CN117925655A - Upland cotton GhPIP5K2 and GhPIP5K22 genes and application thereof - Google Patents

Abstract

The invention discloses upland cotton GhPIP5K2 and GhPIP5K22 genes and application thereof, and belongs to the technical field of genetic engineering. The nucleotide sequence of the GhPIP5K2 gene is shown as SEQ ID NO:1, the nucleotide sequence of the GhPIP5K22 gene is shown as SEQ ID NO: shown at 9. The invention also discloses application of the GhPIP5K2 and GhPIP5K22 genes in improving the stress resistance of cotton to abiotic stress. According to the invention, the target gene is silenced in cotton, and after abiotic stress treatment, the target gene is silenced to cause plant leaf wilting, the activity of antioxidant enzyme is reduced, the MDA content is increased and the expression of the gene related to adversity stress is reduced. Namely, the silencing target gene weakens the stress resistance of the cotton to the abiotic stress, and the positive regulation of the stress resistance of the cotton to the abiotic stress by the target gene is proved. The invention provides important gene resources for high stress resistance breeding of upland cotton.

Description

Upland cotton GhPIP5K2 and GhPIP5K22 genes and their applications

Technical Field

The invention relates to the technical field of genetic engineering, in particular to upland cotton GhPIP5K2 and GhPIP5K22 genes and applications thereof.

Background technique

Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) is produced by phosphorylation of phosphatidylinositol-4-phosphate (PtdIns(4)P) or phosphatidylinositol-5-phosphate (PtdIns(5)P) by phosphatidylinositol phosphokinase (PIPkinase). As a signaling molecule, it also plays a crucial role in resistance to salt and osmotic stress, vesicle trafficking, actin organization, and regulation of plasma membrane and ion channel activity by vanadate-sensitive H ⁺ -ATPase. Phosphatidylinositol-4,5-bisphosphate is both a lipid signal that interacts with effector proteins and a substrate in the phospholipase C signaling pathway. This signaling pathway regulates multiple intracellular processes, including cytoskeletal organization, membrane trafficking, guard cell motility, and pollen tube growth. The relevance of PtdIns(4,5)P2 signaling functions largely depends on its spatiotemporal distribution, which is mainly determined by its metabolic activity within specific membrane regions. The spatiotemporal structure of PtdIns(4,5)P2 is mainly regulated by enzymes involved in its synthesis and decomposition. In these processes, phosphatidylinositol 4-phosphate 5-kinase (PIP5K) of the PIPKs family plays a crucial role in the formation of PtdIns(4,5)P2 patterns. Compared with animal and fungal genomes, higher plant genomes contain a larger number of genes encoding PIP5K, and PIP5K genes have been found in model plants such as Arabidopsis and rice. Arabidopsis has a total of 11 PIP5K genes, and their structure is highly similar to that of animal-derived type I PtdInsP enzymes.

In recent years, some studies have discovered the biological functions of some PIP5Ks. For example, in Arabidopsis, external stimuli such as drought, salt and ABA (abscisic acid) can rapidly induce the expression of AtPIP5K1, and its regulation is also related to soluble protein kinases. AtPIP5K1 and AtPIP5K2 are involved in pollen development. They play an important role in vacuole formation and pollen development. In the pollen grains of pip5k1 and pip5k2 deletion mutants, the loss of function leads to vacuole defects and impaired outer wall formation. Studies have found that AtPIP5K4 and AtPIP5K5 contribute to pollen germination and pollen tube elongation. AtPIP5K4, AtPIP5K5 and AtPIP5K6 are redundantly involved in pollen germination. In addition, AtPIP5K3 regulates the elongation of its root hairs through specific expression in roots. AtPIP5K9 interacts with the cytoplasmic enzyme CINV1 and inhibits root cell elongation under sugar regulation. AtPIP5K7, AtPIP5K8, and AtPIP5K9 are redundantly involved in root growth under osmotic stress. Studies have found that a single gene, OsPIP5K1, plays a key role in the heading process of rice. PIP5K genes are involved in pollen development in wheat and pepper. The soybean GmPIP5K gene enhances drought stress tolerance in overexpressed Arabidopsis plants. These results indicate that PIP5K plays a vital role in plant adaptation to abiotic environments and growth and development.

Cotton (Gossypium spp.) reproduction is susceptible to a variety of adverse conditions, resulting in low yields. Compared with other crops, cotton has better tolerance to abiotic stresses, but its yield is still threatened under extreme temperature, salinity, and drought stress. The PIP5K gene is a key gene that regulates stress responses and pollen production and is an important component of the signaling pathway. However, the exact role of GhPIP5K in cotton stress response and growth regulation remains unclear.

Summary of the invention

The purpose of the present invention is to provide GhPIP5K2 and GhPIP5K22 genes of upland cotton and their applications to solve the problems existing in the above-mentioned prior art. GhPIP5K2 and GhPIP5K22 genes of upland cotton positively regulate cotton's response to abiotic stress, and up-regulating expression in cotton can improve its resistance to abiotic stress.

To achieve the above object, the present invention provides the following solutions:

The present invention provides an application of a GhPIP5K2 gene of upland cotton in regulating the resistance of upland cotton to abiotic stress. The nucleotide sequence of the GhPIP5K2 gene of upland cotton is shown in SEQ ID NO:1.

The present invention also provides the use of a recombinant vector comprising the upland cotton GhPIP5K2 gene in regulating the resistance of upland cotton to abiotic stress.

The present invention also provides the use of an engineered bacterium comprising the above-mentioned recombinant vector in regulating the resistance of upland cotton to abiotic stress.

Furthermore, the expression level of the upland cotton GhPIP5K2 gene is upregulated in upland cotton to improve the resistance of the upland cotton to abiotic stress, wherein the abiotic stress includes high temperature, low temperature, drought and salt stress.

The present invention also provides an application of the upland cotton GhPIP5K22 gene in regulating the resistance of upland cotton to abiotic stress. The nucleotide sequence of the upland cotton GhPIP5K22 gene is shown in SEQ ID NO:9.

The present invention also provides the use of a recombinant vector comprising the upland cotton GhPIP5K22 gene in regulating the resistance of upland cotton to abiotic stress.

The present invention also provides the use of an engineered bacterium comprising the above-mentioned recombinant vector in regulating the resistance of upland cotton to abiotic stress.

Furthermore, the expression level of the upland cotton GhPIP5K22 gene is upregulated in upland cotton to improve the resistance of the upland cotton to abiotic stress, wherein the abiotic stress includes high temperature and low temperature stress.

The present invention also provides a method for improving the resistance of upland cotton to abiotic stress, characterized in that it includes the step of upregulating the expression level of the upland cotton GhPIP5K2 and/or GhPIP5K22 gene in the upland cotton.

The present invention discloses the following technical effects:

The present invention successfully identified 28 genes of the PIP5K family of upland cotton by bioinformatics analysis of upland cotton. The present invention performed transcriptome analysis on the expression pattern of GhPIP5Ks under four abiotic stress treatments of high temperature, low temperature, drought and salt, and found that GhPIP5K family members can respond to abiotic stress; at the same time, it was found that GhPIP5K2 was highly expressed in high temperature, low temperature, drought and salt stress, and GhPIP5K22 was highly expressed in high temperature and low temperature stress. In order to further clarify the molecular mechanism of GhPIP5K2 and GhPIP5K22 genes responding to abiotic stress, the target gene was silenced in cotton plants using VIGS technology, and after the corresponding abiotic stress treatment, it was found that silencing the GhPIP5K2 gene or silencing the GhPIP5K22 gene would cause plant leaf wilt, a decrease in the activity of antioxidant enzymes (CAT, POD and SOD) and an increase in the content of MDA. Silencing GhPIP5K2 reduced the expression of stress-related genes GhHSFB2A, GhDREB2A, GhDREB2C, GhRD20-1, GhRD29A, GhBIN2, GhCBL3, GhNHX1, GhPP2C, GhSnRK2.6 and GhCBF1, while silencing GhPIP5K22 reduced the expression of stress-related genes GhHSFB2B, GhDREB2A, GhDREB2C, GhRD20-1, GhRD29A, GhCBF1 and GhCIPK6. This indicates that silencing GhPIP5K2 and GhPIP5K22 genes weakens cotton's resistance to abiotic stress, and further proves that GhPIP5K2 and GhPIP5K22 genes positively regulate cotton's response to abiotic stress, providing important gene resources for breeding of upland cotton's resistance to abiotic stress.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1 is a map of the vector pEASY-T5 Zero and TRV vector;

Figure 2 shows the phenotypic observation results of control plants and TRV:GhPIP5K2 silenced plants after stress; a. The cotton leaves in the positive control TRV:GhCLA1 showed whitening; b. The silencing efficiency of GhPIP5K2 was detected by qRT-PCR, and the asterisk indicates the significance level compared with the control group (*P<0.05, **P<0.01); c. The effect of heat stress on the phenotype of TRV:00 and TRV:GhPIP5K2; d. The effect of cold stress, drought stress and salt stress on the phenotype of TRV:00 and TRV:GhPIP5K2; e. The number of withered leaves of control plants and silenced plants after heat stress was counted; f. The number of blackened new leaves of control plants and silenced plants after cold stress was counted; g. The number of withered leaves of control plants and silenced plants after drought stress was counted; h. The number of withered leaves of control plants and silenced plants after salt stress was counted;

Figure 3 shows the phenotypic observation results of control plants and TRV:GhPIP5K22 silenced plants after stress; a. The cotton leaves in the positive control TRV:GhCLA1 showed whitening; b. The silencing efficiency of GhPIP5K22 was detected by qRT-PCR, and the asterisk indicates the significance level compared with the control group (*P<0.05, **P<0.01); c. The number of yellowed leaves of control plants and silenced plants was counted after heat stress; d. The number of withered leaves of control plants and silenced plants was counted after cold stress; e. The effects of heat stress and cold stress on the phenotypes of TRV:00 and TRV:GhPIP5K22;

Figure 4 shows the results of physiological and biochemical index detection of TRV:00, TRV:GhPIP5K2 and TRV:GhPIP5K22 plants before and after abiotic stress treatment; a. SOD activity, POD activity, CAT activity and MDA content of TRV:GhPIP5K2; b. SOD activity, POD activity, CAT activity and MDA content of TRV:GhPIP5K22; different letters indicate differences (*P<0.05);

Figure 5 shows the results of stress-related gene expression level detection in TRV:00, TRV:GhPIP5K2 and TRV:GhPIP5K22 plants before and after abiotic stress treatment; a. Expression levels of 11 stress marker genes of TRV:GhPIP5K2; b. Expression levels of 7 stress marker genes of TRV:GhPIP5K22; Compared with the control value, asterisks indicate the significant difference level (*P<0.05, **P<0.01).

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

The inventors successfully identified 28 genes of the PIP5K family of upland cotton by bioinformatics analysis of upland cotton. The present invention conducted transcriptome analysis on the expression patterns of GhPIP5Ks under four abiotic stresses: high temperature, low temperature, drought and salt, and found that GhPIP5K family members can respond to abiotic stresses; at the same time, it was found that GhPIP5K2 was highly expressed in high temperature, low temperature, drought and salt stress, and GhPIP5K22 was highly expressed in high temperature and low temperature stress.

In order to further clarify the molecular mechanism of GhPIP5K2 and GhPIP5K22 genes in response to abiotic stress, the target genes were silenced in cotton plants using VIGS technology, the target gene fragments of GhPIP5K2 and GhPIP5K22 were cloned, and VIGS silencing vectors of the two genes were constructed to study their biological functions under drought and salt stress. The experiment is as follows:

1. Experimental Materials

1.1 Cotton varieties

The present invention takes the upland cotton variety "Xinshi K25" as the research object, and the seeds are provided by Shihezi Agricultural Science Research Institute.

1.2 Vectors and competent cells

The pEASY-T5 Zero cloning vector (CT501-01, containing DH5α Escherichia coli competent cells) and GV3101 Agrobacterium competent cells were purchased from Quanshijin Biotechnology Co., Ltd. and Shanghai Weidi Biotechnology Co., Ltd., respectively. The VIGS vector system for gene silencing (TRV1, TRV2 and TRV-GhCLA1) was donated by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences. The vector map is shown in Figure 1.

2. Experimental Methods

2.1 Primer design

Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used to design cloning primers, qRT-PCR primers, and primers for VIGS silencing vector construction of GhPIP5K2 gene. The primer sequences (SEQ ID NO: 3-8) are shown in Table 1:

Table 1 Primer sequences for cloning, qRT-PCR and VIGS silencing vector construction of GhPIP5K2 gene

Primer-BLAST was used to design cloning primers, qRT-PCR primers, and primers for constructing VIGS silencing vectors for GhPIP5K22 gene. The primer sequences (SEQ ID NOs: 11-16) are shown in Table 2:

Table 2 Primer sequences for cloning, qRT-PCR and VIGS silencing vector construction of GhPIP5K22 gene

2.2 Cotton Planting

a. Fill each pot with an equal amount of nutrient soil (substrate: vermiculite = 1:1), place it in a large basin containing tap water and soak it until the water is absorbed to the surface of the pot for cotton seed germination;

b. Plant the seeds of New Stone K25 in the above pots. To ensure uniform seed germination, the seed planting depth was maintained at 1.5 cm and cultured in an artificial climate incubator (16 h light, 8 h dark, temperature 28 ° C, humidity 70%).

c. When the cotton grows to the four-leaf stage, select young and tender leaves for sampling. The samples are first quickly frozen in liquid nitrogen and then stored at -80℃ for later use.

2.3 Reverse transcription and fluorescence quantitative detection analysis

2.3.1 Total RNA extraction

Total RNA was extracted from cotton leaves according to the instructions of RNAprep pure polysaccharide and polyphenol plant total RNA extraction kit (catalog number DP441, purchased from China Tiangen Biochemical Technology Co., Ltd.).

The RNA quality of all samples was evaluated using a NanoDrop 2000 spectrophotometer from Thermo Scientific, USA. The samples were stored at -80°C.

2.3.2 Reverse transcription

The total RNA obtained above was used as a template, and reverse transcription was performed according to the instructions of FastKing One-Step Genomic cDNA First-Strand Synthesis Premix Kit (Cat. No. KR118, purchased from China Tiangen Biochemical Technology Co., Ltd.) to generate cDNA.

After the reaction was completed, the concentration and purity of cDNA were tested and stored at -20°C for future use.

2.3.3 Fluorescence quantitative detection

Using cDNA as a template, Talent Fluorescence Quantitative Detection Kit (SYBR Green) (Cat. No. FP209, purchased from Tiangen Biochemical Technology Co., Ltd.) was used for fluorescence quantitative detection. The reaction system is shown in Table 3:

Table 3 Fluorescence quantitative detection reaction system

The fluorescence quantitative detection of the GhPIP5K22 gene used the same reaction system as in Table 3, except that the primers were replaced with q-GhPIP5K22-F and q-GhPIP5K22-R primers of qRT-PCR.

The reaction procedure is shown in Table 4:

Table 4 Fluorescence quantitative detection reaction procedure

2.4 Amplification of target gene fragment

2.4.1 Reaction system

Using the cDNA of Neolith K25 as a template, GloriaNova HS2X Master Mix (Cat. No. RK20717, purchased from Ibotek Biotech) was used to amplify the GhPIP5K2 target gene fragment. The system is as follows (Table 5):

Table 5 Target gene fragment amplification reaction system

The amplification of the GhPIP5K22 gene used the same reaction system as in Table 5, except that the primers therein were replaced with GhPIP5K22-F and GhPIP5K22-R primers for amplifying the GhPIP5K22 gene.

2.4.2 Reaction procedure

After adding the reaction solution according to the above system, gently shake to mix, centrifuge briefly, and react according to the reaction procedure in Table 6.

Table 6 Target gene fragment amplification reaction procedure

The target fragment of the GhPIP5K2 gene was amplified by PCR. The target fragment of the GhPIP5K2 gene was 417 bp long and its nucleotide sequence was shown in SEQ ID NO: 2:

GCCTAGGGAGTGTTGCAGAGGAAGAGGAAGATGAAATCACCAACTATCCACAAGGCCTTGTATTGGTCCCTCGTGGAACAGATGACAATAGTGTTGTTGCAGGTTCTCATATACGAGGTCGACGTTTGCGTGCATCAGCTGTAGGTGATGAAGAAGTAGACCTGCTTCTCCCCGGCACGGCAAGACTCCAAATCCAGCTCGGAGTGAACATGCCAGCAAGAGCCGAACAGATTCCAGGAAAAGAAGAAAACATGTTCCATGAATCATATGACGTTGTGTTATATCTGGGAATCATTGACATTTTACAAGAGTATAACATGACTAAGAAGATTGAACATGCCTATAAATCTCTTCAGTTCGATTCACTATCCATATCTGCCGTCGACCCTACGTTTTACTCGCAACGGTTCTTGCAAT.

The full-length nucleotide sequence of the GhPIP5K2 gene is shown in SEQ ID NO: 1 (the underlined portion is the target fragment):

GCCTAGGGAGTGTTGCAGAGGAAG AGGAAGATGAAATCACCAACTATCCACAAGGCCTTGTATTGGTCCCTCGTGGAACAGATGACAATAGTGTTGTTGC AGGTTCTCATATACGAGGTCGACGTTTGCGTGCATCAGCTGTAGGTGATGAAGAAGTAGACCTGCTTCTCCCCGGC ACGGCAAGACTCCAAATCCAGCTCGGAGTGAACATGCCAGCAAGAGCCGAACAGATTCCAGGAAAAGAAGAAAACA TGTTCCATGAATCATATGACGTTGTGTTATATCTGGGAATCATTGACATTTTACAAGAGTATAACATGACTAAGAA GATTGAACATGCCTATAAATCTCTTCAGTTCGATTCACTATCCATATCTGCCGTCGACCCTACGTTTTACTCGCAA CGGTTCTTGCAAT TCATTCAGAAGGTATTTCCTCTGAATTCCATGAAAACTTGA .

The target fragment of the GhPIP5K22 gene was amplified by PCR. The target fragment of the GhPIP5K22 gene is 431 bp long, and its nucleotide sequence is shown in SEQ ID NO: 10 (the underlined part is the target fragment):

AGACGGGTGCATGTACGAAGGAGAGTGGCGTCGTGGGAAAGCCAACGGGAAAGGTAAGTTTTCTTGGCCATCTGGAGCCACTTTTGAAGGTGGTTTCAAGTCGGGTCGGATGGAAGGATTCGGGACGTTTATCGGATCCGACGGCGACACGTACCGTGGGTCGTGGAGCTCCGATCTAAAACACGGCTATGGTCACAAGTGTTACGCAAATGGGGATTACTACGAAGGATCATGGAGGAAAAACCTACAAGAGGGGCACGGCCGTTATGTTTGGAGTAACGGTATCGAATACGTCGGTGAATGGAAAAACGGAGTCATCTCTGGCCGTGGAACCCTGATATGGGCAAATGGAAACGGGTACGATGGGCAATGGGAAAACGGTATGCCCAAAGGAGACGGAGTTTTCTCTTGGCCGGACGGAAGTTGCTATA.

The full-length nucleotide sequence of the GhPIP5K22 gene is shown in SEQ ID NO: 9:

ATGGAGGAAGTAGTGCTCAATGAGCCGAGTGACGTCGTTTTAAACGCCAAGAAGAAGAAATCCGACGAGGAGAAGGACCAGTTGGTGGTTGCTGTTACGACACCTACGGATCACCATCACCGGAGCCGATCTCAAGCTGCCACACGGCGCGTGACTCCCACGACTAACGCCGCGTCTTTCTTCACCATGGGTTCGGGTGATACTGTCGAGAAATTCCTCCCTAACGGTGATC

TTTACATCGGTAGCTTCTCAACCAACGCGCCACACGGATCCGGGAAGTACCTTTGGAA

GACGGGTGCATGTACGAAGGAGAGTGGCGTCGTGGGAAAGCCAACGGGAAAGGTAAG

TTTTCTTGGCCATCTGGAGCCACTTTTGAAGGTGATTTCAAGTCGGGTCGGATGGAAGG

ATTCGGGACGTTTATCGGATCCGACGGCGACACGTACCGTGGGTCGTGGAGCTCCGATC

TAAAACACGGCTATGGTCACAAGTGTTACGCAAATGGGGATTACTACGAAGGATCATGG

AGGAAAAACCTACAAGAGGGGCACGGCCGTTATGTTTGGAGTAACGGTATCGAATACGT

CGGTGAATGGAAAAACGGAGTCATCTCTGGCCGTGGAACCCTGATATGGGCAAATGGAA

ACGGGTACGATGGGCAATGGGAAAACGGTATGCCCAAAGGAGACGGAGTTTTCTCTTG

GCCGGACGGAAGTTGCTATA TCGGAGCATGGAACGGAGATAACATGAAGAAAACTCAA

AAGTTGAACGGGACGTTTTATCACGGGAACGACGGGAAGGAGCATTGCCTGAAAGGAG

GAGAGAGTTTGGTGCTTATGCCGAGGAAAAGATCGTCCGTAGATGGAAGGGGAAGCTT

AGGGGAAAGAAACATGAATTTCCCAAGGATTTGCATATGGGAAAGCGATGGTGAAGCC

GGAGATATCACCTGTGATATAATTGATAATGTGGAAGCTTCGATGATTTACAGAGATGGGT

TTAGGCAATTTAGAAAGAATCCTTGTTGTTTTAGCGGGGAAATTAAGAAGCCAGGACAA

ACTATTTCCAAAGGCCATAAGAATTATGAGTTAATGCTTAACTTGCAATTGGGTATCAGGT

ATTCTGTTGGGAAAGATGCCTCAATTTTGCGCGATTTGAAGCCAAGTGATTTTGATCCCA

AGGAGAAGTTCTGGACCAGGTTTCCTGTTGAAGGATCAAAGCTTACACCTCCTCATCAA

TCTGTGGAGTTCCGGTGGAAGGATTATTGTCCGGTGGTTTTTAGACATTTGAGGGACCTA

TTCCAAGTTGATCCTGCTGATTACATGGTAGCTATTTGCGGTAGTGATGCCCTTAGGGAG

CTTTCTTCCCCGGGGAAGAGTGGAAGCTTCTTTTACCTTACTCAGGATGACAGATTTATG

ATAAAGACAGTAAAGAAATCTGAAGTCAAGGTTCTTATAAGGATGCTTCCAAGTTACTAC

CAACATGTTTCTAAATACGAAAATTCCCTAGTGACAAAATTCTTTGGTGTGCACTGTGTC

AAACCTATAGGTGGCCAAAAGACACGGTTCATTGTGATGGGGAATCTGTTTTGCTCTGA

CTATCGAATTCATAGACGGTTTGACCTGAAAGGATCCTCCCATGGCCGCTCAACTGATAA

GCCGGAAGAGGAAATTGATGAAACCACTACCCTTAAAGACCTGGATCTTAATTATGTGTT

TCGCCTCCAGCGGAATTGGTTCCAAGAGCTTATGAAGCAAATTGATCGAGATTGCGAGT

TCTTGGAGGCTGAGAGAATTATGGATTATAGTCTTTTGGTTGGACTACACTTTCGGGATG

ATAATAGAGGTGATAAAATGGGGTTATCACCGTTTCTGTTGCGCACAGGCAAAAAGGATT

CATATCAGAATGAAAAGTTTATGCGTGGCTGTAGATTCCTTGAAGCTGAGCTACAGGACA

TGGATCGGATTTTAGCTGGCCAGAAACCATTGATACGGCTAGGAGCAAACATGCCAGCA

AGAGCAGTGCGAATGTCTAGAAAAAGCGACTTTGATCAGTATACACAGGGTGGAGTCG

GTCTCTTTTCACATAGTGGCGAAGTTTATGAAGTTGTGTTATACTTTGGCATCATTGACAT

CTTACAAGACTACGACATCAGCAAGAAATTGGAGCATGCCTACAAATCACTACAAGCTG

ATCCTTCTTCAATATCAGCTGTTGGTCCAAAACTCTACTCGAAGAGGTTTCGGGATTTTATAGGAAGAATTTTCATTGAAGACGAGTAG.

2.5 Construction of vector and VIGS silencing of target gene

2.5.1 Construction of silencing vector

(1) Ligation of the target gene fragment and the cloning vector pEASY-T5 Zero

a. Take out the pEASY-T5 Zero vector from the -80℃ freezer and thaw it on ice.

b. Calculate the volume of the added target fragment so that the molar ratio of the vector to the target fragment is 1:5. On ice, add the ingredients shown in Table 7 to a sterile 1.5 mL centrifuge tube:

Table 7 Connection system

c. Shake gently to mix, centrifuge briefly, and connect at 25°C for 5 minutes.

(2) Transformation of DH5α E. coli competent cells

The heat shock method was used to transform into DH5α Escherichia coli competent cells, and PCR verification and sequencing were performed using primers of the target gene sequence (completed by Shanghai Shenggong Biotechnology Co., Ltd.).

(3) Construction of silencing vector

a. The positive plasmid sequenced successfully in (2) was used as a template, and the silencing fragment was amplified by adding restriction enzyme EcoRI and XhoI restriction sites and primers with protective bases, and the fragments of GhPIP5K2 and GhPIP5K22 were inserted into the TRV2 (pYL156) silencing vector by double enzyme digestion to construct TRV:GhPIP5K2 and TRV:GhPIP5K22 silencing vectors. The specific enzyme digestion system is as follows:

Table 8 Enzyme Digestion System

b. After 3 hours of restriction digestion at 37°C, add 10× Loading Buffer to stop the reaction, recover the target gene fragment PCR product by gel, and recover the large restriction fragment by vector. After the restriction product is recovered, connect the target fragment with the silencing vector, transform the ligation product into E. coli competent cells for blue-white screening experiment, perform bacterial liquid PCR and double restriction digestion identification, and after completion, sequence the positive plasmid (Shanghai Shenggong Biotechnology Co., Ltd.), and transfer it into Agrobacterium GV3101 competent cells to obtain the silencing vector.

2.5.2 VIGS silencing target gene

VIGS silencing was performed on the seedlings of Xinshi 25K. The specific method is as follows:

a. Plant the seeds of Upland Cotton "Xinshi 25K" according to the method in 2.2. When the seeds grow to the 7th day and the cotyledons are fully expanded, soak them in water until the nutrient soil in the flowerpot absorbs the water to the surface. Then stop soaking and set aside.

b. Add Kan ⁺ and Rif to LB liquid medium for later use, where the final concentrations of Kan ⁺ and Rif are 50 μg/mL and 25 μg/mL respectively. Thaw the VIGS vector system and target gene bacterial solution taken out from -80℃ on ice, and activate at 28℃, 200rpm for 12-16h (bacterial solution: LB liquid medium = 1:10). After activation, expand the culture in the same ratio.

c. After the bacterial liquid is expanded, centrifuge at 5000 rpm for 10 minutes, pour off the supernatant, retain the bacteria, and use a spectrophotometer to suspend the bacteria with a resuspension solution. The _OD600 should be between 0.8-1.0.

d. After resuspension, place in the dark for 3 hours to allow the bacteria to recover, then mix pYL192 (TRV1) with the bacterial resuspension containing TRV:00 (blank control group), TRV:GhCLA1 (positive control group), TRV:GhPIP5K2 (experimental group 1), and TRV:GhPIP5K22 (experimental group 2) in a 1:1 ratio, mix them thoroughly, and treat them in the dark for 3 hours.

e. On the 7th day of the growth of the cotton seedlings, soak them in water according to the method of step a. On the 8th day of the growth of the cotton seedlings, VIGS injection is performed on them. The specific operation is: use a 1mL syringe needle to cut the back of the cotyledon, and inject the mixed bacterial solution in step d into the cotton cotyledon, so that the bacterial solution fills the entire cotyledon as much as possible.

f. After the injection, wrap it in a plastic bag, culture it in the dark at 25°C for 24 hours, and then culture it under normal growth conditions.

g. After the positive control cotton seedlings turned white, young cotton leaves from the experimental group and the blank control group were taken to detect their gene expression levels and silencing efficiency by fluorescence quantitative PCR.

2.6 Abiotic stress treatment

After silencing, the injected positive cotton seedlings turned albino, and the cotton seedlings grown to four weeks old from TRV:00 empty vector plants (blank control group) and TRV:GhPIP5K2 plants were subjected to high temperature (42°C), low temperature (12°C), drought (15% PEG) and salt (200mmol/LNaCl) stress treatments, respectively. Phenotypic observations were performed.

At the same time, the injected positive cotton seedlings showed albino coloration, and the TRV:GhPIP5K22 plants were grown to four-week-old cotton seedlings and subjected to high temperature (42°C) and low temperature (12°C) stress treatments to observe the phenotype.

2.7 Stress resistance test

2.7.1 Determination of physiological and biochemical indicators in target gene silenced plants

After 10 days of stress treatment, the activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), three antioxidant enzymes and the content of malondialdehyde (MDA) in the leaves of TRV:00 empty vector plants, TRV:GhPIP5K2 plants and TRV:GhPIP5K22 plants were detected according to conventional methods.

2.7.2 Fluorescence quantitative detection of stress-related genes in target gene silenced plants

Before and after the stress treatment, young leaves of the gene-silenced strains were picked, and RNA was extracted using the RNAprep pure polysaccharide and polyphenol plant total RNA extraction kit (catalog number DP441, purchased from Tiangen Biochemical Technology Co., Ltd.), and reverse transcribed using the FastKing one-step genomic cDNA first-strand synthesis premix kit (catalog number KR118, purchased from Tiangen Biochemical Technology Co., Ltd.). Finally, the expression levels of stress-related genes were detected using the Talent fluorescent quantitative detection kit (SYBR Green) (catalog number FP209, purchased from Tiangen Biochemical Technology Co., Ltd.). For specific methods, refer to the instructions.

Among them, the stress-related genes detected in TRV:GhPIP5K2-silenced plants were GhHSFB2A, GhDREB2A, GhDREB2C, GhRD20-1, GhRD29A, GhBIN2, GhCBL3, GhNHX1, GhPP2C, GhSnRK2.6 and GhCBF1.

The stress-related genes detected in TRV:GhPIP5K22-silenced plants were GhHSFB2B, GhDREB2A, GhDREB2C, GhRD20-1, GhRD29A, GhCBF1 and GhCIPK6.

3. Results and Analysis

3.1 Phenotypic observations

On the 9th day after infection, cotton leaves showed a whitening phenotype (Figure 2a and Figure 3a). The positive control TRV:GhCLA1 verified the effectiveness of the VIGS technology. Combined with qRT-PCR to detect the silencing efficiency, VIGS significantly reduced the expression level of the target gene (Figure 2b and Figure 3b).

Regarding the function of GhPIP5K2 under four abiotic stress conditions (high temperature, low temperature, drought and salt), the TRV:GhPIP5K2 plants with silenced and normal plants showed characteristics such as wilting, yellowing of leaves and water shortage (Figure 2c and d). Furthermore, compared with the control plants, the new leaves of the TRV:GhPIP5K2 plants with silenced turned black under cold stress (Figure 2d). According to statistics, the number of TRV:GhPIP5K2 plants with yellowing or wilting leaves after the four abiotic stress treatments was significantly higher than that of normal plants (Figure 2e, f, g and h).

Regarding the function of GhPIP5K22 under two abiotic stress conditions (high temperature and low temperature), the silenced TRV:GhPIP5K22 plants and normal plants were compared, and the TRV:GhPIP5K22 plants showed characteristics such as wilting, yellowing of leaves, and water shortage (Figure 3e). According to statistics, after the two abiotic stress treatments, the number of TRV:GhPIP5K22 plants with yellowing or wilting leaves was significantly higher than that of normal plants (Figure 3c and d).

These results suggest that GhPIP5K2 and GhPIP5K22 play active roles in cotton abiotic stress processing.

3.2 Results of physiological and biochemical indexes

In order to explore the effects of abiotic stress on GhPIP5K2 and GhPIP5K22, the present invention detected the changes in SOD, POD, and CAT activities and MDA content in TRV:GhPIP5K2 and TRV:GhPIP5K22 silenced cotton plants. The results showed that compared with TRV:00 plants, the antioxidant enzyme activities (SOD, POD, and CAT) of silenced plants TRV:GhPIP5K2 and TRV:GhPIP5K22 were significantly reduced; on the contrary, the MDA content was significantly increased (Figure 4).

These findings suggest that silencing GhPIP5K2 and GhPIP5K22 impairs cotton tolerance to abiotic stress.

3.3 Results of fluorescence quantitative detection of genes related to stress

Compared with the control plants, the expression of stress-related genes in TRV:GhPIP5K2 and TRV:GhPIP5K22 plants changed significantly before and after stress treatment.

In TRV:GhPIP5K2 plants, the expression of GhBIN2, GhCBL3, and GhNHX1 was upregulated in the control plants, but significantly downregulated in the silenced plants after stress treatment. The expression levels of GhDREB2A, GhHSFB2C, GhRD20-1, GhPP2C, and GhSnRK2.6 in the silenced plants TRV:GhPIP5K2 were lower than those in the control plants before and after treatment. The expression levels of GhHSFB2A, GhDR29A, and GhCBF1 were downregulated in the silenced plants compared with the control plants before treatment, while the expression levels in the silenced plants were significantly reduced under at least one stress treatment after treatment compared with the control plants (Fig. 5a). The results suggest that GhPIP5K2 may have a positive regulatory effect on abiotic stress.

In TRV:GhPIP5K22 plants, the expression of GhHSFB2B was upregulated in control plants, but significantly downregulated in silenced plants after heat stress treatment. Similarly, the other three stress-related genes (GhDREB2A, GhDREB2C, and GhRD29A) showed a similar pattern. In contrast, in the silenced plants TRV:GhPIP5K22, lower expression levels of GhRD20-1, GhCIPK6, and GhCBF1 were observed before and after treatment than in control plants (Figure 5b). These findings suggest that silencing GhPIP5K22 may directly affect the expression levels of GhRD20-1, GhCIPK6, and GhCBF1. In summary, we speculate that GhPIP5K22 may play an active regulatory role in the temperature stress response process.

In summary, the upland cotton with VIGS silencing GhPIP5K2 or silencing GhPIP5K22 is more sensitive to abiotic stress than the control group, indicating that the stress resistance of cotton is reduced, thus proving that the above genes positively regulate the response of cotton to abiotic stress. The present invention provides important gene resources for breeding of upland cotton with high stress resistance to abiotic stress.

The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (9)

1. The application of the upland cotton GhPIP5K2 gene in regulating and controlling the stress resistance of the upland cotton to abiotic stress is characterized in that the nucleotide sequence of the upland cotton GhPIP5K2 gene is shown as SEQ ID NO: 1.

2. Use of a recombinant vector comprising the upland cotton GhPIP5K2 gene of claim 1 for regulating and controlling stress resistance of upland cotton to abiotic stress.

3. Use of an engineering bacterium comprising the recombinant vector of claim 2 for regulating stress resistance of upland cotton to abiotic stress.

4. The use according to any one of claims 1 to 3, wherein the upland cotton is raised in stress resistance to abiotic stress by up-regulating the expression level of the upland cotton ghip 5K2 gene in upland cotton;

the abiotic stresses include high temperature, low temperature, drought and salt stresses.

5. The application of the upland cotton GhPIP5K22 gene in regulating and controlling the stress resistance of the upland cotton to abiotic stress is characterized in that the nucleotide sequence of the upland cotton GhPIP5K22 gene is shown as SEQ ID NO: shown at 9.

6. Use of a recombinant vector comprising the upland cotton GhPIP5K22 gene of claim 6 for regulating and controlling stress resistance of upland cotton to abiotic stress.

7. Use of an engineering bacterium comprising the recombinant vector of claim 7 for regulating stress resistance of upland cotton to abiotic stress.

8. The use according to any one of claims 6 to 8, wherein the upland cotton is raised in stress resistance to abiotic stress by up-regulating the expression level of the upland cotton ghip 5K22 gene in upland cotton;

The abiotic stress includes high and low temperature stress.

9. A method for increasing stress resistance of upland cotton to abiotic stress, comprising the step of up-regulating the expression level of the upland cotton ghip 5K2 and/or ghip 5K22 gene in upland cotton.