CN117925655A

CN117925655A - Upland cotton GhPIP5K2 and GhPIP5K22 genes and application thereof

Info

Publication number: CN117925655A
Application number: CN202410093141.6A
Authority: CN
Inventors: 宿俊吉; 王彩香; 令萍洁; 杨军宁; 郭学峰; 李丹丹; 张雪利
Original assignee: Gansu Agricultural University
Current assignee: Gansu Agricultural University
Priority date: 2024-01-23
Filing date: 2024-01-23
Publication date: 2024-04-26

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 application thereof

Technical Field

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

Background

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 signal molecule, it also plays a critical role in regulating plasma membrane and ion channel activities in terms of salt and permeation stress resistance, vesicle transport, actin organization, vanadate-sensitive H ⁺ -ATPase, and the like. Phosphatidylinositol-4, 5-bisphosphate is a lipid signal that interacts with effector proteins as well as a substrate in the phospholipase C signal transduction pathway. The signaling pathway is through the regulation of a variety of processes within the cell, including cytoskeletal organization, membrane transport, guard cell movement, and pollen tube growth. The correlation of PtdIns (4, 5) P2 signaling function depends to a large extent on its spatiotemporal distribution, which is determined primarily by its metabolic activity in 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-phospho5-kinase (PIP 5K) of the PIPKs family plays a vital role in the formation of PtdIns (4, 5) P2 pattern. Compared with animal and fungal genomes, the higher plant genome has a large number of genes encoding PIP5K, and PIP5K genes are found in pattern plants such as Arabidopsis and rice. Arabidopsis thaliana has a total of 11 PIP5K genes, and its structure is highly similar to that of animal-derived type I PtdInsP enzyme.

In recent years, some studies have found the biological function of part of PIP5 Ks. For example, in arabidopsis, drought, salt, ABA (abscisic acid) and other external stimuli rapidly induce AtPIP K1 expression, which is also regulated by soluble protein kinases. AtPIP5K1 and AtPIP K2 are involved in pollen development, they play an important role in vacuole formation and pollen development, and in the pip5K1 and pip5K2 deletion mutant pollen grains, vacuole defects and outer wall formation are impaired due to the loss of function. It was found that AtPIP K4 and AtPIP K5 contributed to pollen germination and pollen tube elongation. AtPIP5K4, atPIP5K5 and AtPIP K6 are redundantly involved in pollen germination. Furthermore AtPIP K3 regulates the elongation of its root hairs by specific expression in the root. AtPIP5K9 interacts with the cytoplasmic enzyme CINV1 and produces an inhibitory effect on root cell elongation under sugar control. AtPIP5K7, atPIP5K8 and AtPIP K9 are redundantly involved in root growth under osmotic stress. It was found that a single gene OsPIP K1 plays a key role in the heading process of rice. The PIP5K gene is involved in pollen development of wheat and capsicum. The soybean GmPIP K gene enhances drought stress tolerance in the overexpressed plants of arabidopsis. These results indicate that PIP5K plays a critical role in plant adaptation to abiotic environments and in the growth and development process.

Cotton (Gossypium spp.) breeding is susceptible to a variety of adverse conditions, resulting in low yields. Cotton has better tolerance to abiotic stress than other crops, but its yield is still compromised under extreme temperatures, salt and drought stresses. The PIP5K gene is a key gene regulating stress response and pollen production and is an important component in the signaling pathway. However, the exact role of GhPIP5K in cotton stress response and growth regulation is still unclear.

Disclosure of Invention

The invention aims to provide upland cotton GhPIP5K2 and GhPIP5K22 genes and application thereof, so as to solve the problems in the prior art. The cotton responses to abiotic stress by forward regulation of the GhPIP5K2 and GhPIP5K22 genes of upland cotton, and up-regulation of expression in cotton can improve the stress resistance of the cotton to abiotic stress.

In order to achieve the above object, the present invention provides the following solutions:

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

The invention also provides application of the recombinant vector comprising the upland cotton GhPIP5K2 gene in regulating and controlling the stress resistance of upland cotton to abiotic stress.

The invention also provides application of the engineering bacteria comprising the recombinant vector in regulating and controlling the stress resistance of upland cotton to abiotic stress.

Further, by up-regulating the expression level of the upland cotton GhPIP5K2 gene in upland cotton, stress resistance of the upland cotton to abiotic stresses including high temperature, low temperature, drought and salt stress is improved.

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

The invention also provides application of the recombinant vector comprising the upland cotton GhPIP5K22 gene in regulating and controlling the stress resistance of upland cotton to abiotic stress.

Further, by up-regulating the upland cotton GhPIP5K22 gene expression level in upland cotton, the stress resistance of the upland cotton to abiotic stress including high and low temperature stress is improved.

The invention also provides a method for improving the stress resistance of upland cotton to abiotic stress, which is characterized by comprising the step of up-regulating the expression level of GhPIP5K2 and/or GhPIP5K22 genes in upland cotton.

The invention discloses the following technical effects:

the 28 genes of the PIP5K family of the upland cotton are successfully identified by carrying out bioinformatics analysis on the upland cotton. The invention carries out transcriptome analysis on the expression mode of GhPIP5Ks under the treatment of four abiotic stresses of high temperature, low temperature, drought and salt, and discovers that GhPIP5K family members can respond to the abiotic stresses; meanwhile, the GhPIP5K2 is found to be highly expressed in high temperature, low temperature, drought and salt stress, and the GhPIP5K22 is found to be highly expressed in high temperature and low temperature stress. In order to further define the molecular mechanism of GhPIP5K2 and GhPIP5K22 genes responding to abiotic stress, the VIGS technology is utilized to silence target genes in cotton plants, and after the corresponding abiotic stress treatment, the silence GhPIP5K2 gene or the silence GhPIP5K22 gene is found to cause wither leaves of plants, reduced activity of antioxidant enzymes (CAT, POD and SOD) and increased MDA content. Silencing GhPIP5K2 reduces expression of adversity stress related genes GhHSFB2A, ghDREB2A, ghDREB2C, ghRD-1, ghRD29A, ghBIN2, ghCBL3, ghNHX1, ghPP C, ghSnRK2.6 and GhCBF1, and silencing GhPIP5K22 reduces expression of adversity stress related genes GhHSFB2B, ghDREB2A, ghDREB2C, ghRD-1, ghRD29A, ghCBF1 and GhCIPK 6. The method shows that the silence GhPIP5K2 and GhPIP5K22 genes weaken the stress resistance of cotton to abiotic stress, and further proves that the GhPIP5K2 and GhPIP5K22 genes positively regulate cotton to respond to abiotic stress, and provide important gene resources for the stress resistance breeding of upland cotton to abiotic stress.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a vector pEASY-T5 Zero and TRV vector map;

FIG. 2 is a graph showing phenotypic observations of control plants and TRV GhPIP5K 2-silenced plants after stress; wherein, a, the cotton leaf in the positive control TRV GhCLA shows whitening phenomenon; b. using qRT-PCR to detect the silencing efficiency of ghip 5K2, asterisks indicate the level of significance (< 0.05, <0.01, < P) compared to the control group; c. effects of heat stress on TRV 00 and TRV GhPIP5K2 phenotypes; d. effects of Cold stress, drought stress and salt stress on TRV 00 and TRV GhPIP5K2 phenotypes; e. counting the quantity of wilt leaves of the control plant and the silent plant after heat stress; f. counting the blackening quantity of new leaves of a control plant and a silent plant after cold stress; g. counting the quantity of wilt leaves of a control plant and a silent plant after drought stress; h. counting the quantity of wilted leaves of a control plant and a silent plant after salt stress;

FIG. 3 is a phenotypic observation of control plants and TRV GhPIP5K22 silenced plants after stress; wherein, a, the cotton leaf in the positive control TRV GhCLA shows whitening phenomenon; b. using qRT-PCR to detect the silencing efficiency of ghip 5K22, asterisks indicate the level of significance (< 0.05, <0.01, < P) compared to the control group; c. counting the number of yellowing leaves of the control plants and the silent plants after heat stress; d. counting the quantity of wilt leaves of a control plant and a silent plant after cold stress; e. effects of heat and Cold stress on the TRV:00 and TRV:GhPIP5K22 phenotypes;

FIG. 4 shows the results of physiological and biochemical index detection of plants with TRV 00, TRV GhPIP5K2 and TRV GhPIP5K22 before and after abiotic stress treatment; wherein, TRV is GhPIP5K2 SOD activity, POD activity, CAT activity and MDA content; TRV: SOD activity, POD activity, CAT activity and MDA content of GhPIP5K 22; different letters represent differences (< 0.05 by P);

FIG. 5 shows the results of stress-related gene expression level detection of plants TRV 00, TRV GhPIP5K2 and TRV GhPIP5K22 before and after abiotic stress treatment; expression level of 11 stress marker genes of TRV GhPIP5K 2; expression level of 7 stress marker genes of TRV GhPIP5K 22; asterisks indicate the level of difference significance (< P <0.05, < P < 0.01) compared to control values.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

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

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The inventor successfully identifies 28 genes of the PIP5K family of upland cotton by carrying out bioinformatics analysis on the upland cotton. The invention carries out transcriptome analysis on the expression mode of GhPIP5Ks under the treatment of four abiotic stresses of high temperature, low temperature, drought and salt, and discovers that GhPIP5K family members can respond to the abiotic stresses; meanwhile, the GhPIP5K2 is found to be highly expressed in high temperature, low temperature, drought and salt stress, and the GhPIP5K22 is found to be highly expressed in high temperature and low temperature stress.

In order to further define the molecular mechanism of GhPIP5K2 and GhPIP5K22 genes responding to abiotic stress, the target genes are silenced in cotton plants by utilizing the VIGS technology, ghPIP5K2 and GhPIP5K22 target gene fragments are cloned, VIGS silencing vectors of the two genes are respectively constructed, and biological functions under drought and salt stress are studied. The experiment is as follows:

1. Experimental materials

1.1 Cotton varieties

The invention takes a upland cotton variety 'new stone K25' as a research object, and seeds are provided by stone river seed agricultural science institute.

1.2 Vectors and competent cells

PEASY-T5 Zero cloning vectors (CT 501-01, containing DH 5. Alpha. E.coli competent cells) and GV3101 Agrobacterium competent cells were purchased from full gold Biotechnology Inc. and Shanghai Biotechnology Inc., respectively, and the VIGS vector systems for gene silencing (TRV 1, TRV2 and TRV-GhCLA 1) were given away by cotton institute of the national academy of sciences, the vector maps are shown in FIG. 1.

2. Experimental method

2.1 Primer design

Cloning primers, qRT-PCR primers, and primers for construction of the VIGS silencing vector for the GhPIP5K2 gene were designed using Primer-BLAST (https:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST /), the Primer sequences (SEQ ID NOS: 3-8) are shown in Table 1:

TABLE 1 cloning of GhPIP5K2 Gene, qRT-PCR, construction of the primers sequence for the VIGS silencing vector

Cloning primers, qRT-PCR primers, and primers for construction of the VIGS silencing vector for the GhPIP5K22 gene were designed using Primer-BLAST, and the Primer sequences (SEQ ID NOS: 11-16) are shown in Table 2:

TABLE 2 cloning of GhPIP5K22 Gene, qRT-PCR, construction of primer sequence for VIGS silencing vector

2.2 Cotton planting

A. An equal amount of nutrient soil (substrate: vermiculite=1:1) was placed in each pot and immersed in a large pot containing tap water until water was absorbed onto the pot surface for cotton seed germination;

b. Planting the seeds of the neolith K25 in the flowerpot, and culturing in a climatic incubator (16 h illumination, 8h darkness, temperature 28 ℃ and humidity 70%) to ensure that the emergence of the seeds is consistent and the planting depth of the seeds is kept at 1.5 cm;

c. when cotton grows to the four-leaf stage, tender leaves are selected for sampling, and the sampled samples are quickly frozen in liquid nitrogen and then stored at the temperature of minus 80 ℃ for standby.

2.3 Reverse transcription and fluorescent quantitative detection assays

2.3.1 Extraction of Total RNA

Total RNA was extracted from cotton leaves according to RNAprep pure polysaccharide polyphenol plant total RNA extraction kit (cat No. DP441, available from China Tiangen Biochemical technology Co., ltd.).

All samples were assessed for RNA quality using a NanoDrop 2000 spectrophotometer from Thermo Scientific. The samples were stored at-80℃low temperature.

2.3.2 Reverse transcription

The total RNA obtained above was used as a template, and was subjected to reverse transcription according to FastKing one-step method to remove the first strand of genomic cDNA to prepare a premix kit (product number KR118, purchased from China root Biochemical technology Co., ltd.) to generate cDNA.

After the reaction was completed, the concentration and purity of cDNA were measured and stored at-20℃for further use.

2.3.3 Fluorescent quantitative detection

Fluorescent quantitative detection was performed using Talent fluorescent quantitative detection kit (SYBR Green) (cat. No. FP209, available from Tiangen Biochemical technologies Co., ltd.) as a template. The reaction system is shown in Table 3:

table 3 fluorescent quantitative determination reaction system

The same reaction system as in Table 3 was used for the fluorescent quantitative detection of the GhPIP5K22 gene, with the primers replaced with q-GhPIP5K22-F and q-GhPIP5K22-R primers of qRT-PCR.

The reaction procedure is shown in table 4:

TABLE 4 fluorescent quantitative determination reaction procedure

2.4 Amplification of the Gene fragment of interest

2.4.1 Reaction System

The amplification of the GhPIP5K2 gene fragment was performed using GloriaNova HS X Master Mix (cat No. RK20717, available from Aibotake BioCo.) using the cDNA of Xinshi K25 as a template, and the system was as follows (Table 5):

TABLE 5 amplification reaction System of target Gene fragment

Amplification of the GhPIP5K22 Gene Using the same reaction system as in Table 5, the primers were replaced with the GhPIP5K22-F and GhPIP5K22-R primers for amplifying the GhPIP5K22 gene.

2.4.2 Reaction procedure

After the reaction solution was added according to the above system, the mixture was gently mixed, centrifuged briefly, and reacted according to the reaction procedure shown in Table 6.

TABLE 6 amplification reaction procedure for target Gene fragment

Amplifying the GhPIP5K2 gene target fragment by adopting a PCR method, wherein the length of the GhPIP5K2 gene target fragment is 417bp, and the nucleotide sequence is shown as SEQ ID NO:2 is shown as follows:

GCCTAGGGAGTGTTGCAGAGGAAGAGGAAGATGAAATCACCAACTATCCACAAGGCCTTGTATTGGTCCCTCGTGGAACAGATGACAATAGTGTTGTTGCAGGTTCTCATATACGAGGTCGACGTTTGCGTGCATCAGCTGTAGGTGATGAAGAAGTAGACCTGCTTCTCCCCGGCACGGCAAGACTCCAAATCCAGCTCGGAGTGAACATGCCAGCAAGAGCCGAACAGATTCCAGGAAAAGAAGAAAACATGTTCCATGAATCATATGACGTTGTGTTATATCTGGGAATCATTGACATTTTACAAGAGTATAACATGACTAAGAAGATTGAACATGCCTATAAATCTCTTCAGTTCGATTCACTATCCATATCTGCCGTCGACCCTACGTTTTACTCGCAACGGTTCTTGCAAT.

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

ATGTCTGGCCTTGTGGTCACTGTTGGTAACGTGGAAGAAGTACTTTCTCGCGCAGAAAGAACTAAATCTCTTGATGCCATCATTGACAAGGACAACGGATGTATACTAACTAATGGTGATGCTAACCATAGTTCCGAAACAGCTGGATTTAGAGTTGGAGAACTCTTGCTGCCGAATGGGGACTCTTATTCCGGGTCATTGCTCGGAAACATGCCAGAGGGTCAAGGGAAATATGTTTGGCAAGGTGGTTGTGTGTATGAAGGAGAATGGAGACGTGGGATGAGGCAAGGGATTGGCAAAATACAATGGCCTTCTGGAACTGTTTATGATGGTGAATTCTCAGGTGGATATATGCATGGTACTGGGACATATATTGGCTCTAATAAATTGACTTATAAGGGGAGATGGAAATTGAGTCTCAAACATGGTTTAGGATACCAAGTTTATCCTAATGGAGATGTGTTTGAAGGCTCCTGGATGCAGGGAACACCGGAAGGTCCTGGAAAATATACTTGGGCCAATGGAAATGTTTATCTAGGGAATATGAAGGGTGGAAAAATGTCAGGCAAAGGAACTCTCACTTGGACAAATGGAGACTCCTTTGAAGGAAGCTGGTTAAATGGAATGATACACGGATTTGGAGTGTATACTTGGAGAGATGGTGGTTGCTATGTAGGAACTTGGACACGGGGTTTAAAGGATGGAAAAGGATCATTTTATCCCCAAGGCAACCGGCTTCCAGCCTCACAAGAAGTTTACCTCAATGCTCTCAGAAAAAGAGGATTGTTACCAGATTTGAGAAAACAGAATCATTCTCATATCCACCATGCTGCTTCTGTGGACATGGGAAGTGTCAAGGTTGGTGGCAACCGGGTATCTGATCGTAATTCTGATAAGCTATCAGAAGGAAACTTATTAAATCTACAACAGTCTCGCAACAGAAATGTTTCCTTGGAAAGACGTTGGAGTCTGGAGGTATCCATTGAGAAAGTGATTGGGCACGATTCGTCATTAGAGTTATCTGATTCTTTTAAGGAAGGGAGAGAAAACGGAAGTGAAACAAATGCTCCAATCTTAGAACGTGAATACATGCAAGGTGTCTTAATTAGTGAGCTTGTGTTGAATAATAGTTTTTCACCACCATCTAGAAGAGCGAAGCGGAGACACAAAAAGTTAGCAAAAGAGGTTAAGAGGCCTGGAGAAGCAATCATTAAAGGTCACAGGAGTTATGATTTAATGCTTAGTTTGCAGCTTGGAATCAGATACACTGTGGGGAAAATTACACCTGTGCAACGACGAGAGGTTAGAGCATCAGACTTTGGCCCCCGAGCAAGCTTTTGGATGAATTTTCCTAAAGTGGGATCACAATTGACACCTACCCATCAGTCTGATGATTTTAAGTGGAAAGATTACTGCCCAATGGTTTTCAGGAATCTAAGGGAGATGTTCAAGATTGATGCTGCCGACTACATGATGTCCATTTGTGGAAATGATGCTCTCAGGGAACTTTCTTCTCCTGGGAAAAGTGGTAGTATCTTCTTTCTGTCTCAAGATGATCGTTTCATGATTAAGACACTCCGGAAATCTGAAGTAAAGGTTCTTCTAAGAATGCTTCCCAACTATCATCATCACGTGAGATCATATGAGAACACACTCATCACAAAGTTCTTTGGGCTTCACAGAATCAAACCATCTAGTGGTCAGAAGTTTCGCTTTGTAGTAATGGGAAATATGTTTTGCACCGAGTTAAGGATTCATAGAAGATATGACTTGAAAGGATCATCACAAGGGCGTTCTGCTGATAATGTTGAAATTGATGAGAACACAACGCTTAAAGATCTGGATCTCAACTACTGCTTTTATTTGGAACCTTCTTGGCGAGATGCTTTATTAAGGCAAATAGAGATTGATAGTAAATTTTTGGAAGCACAATGCATTATGGATTATAGCCTTTTGCTTGGTGTGCATTATCGGGCACCCCAGCATTTGAGGTCTCTCATGTCCTACAACAGAACGGACGGCCTAGGGAGTGTTGCAGAGGAAGAGGAAGATGAAATCACCAACTATCCACAAGGCCTTGTATTGGTCCCTCGTGGAACAGATGACAATAGTGTTGTTGCAGGTTCTCATATACGAGGTCGACGTTTGCGTGCATCAGCTGTAGGTGATGAAGAAGTAGACCTGCTTCTCCCCGGCACGGCAAGACTCCAAATCCAGCTCGGAGTGAACATGCCAGCAAGAGCCGAACAGATTCCAGGAAAAGAAGAAAACATGTTCCATGAATCATATGACGTTGTGTTATATCTGGGAATCATTGACATTTTACAAGAGTATAACATGACTAAGAAGATTGAACATGCCTATAAATCTCTTCAGTTCGATTCACTATCCATATCTGCCGTCGACCCTACGTTTTACTCGCAACGGTTCTTGCAATTCATTCAGAAGGTATTTCCTCTGAATTCCATGAAAACTTGA.

amplifying a GhPIP5K22 gene target fragment by adopting a PCR method, wherein the GhPIP5K22 gene target fragment is 431bp long, and the nucleotide sequence of the GhPIP5K22 gene target fragment is shown as SEQ ID NO:10 (underlined is the target fragment):

AGACGGGTGCATGTACGAAGGAGAGTGGCGTCGTGGGAAAGCCAACGGGAAAGGTAAGTTTTCTTGGCCATCTGGAGCCACTTTTGAAGGTGGTTTCAAGTCGGGTCGGATGGAAGGATTCGGGACGTTTATCGGATCCGACGGCGACACGTACCGTGGGTCGTGGAGCTCCGATCTAAAACACGGCTATGGTCACAAGTGTTACGCAAATGGGGATTACTACGAAGGATCATGGAGGAAAAACCTACAAGAGGGGCACGGCCGTTATGTTTGGAGTAACGGTATCGAATACGTCGGTGAATGGAAAAACGGAGTCATCTCTGGCCGTGGAACCCTGATATGGGCAAATGGAAACGGGTACGATGGGCAATGGGAAAACGGTATGCCCAAAGGAGACGGAGTTTTCTCTTGGCCGGACGGAAGTTGCTATA.

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

ATGGAGGAAGTAGTGCTCAATGAGCCGAGTGACGTCGTTTTAAACGCCAAGAAGAAGAAATCCGACGAGGAGAAGGACCAGTTGGTGGTTGCTGTTACGACACCTACGGATCACCATCACCGGAGCCGATCTCAAGCTGCCACACGGCGCGTGACTCCCACGACTAACGCCGCGTCTTTCTTCACCATGGGTTCGGGTGATACTGTCGAGAAATTCCTCCCTAACGGTGATC

TTTACATCGGTAGCTTCTCAACCAACGCGCCACACGGATCCGGGAAGTACCTTTGGAAA

GACGGGTGCATGTACGAAGGAGAGTGGCGTCGTGGGAAAGCCAACGGGAAAGGTAAG

TTTTCTTGGCCATCTGGAGCCACTTTTGAAGGTGATTTCAAGTCGGGTCGGATGGAAGG

ATTCGGGACGTTTATCGGATCCGACGGCGACACGTACCGTGGGTCGTGGAGCTCCGATC

TAAAACACGGCTATGGTCACAAGTGTTACGCAAATGGGGATTACTACGAAGGATCATGG

AGGAAAAACCTACAAGAGGGGCACGGCCGTTATGTTTGGAGTAACGGTATCGAATACGT

CGGTGAATGGAAAAACGGAGTCATCTCTGGCCGTGGAACCCTGATATGGGCAAATGGAA

ACGGGTACGATGGGCAATGGGAAAACGGTATGCCCAAAGGAGACGGAGTTTTCTCTTG

GCCGGACGGAAGTTGCTATATCGGAGCATGGAACGGAGATAACATGAAGAAAACTCAA

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 vectors and VIGS silencing of genes of interest

2.5.1 Construction of silencing vector

(1) Ligation of the Gene fragment of interest to the cloning vector pEASY-T5 Zero

A. the pEASY-T5 Zero vector was removed from the-80℃refrigerator and thawed on ice.

B. the volume of the target fragment added was calculated such that the molar ratio of carrier to target fragment=1:5, and the ingredients shown in table 7 were added to a sterile 1.5mL centrifuge tube on ice:

Table 7 connection system

C. the mixture was gently mixed, centrifuged briefly, and then connected at 25℃for 5 minutes.

(2) Transformation of DH5 alpha E.coli competent cells

The gene sequence primer is used for PCR verification and sequencing (completed by Shanghai Bioengineering Co., ltd.) after being transformed into DH5 alpha escherichia coli competent cells by a heat shock method.

(3) Construction of silencing vector

A. The positive plasmid which is sequenced successfully in the step (2) is used as a template, a silencing fragment is amplified by adding primers of restriction enzyme EcoRI and XhoI cleavage sites and protective bases, and fragments of GhPIP5K2 and GhPIP5K22 are respectively inserted into a TRV2 (pYL) silencing vector by a double-enzyme digestion method, so that a TRV:GhPIP5K2 silencing vector and a TRV:GhPIP5K22 silencing vector are constructed, wherein the specific enzyme digestion system is as follows:

Table 8 enzyme digestion System

B. after enzyme digestion for 3 hours at 37 ℃, 10X Loading Buffer is added to stop the reaction, the PCR product of the target gene fragment is recovered by glue, and the enzyme digestion large fragment is recovered by the carrier. After the enzyme digestion product is recovered, the target fragment is connected with a silencing vector, the connection product is transformed into escherichia coli competent cells for blue and white spot screening experiments, bacterial liquid PCR and double enzyme digestion identification are carried out, positive plasmid sequencing (Shanghai biological engineering Co., ltd.) is carried out after the completion, and the positive plasmid is transferred into agrobacterium GV3101 competent cells, so that the silencing vector is obtained.

2.5.2VIGS silencing of genes of interest

VIGS silencing was performed on new 25K seedlings as follows:

a. Planting upland cotton 'new stone 25K' seeds according to the method of 2.2, soaking the seeds until the seeds grow to 7 days and cotyledons are fully unfolded until nutrient soil in the flowerpot absorbs water to the surface, stopping soaking, and standing for later use.

B. Kan ⁺ and Rif were added to LB liquid medium for use, wherein the final concentrations of Kan ⁺ and Rif were 50. Mu.g/mL and 25. Mu.g/mL, respectively. The VIGS vector system and the objective gene bacterial liquid taken out from-80 ℃ were thawed on ice, and activated at 28 ℃ and 200rpm for 12-16 hours (bacterial liquid: LB liquid medium=1:10). And after the activation is finished, the propagation is carried out according to the same proportion.

C. After the bacterial liquid is propagated, centrifuging for 10min at 5000rpm, pouring out supernatant, retaining bacterial cells, suspending the bacterial cells by using a spectrophotometry heavy suspension, and obtaining the bacterial cell with OD ₆₀₀ between 0.8 and 1.0.

D. After resuspension was completed, the cells were left to stand in the dark for 3 hours, and pYL (TRV 1) was mixed with bacterial body weight suspensions 1:1 containing TRV:00 (blank control group), TRV: ghCLA1 (positive control group), TRV: ghPIP5K2 (experimental group 1) and TRV: ghPIP5K22 (experimental group 2), respectively, and the mixture was thoroughly mixed and treated in the dark for 3 hours.

E. On day 7 of cotton seedling growth, it was immersed in water according to the method of step a. The cotton seedlings were subjected to VIGS injections on day 8 of growth, with specific manipulations: and d, cutting the back of the cotyledon by using a 1mL syringe needle, and injecting the mixed bacterial liquid obtained in the step d into the cotton cotyledon to fill the whole cotyledon with the bacterial liquid as much as possible.

F. after injection, the cells were wrapped with plastic bags and incubated in the dark at 25℃for 24h, followed by incubation under normal growth conditions.

G. After the positive control cotton seedlings whiten, the cotton young leaves of the experimental group and the blank control group are adopted to carry out fluorescent quantitative PCR to detect the gene expression level and silencing efficiency.

2.6 Abiotic stress treatment

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

Meanwhile, the positive cotton seedlings to be injected are whitened, and the cotton seedlings growing to four-week-old from the TRV: ghPIP5K22 plants are subjected to high-temperature (42 ℃) and low-temperature (12 ℃) stress treatment. Phenotypic observations were performed.

2.7 Stress resistance detection

2.7.1 Determination of physiological and Biochemical indicators in Gene-silenced plants

After stress treatment for 10 days, the Catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) of the leaves of the TRV:00 empty carrier plant, the TRV:GhPIP5K2 plant and the TRV:GhPIP5K22 plant were detected respectively according to the conventional method, and the activities of the three antioxidant enzymes and the content of Malondialdehyde (MDA) were detected.

2.7.2 Quantitative detection of adversity stress related Gene fluorescence in Gene-silenced plants of interest

Young leaves of the gene silencing line were picked before and after stress treatment, RNA was extracted by using a RNAprep pure polysaccharide polyphenol plant total RNA extraction kit (cat No. DP441, purchased from the physcolio Biotechnology Co., ltd.), reverse transcription was performed by using a FastKing one-step method to remove the genomic cDNA first strand synthesis premix kit (cat No. KR118, purchased from the physcolio Biotechnology Co., ltd.), and finally expression level detection of the stress-related gene was performed by using a Talent fluorescent quantitative detection kit (SYBR Green) (cat No. FP209, purchased from the physcolothio Biotechnology Co., ltd.) for specific methods.

Wherein, the stress related genes detected by the TRV GhPIP5K2 silent plants are GhHSFB2A, ghDREB2A, ghDREB2C, ghRD-1, ghRD29A, ghBIN2, ghCBL3, ghNHX1, ghPP C, ghSnRK2.6 and GhCBF1.

The stress related genes detected by the GhPIP5K22 silent plants are GhHSFB2B, ghDREB2A, ghDREB2C, ghRD-1, ghRD29A, ghCBF1 and GhCIPK6.

3. Results and analysis

3.1 Phenotypic observations

On day 9 post infection, cotton leaves exhibited a albino phenotype (a in FIG. 2 and a in FIG. 3), and the positive control TRV GhCLA1 validated the effectiveness of the VIGS technique, and the expression level of the gene of interest was significantly reduced by the VIGS in combination with qRT-PCR detection of silencing efficiency (b in FIG. 2 and b in FIG. 3).

For GhPIP5K2 function under four abiotic stress conditions (high temperature, low temperature, drought and salt), the silenced TRV: ghPIP5K2 plants and normal plants were compared, and the TRV: ghPIP5K2 plants exhibited characteristics of withering, yellowing of leaves, and water shortage (FIGS. 2c and d). Further, the silenced TRV: ghPIP5K2 plants had new leaves blackened under cold stress (d in FIG. 2) compared to control plants. After four abiotic stress treatments, the number of TRV: ghPIP5K2 plants with yellowing or withering leaves was significantly greater than that of normal plants (e, f, g and h in FIG. 2).

For GhPIP5K22 function under two abiotic stress conditions (high temperature, low temperature), the silenced TRV: ghPIP5K22 plants and normal plants were compared, and the TRV: ghPIP5K22 plants exhibited characteristics of wither, yellowing of leaves, lack of water, and the like (FIG. 3 e). After statistical treatment with two abiotic stresses, the number of TRV: ghPIP5K22 plants with yellowing or withering leaves was significantly greater than that of normal plants (c and d in FIG. 3).

These results indicate that GhPIP5K2 and GhPIP5K22 play a positive role in cotton abiotic stress treatment.

3.2 Physiological and Biochemical index detection results

In order to explore the effect of abiotic stress on GhPIP5K2 and GhPIP5K22, the invention detects the change of SOD, POD, CAT activity and MDA content in the TRV: ghPIP5K2 and TRV: ghPIP5K22 silencing cotton plants. The results show that compared with the TRV:00 plants, the antioxidant enzyme activities (SOD, POD and CAT) of the silencing plants TRV:GhPIP5K2 and TRV:GhPIP5K22 are obviously reduced; in contrast, the MDA content was significantly increased (FIG. 4).

The findings above indicate that silencing GhPIP5K2 and GhPIP5K22 weakens cotton's tolerance to abiotic stress.

3.3 Fluorescent quantitative detection results of adversity stress related genes

Compared with the control plants, the expression of the stress related genes of the TRV: ghPIP5K2 and the TRV: ghPIP5K22 plants is obviously changed before and after the stress treatment.

In TRV GhPIP5K2 plants, ghBIN, ghCBL and GhNHX1 expression was up-regulated in control plants, while in plants silenced after stress treatment was significantly down-regulated. The expression level of GhDREB.sup. 2A, ghHSFB.sup. 2C, ghRD20-1, ghPP.sup.C and GhSnRK2.6 in the silenced plant TRV: ghPIP5K2 was lower than in the control plants before and after the treatment. The expression levels of GhHSFB A, ghDR a and GhCBF1 were down-regulated in the silenced plants compared to the pre-treated control, while the expression levels of the silenced plants were significantly reduced under at least one stress treatment after treatment compared to the control (fig. 5 a). The results indicate that GhPIP5K2 may have a positive regulatory effect on abiotic stress.

In the TRV GhPIP5K22 plants, ghHSFB B expression was up-regulated in control plants, while in the silent plants after high temperature stress treatment was significantly down-regulated. Likewise, the other three stress-related genes (GhDREB, A, ghDREB, 2C and GhRD, 29A) also exhibited similar patterns. In contrast, in the silencing plant TRV: ghPIP5K22, it was observed that GhRD-1, ghCIPK6 and GhCBF1 were expressed at a lower level before and after the treatment than the control plant (b in FIG. 5). These findings suggest that silencing of GhPIP5K22 may directly affect the expression levels of GhRD20-1, ghCIPK6 and GhCBF 1. Taken together, we speculate that GhPIP5K22 may play a positive regulatory role in the temperature stress response process.

In conclusion, upland cotton with VIGS silencing of ghip 5K2 or ghip 5K22 is more sensitive to abiotic stress than control group, demonstrating that cotton stress resistance is reduced, thus proving that the gene positively regulates cotton response to abiotic stress response. The invention provides important gene resources for high stress resistance breeding of upland cotton against abiotic stress.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

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.