amount of sterile water is left in the triangular flask. Put it on a shaker (30°C, 100rpm), change the sterile water every 8 hours, wait for the radicle to grow about 1cm (about 36-48h), gently insert the radicle into the germination medium, 30°C Culture in dark until the hypocotyl elongates to about 3cm (about 48h). About 20 hours before the infection, a single colony of Agrobacterium carrying the genetic transformation vector was inoculated in liquid YEB medium containing 50 mg/L Km and 125 mg/L Sm, placed on a shaker (200 rpm) at 28°C, and measured after culturing for about 20 hours The OD value of the bacterial solution (OD ₆₀₀ ), the OD ₆₀₀ is suitable for transformation in the range of 0.8-1.0. Collect the activated Agrobacterium liquid, separate it at 8000rpm for 1min, discard the supernatant, and resuspend the bacteria with a 1:1 volume ratio containing co-cultivation liquid medium (containing 100 μmol/L AS, acetosyringone) and collect the resuspension in Place a 100mL Erlenmeyer flask on a shaker (30°C, 100rpm) and incubate for about 20min. Cut the hypocotyls into small pieces about 1 cm long, place them in the resuspension liquid and infect them on a shaker (30°C, 100 rpm) for 50 min, discard the liquid, take out the hypocotyl segments, and gently put them on the surface of the solid co-culture medium, dark Cultivate for about 48h. After dark culture, transfer the hypodermal segment to solid selection medium for culture (30°C, 16h light/8h dark, the same below) for 15 days, then transfer to solid hypodermal growth medium, and subculture once every 15 days or so until healed. The wound tissue was obviously formed, and the callus was transferred to solid callus medium for culture. Transfer the embryogenic callus in a good state to a liquid suspension medium and place it on a shaker (30°C, 100rpm) for suspension culture for about 10 days, filter through a sieve, spread the small somatic embryos in the somatic embryo elongation medium until The green somatic embryos grow out, pick the green somatic embryos into the somatic embryo elongation medium and continue to cultivate until the length is about 1 cm, and insert them into the rooting medium until the seedlings grow out. The above operations must be completed under strict sterile conditions.

The vigorously grown regenerated cotton seedlings were transplanted into planting pots, and were routinely managed in the greenhouse until the cotton fibers and seeds matured. Harvest the transgenic cotton seeds of the T0 generation, continue to plant the T1 generation and harvest the seeds, and perform GUS tissue staining on the germinated T2 generation seedlings after sowing the T1 generation seeds (see the routine operation method), and screen out the homozygous transgenic T2 generation lines (all are GUS-positive or GUS-negative plants), transplant T2 generation homozygous lines, and detect the expression level of the target gene GhGAIs and compare the changes in fiber yield and quality traits.

The substratum for the genetic transformation of cotton mediated by Agrobacterium tumefaciens in table 3

MS: Murashige & Skoog, 1962; B5: Gamborg, 1986; Gelrite: Sigma, Cat. No.: G1910; SH: Schenk & Hildebrandt, 1972.

Example 4 Detection of cotton GhGAIs gene transcription level

The RNA of control and transgenic cotton fibers 18 days after flowering was extracted, reverse-transcribed to synthesize a strand of cDNA, which was used as a template for quantitative PCR detection. The specific operation steps are as follows: a cDNA one-strand synthesis kit (MBI company) is used to synthesize one-strand cDNA of various RNAs, and the operations are all carried out according to the instructions of the kit. Quantitative PCR was performed on the CFX96 quantitative PCR detection system (Bio-Rad). The 25 μL reaction system included 12.5 μL 2×Super Mix (Bio-Rad), 0.2 μmol/L of upstream and downstream primers and 1 μL of one-strand cDNA. The temperature cycle parameters were pre-denaturation at 95°C for 2 min; 95°C for 10 sec, 57°C for 20 sec, and 40 cycles of amplification. Cotton Actin4 gene was used as internal standard. The relative expression of each gene was calculated using the analysis software Bio-Rad CFX Manager 2.0 that comes with the quantitative PCR instrument. The sequences of all quantitative PCR primers are listed in Table 4.

As shown in Figure 3, the transcription levels of the four pairs of target genes GhGAIs in the fiber of GAIsRNAi transgenic cotton 18 days after flowering were generally lower than those of non-transgenic lines, indicating that the GAIsRNAi element initiated by pFbl2 can successfully inhibit the expression of DELLA protein gene.

Table 4 Quantitative PCR primer sequences

Primer Base sequence (5'---3') use GhGAI1A-F GGACCACCTCAACCCGATG GhGAI1A upstream primer GhGAI1A-R CGATGCGTTCGGCCAATTC GhGAI1A downstream primer GhGAI1D-F GGACCGCCTCAACCGGATA GhGAI1D upstream primer GhGAI1D-R CGATGCGTTCGGCCAATTG GhGAI1D downstream primer GhGAI2A-F CACCCACCAACGTTAAATCT GhGAI2A upstream primer GhGAI2A-R ACCACGGGACGAGTTGAAG GhGAI2A downstream primer GhGAI2D-F CACCCACCAACGTTAAATCC GhGAI2D upstream primer GhGAI2D-R ACCACGGGACGAGTTGAAT GhGAI2D downstream primer GhGAI3A-F TTCTTTAGAAGCTTGCAGGG GhGAI3A upstream primer GhGAI3A-R CCAATGGCTCGTGCCTTTCT GhGAI3A downstream primer GhGAI3D-F TTCTTTAGAAGCTTGCAGGA GhGAI3D upstream primer GhGAI3D-R CAATGGCTCGTGCCTTTCC GhGAI3D downstream primers GhGAI4A-F GTTCATCATGGATCCTGTAAG GhGAI4A upstream primer GhGAI4A-R GAATCTTCCTCCATTGCTGG GhGAI4A downstream primer GhGAI4D-F GTTCATCATGGATCCTGAAAC GhGAI4D upstream primer GhGAI4D-R GAATCTTCCTCCATTGCTGT GhGAI4D downstream primer GhAct4-F TTGCAGACCGTATGAGCAAG GhActin4 upstream primer GhAct4-R ATCCTCCGATCCAGACACTG GhActin4 downstream primer

Example 5 Investigation of yield and fiber quality traits of GAIsRNAi transgenic cotton

Yield content and cotton index are both important yield traits of cotton. Clothing percentage refers to the ratio of the fiber weight on the seed cotton to the weight of the seed cotton, expressed as a percentage; that is, the ratio of the fiber weight to the total weight of the whole seed and fiber. Clothing refers to the weight of fibers on a hundred cottonseeds. Table 5 shows the results of the analysis of the cotton of the T2 generation GAIsRNAi transgenic cotton on the coat fraction and coat finger. Compared with the control, the transgenic cotton single plant has a 4-24% increase in the coat content and a 20-80% increase in the coat index, which shows that the GAIsRNAi transgenic cotton has a high yield-increasing potential.

Table 5 Fiber yield traits of GAIsRNAi transgenic cotton

sample Clothes (%) Clothing score increased (%) compared to the control Clothes finger (g) Clothes finger increased (%) compared with the control WT 38.6 5.5

FGi01-10 48.1 24.61 9.9 80.0 Fgi02-2 44.2 14.51 6.6 20.0 FGi02-3 43.5 12.69 7.7 40.0 FGi04-2 40.2 4.15 6.6 20.0 FGi05-1 42.1 9.07 7.1 29.1 FGi06-13 42.0 8.81 7.2 30.9 FGi07-1 40.8 5.70 6.6 20.0 FGi08-7 42.5 10.10 7.3 32.7

Note: WT in the above table refers to non-transgenic cotton. FGi is GAIsRNAi transgenic cotton, and the numbers behind it show different transformants and homozygous lines.

The cotton fiber samples of GAIsRNAi transgenic cotton and control were sent to the Cotton Quality Supervision and Testing Center (Anyang) of the Ministry of Agriculture. Under environmental conditions, five indicators including the average length of the upper half, uniformity index, specific strength at break, elongation, and micronaire value were tested. The results are shown in Table 6, which shows that the fiber quality of the GAIsRNAi transgenic cotton has no significant change compared with the control.

Table 6 Fiber quality traits of GhGAIsRNAi transgenic cotton

Note: WT in the above table refers to non-transgenic cotton. FGi is GAIsRNAi transgenic cotton, and the numbers behind it show different transformants and homozygous lines.

The above examples show that the method for improving cotton of the present invention can specifically regulate the expression of 4 pairs of cotton DELLA protein genes during the synthesis phase of the fiber secondary wall, and achieve the purpose of improving cotton fiber yield (cloth fraction and cotton finger).

references:

1. Yue-Hua Xiao, Meng-Hui Yin, Lei Hou, and Yan Pei (2006) Direct amplification of intron-containing hairpin RNA construct from genomic DNA, BioTechniques 41:548-552 (November 2006).

Claims (7)

1. A plant expression vector for regulating the expression of 4 pairs of cotton DELLA protein genes GhGAIs, wherein the 4 pairs of genes are GhGAI1A and GhGAI1D, GhGAI2A and GhGAI2D, GhGAI3A and GhGAI3D, GhGAI4A and GhGAI4D; The vector comprises RNAi elements targeting the four pairs of genes in series; the RNAi elements have a nucleotide sequence as shown in SEQ ID No.5; The vector includes a promoter that regulates the RNAi element; the promoter is a specific promoter for the synthesis phase of the fiber secondary wall; the promoter is a cotton FbLate-2 gene promoter, and its nucleotide sequence is as follows: Shown in SEQ ID No.6. 2. The vector according to claim 1, wherein the plant expression vector has a nucleotide sequence as shown in SEQ ID No.7. 3. A host cell containing the vector of claim 1, which is a non-plant cell. 4. The host cell according to claim 3, characterized in that: the host cell is Agrobacterium tumefaciens. 5. The substance for improving cotton fiber yield, characterized in that: its main active ingredient is the carrier according to claim 1, or the host cell according to claim 3 or 4. 6. The use of the carrier according to claim 1 in increasing cotton fiber yield. 7. Use of the host cell according to claim 3 or 4 in increasing cotton fiber yield.