Cotton GhCYP71A1 gene and application of protein coded by cotton GhCYP71A1 gene in cold resistance regulation

Abstract

The invention relates to the technical field of plant molecular biology, in particular to a cotton GhCYP71A1 gene and application of protein coded by the cotton GhCYP71A1 gene in cold resistance regulation and control. A gene related to the cold resistance of a cotton CYP family is excavated, it is found that the gene can negatively regulate and control the cold resistance of cotton and can serve as a target gene for cotton cold-resistant breeding, the molecular mechanism of the GhCYP71A1 for regulating and controlling the low-temperature response in the seedling emergence stage of the cotton is further clarified, material support is provided for low-temperature-resistant breeding of the cotton, and the application of the GhCYP71A1 to the low-temperature-resistant breeding of the cotton is promoted. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of plant molecular biology, in particular to cotton GhCYP71A1 Application of genes and their encoded proteins in the regulation of cold tolerance. Background Art

[0002] In the textile industry, cotton is an important raw material. However, low temperatures can damage cotton quality and reduce yield. Therefore, cultivating low-temperature resistant materials is the most economical and effective way to solve the harm of low temperatures during the cotton seedling period. With the development of molecular biology technology, in-depth analysis of the low-temperature regulation mechanism of cotton seedlings and the discovery of key genes are key issues in stress-resistant breeding.

[0003] Cold stress on cotton can be divided into low temperature stress and freezing stress. Low temperature stress refers to the temperature range of 0 to 15 °C, while freezing stress refers to the temperature range below 0 °C. Under low temperature conditions, the fluidity of the cell membrane in cotton decreases, affecting the activity of multiple enzymes, which seriously affects its physiological homeostasis and ultimately has an adverse effect on cotton yield and fiber quality. Cytochrome P450 (CYP) monooxygenases are an important family in plants. These enzymes catalyze multiple reactions in biochemical pathways involving primary and secondary metabolic pathways. They are called cytochrome P450 because they have the highest absorbance at 450 nm after binding to reduced CO. In addition, most plant P450s are endoplasmic reticulum-bound proteins, with a hydrophobic helix at the amino terminus that anchors them to the endoplasmic reticulum membrane and locates most of the rest of the protein on the outer cytoplasmic surface of the endoplasmic reticulum membrane. Adjacent to the hydrophobic helix at the amino terminus is a proline (P)-rich conserved region containing a conserved (P / I)PGPx(G / P)xP sequence. Cytochrome P450 is one of the most widely used enzymes in nature. It not only plays a vital role in many biological functions such as growth, nutrition, development, and detoxification, but also participates in the low temperature response process of many organisms. P450 genes are also involved in the homeostasis of plant hormones and are generally related to the biosynthesis and catabolism of secondary metabolites, plant hormones, etc. Previously, a member of the rice CYP450 family (OsCYP71D8L) has been shown to play a key role in responding to abiotic stresses by maintaining the homeostasis of gibberellins (GA) and cytokinins (CK), and CYP82 family genes are also involved in plant-pathogen interactions. Cotton CYP82D can regulate plant cell death by regulating the octadecane-like pathway, and negatively regulates cotton resistance to dahlia Verticillium wilt by controlling jasmonic acid (JA) biosynthesis. Arabidopsis overexpression AtCYP79B2 The results showed that transgenic Arabidopsis thaliana showed dwarfism and sterility, which are also typical traits of large accumulation of auxin, further confirming AtCYP79B2 Participates in the catalytic synthesis of auxin. P450s under low temperature stress, CYP genes in ryegrass and tall fescue ( CYP73A , CYP75A and CYP75B ) expression level is upregulated, and it is involved in the synthesis of flavonoids and regulates its cold tolerance. CYP707A The gene is upregulated under low temperature stress and positively regulates the cold tolerance of Arabidopsis through an ABA-dependent pathway; CYP735As It can positively regulate low temperature by promoting the synthesis of cytokinin (CK). CYP709B3 Overexpression of the gene enhanced the salt tolerance of transgenic Arabidopsis. PgCYP736B Overexpression of the gene enhanced resistance to salt stress by reducing the production of hydrogen peroxide accumulation and increasing carotenoid levels and the expression of abscisic acid biosynthesis genes.

[0004] Although the research on cold tolerance of CYP-related genes in plants has made some progress, the effects and pathways of action in different plants are different. At the same time, there are few studies on CYP-related genes in cotton, and only CYP There are reports on genes involved in cotton's response to salt stress and disease stress, but their regulatory pathways under low temperature stress in cotton remain unclear.

[0005] Therefore, we should explore the important genes regulating cotton cold tolerance and analyze CYP The role of genes in cotton's low temperature response and their regulatory pathways have become technical challenges that need to be urgently addressed in this field. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a cytochrome P450 family gene related to cotton cold tolerance GhCYP71A1 , this gene has tissue expression specificity, with the highest expression level in cotton roots. This gene is induced by low temperature stress, negatively regulates cotton cold tolerance, reduces the degree of inhibition of cotton roots under low temperature stress, and thus accumulates relatively more biomass. The amino acid sequence of the protein encoded by this gene is shown below (1) or (2): (1) As shown in SEQ ID NO.1; (2) An amino acid sequence derived from (1) with equivalent function, wherein one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO.1.

[0007] The protein has an isoelectric point of 7.66 and a molecular weight of 58.26 kD, which is a transmembrane protein. Multiple amino acid sequence alignment revealed that the protein has multiple conserved domains, including the C-terminal heme binding domain, K helix and PERF domain, and the protein is located on the endoplasmic reticulum membrane.

[0008] Furthermore, the present invention provides cotton encoding the protein GhCYP71A1Gene.

[0009] Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0010] Furthermore, the present invention provides biological materials containing the gene.

[0011] Preferably, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or non-renewable plant cells or tissues.

[0012] Furthermore, the present invention provides the use of the protein, the gene or the biological material in regulating the cold tolerance of cotton.

[0013] Preferably, the regulation is negative regulation.

[0014] Preferably, by inhibiting or eliminating the expression or activity of the protein or gene, the cold tolerance of cotton is improved, the root growth of cotton is promoted, the survival rate of cotton is improved, or the accumulation of organic matter in cotton is promoted.

[0015] Furthermore, the present invention provides the use of the protein, the gene or the biological material in the preparation of transgenic cotton.

[0016] Furthermore, the present invention provides the use of the protein, the gene or the biological material in cotton variety improvement.

[0017] Furthermore, the present invention provides a method for improving the cold tolerance of cotton, comprising: using genetic engineering means to inhibit or eliminate the expression level or activity of the protein or the gene in cotton.

[0018] In the specific implementation process, the genetic engineering method includes gene silencing technology.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention has discovered a gene in the cotton CYP family that is related to cold tolerance, and found that the gene can negatively regulate the cold tolerance of cotton and can be used as a target gene for cotton cold tolerance breeding. GhCYP71A1 The molecular mechanism that regulates low temperature response during cotton emergence provides material support for cotton breeding for low temperature resistance and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 1 is the analysis of associated loci on the 5.42 Mb~5.52 Mb region of cotton A10 chromosome, where A is the Manhattan plot; B is the LD heat map of the localization region, and the dotted line represents the candidate region between 5.42 Mb and 5.52 Mb; C is the difference analysis of dry weight among the three haplotypes; D is the expression level of the candidate gene; E is GhCYP71A1 Coding region variant sites.

[0021] Figure 2 It is the signal peptide prediction result of GhCYP71A1, where C-score (raw cleavage site score) is used to distinguish whether it is a cleavage site, and the highest peak is the first amino acid after the cleavage point; S-score (singalpeptide score) is used to distinguish whether the corresponding position is a signal peptide region; Y-score (combined cleavagesite score) is the geometric mean of C-score and S-score, which is used to avoid the influence of multiple high-scoring C-score values ​​on the results.

[0022] Figure 3 This is the predicted result of the transmembrane domain of GhCYP71A1, where purple represents the membrane, blue represents the inside of the membrane, and yellow represents the outside of the membrane.

[0023] Figure 4 This is the phylogenetic tree of GhCYP71A1 homologous proteins.

[0024] Figure 5 This is a partial result of the sequence alignment analysis of GhCYP71A1, where dots represent gaps in sequence alignment, blue boxes represent conserved domains, and red covered areas are highly conserved sites.

[0025] Figure 6 This is a partial result of the sequence alignment analysis of GhCYP71A1, where dots represent gaps in sequence alignment, blue boxes represent conserved domains, and red covered areas are highly conserved sites.

[0026] Figure 7 This is the analysis result of the promoter region.

[0027] Figure 8 yes GhCYP71A1 Expression pattern analysis of the cells, where A represents 0 h of low-temperature treatment at 4 ℃, and B represents 24 h of low-temperature treatment at 4 ℃.

[0028] Fig. 9 yes GhCYP71A1 The subcellular localization results.

[0029] Fig.10 This is the result of LUC dual luciferase interaction verification, where (1) pGreenⅡ62-SK+pGreenⅡ0800-LUC; (2) pGreenⅡ62-SK- GhERF105 +pGreenⅡ0800-LUC; (3)pGreenⅡ62-SK+pGreenⅡ0800-LUC- GhCYP71A1; (4) pGreenⅡ62-SK- GhERF105 +pGreenⅡ0800-LUC- GhCYP71A1 .

[0030] Fig.11 is the result of yeast one-hybrid interaction test, among which pGADT7-p53+pHIS2 represents negative control; pGADT7-p53+pHIS2-53m represents positive control; pGADT7-GhERF105+pHIS2-GhCYP71A1 represents test group; 10 -1 , 10 -2 , 10 -3 It means that the yeast solution was diluted 10 times, 100 times, and 1000 times respectively.

[0031] Fig.12 Albino phenotype and gene silencing GhCYP71A1 The expression levels of A and B are shown in Table 1. GhCYP71A1 The expression level of .

[0032] Fig.13 These are the phenotypic results of silenced plants and control plants before and after low temperature treatment.

[0033] Fig.14 It is the survival rate of gene silenced plants and control plants after low temperature cold treatment.

[0034] Fig.15 These are the test results of root-related traits of gene-silenced plants and control plants before and after low-temperature cold treatment, where A is aboveground dry weight (SDW), B is total root length (TRL), C is total root projected area (TRPA), D is total root surface area (TRSA), E is primary root average diameter (PRAD), and F is total root volume (TRV). DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0037] The present invention relates to molecular biology experiments. If not otherwise specified, reference can be made to the book Molecular Cloning (J. Sambrook, EF Fritsch, T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used reference books for technicians in the field of molecular biology when conducting experimental operations related to molecular biology. In addition, according to different experimental purposes, technicians in the field of molecular biology complete corresponding experiments under the guidance of the operating manuals attached to various commercial kits or entrust specialized companies to conduct experiments, such as gene sequencing, plasmid sequencing, and molecular weight determination.

[0038] The test material in the following examples is Liaomian No. 6 (upland cotton). Full cotton seeds are selected, sown in a mixture of nutrient soil and vermiculite, and placed in a constant temperature and light room (28°C) for cultivation; Nicotiana benthamiana is placed in a constant temperature and light culture room (22°C) for cultivation.

[0039] The primer sequences used in the following examples are shown in Table 1, and the sequence numbers are shown as SEQ ID No.4 to SEQ ID No.21.

[0040] Table 1 Primer sequences

[0041] Example 1 Mining of cotton cold tolerance genes In order to identify genes related to cold tolerance in cotton seedling stage, 14 cold tolerance index data of 200 natural populations of upland cotton were obtained under two types of cold stress (constant low temperature and diurnal temperature variation), and genome-wide association analysis (GWAS) was performed using SNPs and insertion / deletion markers (InDels) of the population. On chromosome A10, there are 13 genes in the linkage region of 5.42 Mb~5.52 Mb ( Gh_A10G0493 - Gh_A10G0505 )( Figure 1 A and Figure 1 B). Gh_ A10G0500 The expression levels were high in both cold-tolerant and cold-sensitive cotton and the differences were significant ( Figure 1 D), and Arabidopsis AT5G07990 The similarity is 98.43%. It is involved in the synthesis of flavonoids and is named after the homologous gene in Arabidopsis. GhCYP71A1 Further research found that GhCYP71A1 The coding regions of the gene had non-synonymous mutations at 5440164 bp, 5440494 bp and 5440711 bp. Gh_A10G0500The dry weight accumulation of germplasm carrying HapC was 6.69% and 8.13% higher than that of HapA and HapB. The dry weight also increased by 6.32% and 9.22% under constant low temperature conditions, which was significantly different ( Figure 1 C and Figure 1 E). In summary, GhCYP71A1 It may be a key gene regulating dry matter weight under low temperature conditions.

[0042] Example 2 Amino Acid Sequence Analysis In this example, bioinformatics methods such as NCBI-Blast and TBtools were used to analyze GhCYP71A1 The amino acid sequence was analyzed as follows: Obtained from Cottonfgd (https: / / cottonfgd.org / ) GhCYP71A1 The CDS sequence and promoter sequence of the cytokine were determined, and the full-length amplification primers Gh CYP71A1-F / Gh CYP71A1-R were designed (Table 1). The cDNA of Liaomian No. 6 was used as a template for PCR amplification. The signal peptide prediction tool SignaIP4.1 (http: / / www.cbs.dtu.dk / services / SignalP-4.1 / ) and the transmembrane domain prediction tool TMHMM-2.0 (TMHMM2.0-DTU Health Tech-Bioinformatic Services) were used for analysis. GhCYP71A1 Signal peptide and transmembrane domain of the encoded protein. In order to study the conserved sequence of GhCYP71A1 protein, the cotton amino acid sequence was aligned with the CYP71A1 amino acid sequences of Arabidopsis, tobacco, peanut, corn, soybean, wheat, and rice using MEGA software, and an evolutionary tree was constructed. The promoter region was analyzed using PLANTCARE (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ).

[0043] The signal peptide prediction results of GhCYP71A1 are as follows Figure 2 As shown in Figure 2, the predicted results of the transmembrane domain of GhCYP71A1 are as follows: Figure 3 The results show that GhCYP71A1 The N-terminus of the protein contains a signal peptide, and the most likely site of cleavage is between the 29th and 30th amino acid residues, and there are amino acids distributed both inside and outside the membrane, indicating that GhCYP71A1 The gene-encoded protein is a secretory protein with a signal peptide and a transmembrane domain.

[0044] Phylogenetic tree analysis of GhCYP71A1 homologous proteins Figure 4As shown, the sequence alignment analysis of GhCYP71A1 is as follows Figure 5 and Figure 6 As shown in the figure, the results showed that the CYP71A1 protein sequence in cotton contained typical P450 protein conserved domains, including PERF domain (PxRx), K helix (ExxR) and C-terminal heme binding domain (FxxGxxxCxG). Different from the typical P450 structure, the amino acid sequence of this gene does not include I helix (A / GGxD / ETT / S), and helix C (WR) is also different from the typical helix C structure (WxxxR). The evolutionary tree shows that although cotton CYP71A1 is closely related to the dicotyledonous plant soybean, it is more distantly related to dicotyledonous plants such as Arabidopsis and tobacco, as well as corn, rice and wheat in monocotyledonous plants. Therefore, in terms of catalytic mode and catalytic activity, the P450 family genes in cotton are different from those in other species, and there are large differences between different species.

[0045] The analysis results of the promoter region are as follows Figure 7 As shown, the results show that GhCYP71A1 In addition to the core promoter components TATA box and CAAT box, the promoter also includes cis-acting elements related to light response, circadian rhythm regulation, and stress response. GhCYP71A1 It can play a stress resistance role by participating in cotton stress signaling pathway.

[0046] Example 3 Analysis of gene expression patterns The roots, stems and leaves of Liaomian 6 cotton varieties at the true leaf stage were sampled and ground, and total RNA was extracted using a polysaccharide and polyphenol plant total RNA extraction kit (Novagen, Nanjing). The RNA was reverse transcribed into cDNA using a HiScript III 1st Strand cDNA Synthesis Kit (Novagen, Nanjing). GhCYP71A1 The expression level of was detected. Roots, stems and leaves of Liaomian 6 cotton were sampled at 0 h and 24 h after cold treatment at the true leaf stage. After reverse transcription of RNA, real-time fluorescence quantitative PCR was used. The reaction system and reaction procedure were carried out according to the instructions of Taq Pro Universal SYBR qPCR Master Mix Fluorescence Quantification Kit (Norway, Nanjing). The quantitative reverse transcription polymerase chain reaction was carried out on the Roche 480 polymerase chain reaction system. The primer-BLAST website (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi) was used to design the primer-BLAST. GhCYP71A1The qRT-PCR primers were GhCYP71A1-RT-F / GhCYP71A1-RT-R (Table 1). The internal reference gene was GhHIS, and each reaction was repeated three times with 2 -ΔΔCt Methods The relative expression of genes was calculated.

[0047] The results are as follows Figure 8 As shown, GhCYP71A1 The gene expression level in the root was the highest and was nearly 10 times higher than that in the stem and leaf, proving that the gene was dominantly expressed in the root of Liaomian 6 ( Figure 8 A), GhCYP71A1 The expression level in roots and stems after 4 ℃ cold treatment for 0 h was 10 times higher than that in roots and stems after 24 h cold treatment, indicating that the expression level of this gene decreased with the extension of cold treatment time ( Figure 8 B) Description GhCYP71A1 Affected by low temperatures and can respond to low temperature stress.

[0048] Example 4 Subcellular localization Homologous recombination technology was used to construct a 35S::GhCYP71A1-GFP fluorescent expression vector. The root cDNA of Liaomian No. 6 was used as a template to amplify the full length of the gene. The amplification primers were GhCYP71A1-OX-F / GhCYP71A1-OX-R (Table 1), connected to the pCAMBIA2300_35S_GFP-HA vector (purchased from Beijing Quanshijin Company) and then transformed into Escherichia coli competent DH5α. The obtained positive clones were sent to Shanghai Biotech Co., Ltd. for sequencing. After the sequencing was correct, the plasmid was extracted and the fusion construct 35S::GhCYP71A1::GFP was transformed into Agrobacterium tumefaciens GV3101 strain and injected into tobacco for transient transformation. The cells were cultured overnight in the dark and observed under light for 24 h, using a laser scanning confocal microscope.

[0049] The results are as follows Fig. 9 Shown, indicating GhCYP71A1 Localized in the endoplasmic reticulum.

[0050] Example 5 LUC dual luciferase assay Will GhCYP71A1 The 2000 bp before the start codon of the coding region was amplified, and the primer sequences were shown in Table 1. The primers were connected to the pGreenII0800-LUC vector (purchased from Beijing Quanshijin Company) and the recombinant vector was transformed with Agrobacterium. GhERF105The CDS sequence of the ERF transcription factor family member was amplified, the primer sequences are shown in Table 1, and homologous recombination was used to connect to the pGreenII 62-SK vector (purchased from Beijing Quanshijin Company). The reporter vector was mixed with the activation vector bacterial solution and injected into tobacco leaves. After 24 h of dark treatment, normal light growth was restored for 24 h, and the fluorescence signal was observed.

[0051] The results are as follows Fig.10 As shown, GreenⅡ62-SK- GhERF105 +pGreenⅡ0800-LUC- GhCYP71A1 The injection area was excited to emit strong light, indicating GhERF105 Can be combined GhCYP71A1 The promoter region of GhCYP71A1 by GhERF105 regulation.

[0052] Example 6 Yeast one-hybrid test To further verify the above results, yeast single hybridization was used for point-to-point verification. GhCYP71A1 The GGC-BOX binding domain of the promoter was recombined in pHis2 vector (purchased from Beijing Quanshijin Company). The plasmid was extracted and transformed according to the instructions (Coolarber, Beijing). After selecting positive single clones, they were spread into SD-TH solid medium containing 3-AT (3-AT concentration gradient was 0-200 mM). The target gene ( GhERF105 ) was amplified by PCR, and the primer sequences are shown in Table 1. It was connected to the pGADT7 vector (purchased from Beijing Quanshijin Company) by homologous recombination. The empty vector was used as a negative control, and the positive colonies were selected to extract the plasmid with the correct sequencing. The yeast expression vector containing the target gene was co-transformed into the yeast strain, and the transformed yeast strain was spread on the selective medium to observe the growth.

[0053] The results of yeast one-hybrid interaction experiments are as follows Fig.11 As shown, pHis2- GhCYP71A1 The applicable concentration of 3-AT is 30mM, and pGADT7-GhERF105+pHis2-GhCYP71A1 can grow normally in SD / -His / -Leu / -Trp medium with 30mM 3-AT. GhCYP71A1 The GGC-BOX on the promoter can be GhERF105 Recognized by upstream transcription factors GhERF105 regulation.

[0054] Example 7 Gene silencing test verification GhCYP71A1 Regulating cotton's cold tolerance Using Liaomian No. 6 as the material, RNA was extracted, and after reverse transcription, PCR was used to amplify a 300 bp specific sequence (shown in SEQ ID No. 3) designed by the gene silencing sequence design website (https: / / VIGS.solgenomics.net / ). The primers are shown in Table 1. The amplified PCR product was run on agarose gel electrophoresis, and the correct band was recovered and purified. The recovered purified product was connected to the pTRVⅡ vector (purchased from Beijing Quanshijin Company) and transferred into Escherichia coli DH5α for colony PCR. The positive plaques were sent to Sangon Biotechnology Company for sequencing. The plasmid was extracted with the correctly sequenced bacterial solution for Agrobacterium (LBA4404) transformation. When the cotton cotyledons were completely flattened, the VIGS injection bacterial solution was prepared. The epidermis was gently punctured on the back of the cotyledons avoiding the main veins with the needle of a syringe. The mixed resuspended bacterial solution was injected into the cotyledons along the injured part of the epidermis with a syringe until the back of the cotyledons was filled with bacterial solution. The injected cotton seedlings were placed in the dark at room temperature for 24 hours and then transferred to a 28℃ greenhouse for continued growth. After about two weeks, when the new leaves of the positive control were completely white, sampling and low-temperature treatment were carried out.

[0055] Albino phenotype Fig.12 As shown in A, then TRVⅡ:00 and TRVⅡ: GhCYP71A1 The roots of the plants were sampled, 10 plants were randomly selected, quickly frozen in liquid nitrogen and stored at -80 °C. GhCYP71A1 The expression of TRVⅡ:00 and TRVⅡ: GhCYP71A1 The roots of the plants were ground to extract RNA, and then reverse transcribed into cDNA for fluorescence quantitative determination. The relative expression of the target gene was measured with 2 –△△CT The result is as follows Fig.12 As shown in B, in the silenced plants, the relative expression of genes in TRVⅡ:00 plants was different from that in TRVⅡ: GhCYP71A1 The relative expression levels of the plants reached a significant difference, so it can be determined GhCYP71A1 Successfully silenced.

[0056] Furthermore, for TRVⅡ:00 plants and TRVⅡ: GhCYP71A1 The plants were cold treated at 4 ℃ for 3 days and recovered at 28 ℃ for 4 days before root sweeping to observe the difference between the two groups of plants and measure the total root length (TRL), total root projection area (TRPA), total root surface area (TRPA), taproot average diameter (PRDA), total root volume (TRV) and other indicators. GhCYP71A1 The plants were treated at 4 ℃ for 3 days and then recovered at 28 ℃ for 4 days. Ten plants with the same growth trend and conditions were selected and their above-ground parts were dried and their dry weight was weighed.

[0057] The results of root sweeping are as follows Fig.13As shown, TRVⅡ: GhCYP71A1 The plant survival phenotype was better than that of TRVⅡ:00 plants. Under the same growth conditions, TRVⅡ: GhCYP71A1 The root system is more developed and the survival rate is higher than TRVⅡ:00 ( Fig.14 ), in summary GhCYP71A1 It has the function of regulating the root development of cotton.

[0058] The test results of root system related traits are as follows Fig.15 As shown, the results showed that TRVⅡ: GhCYP71A1 The aboveground dry matter weight was 80% higher than that of TRVⅡ:00 plants, and the total root length (TRL) was longer. GhCYP71A1 The total root projection area (TRPA), total root surface area (TRSA), and total root volume (TRV) were better than those of TRVⅡ:00 plants, indicating that TRVⅡ: GhCYP71A1 Plants that accumulate more organic matter grow better, indicating GhCYP71A1 It has a negative regulatory effect on low temperature.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Cotton GhCYP71A1 The protein encoded by the gene is characterized in that Its amino acid sequence is shown as follows (1) or (2): (1) As shown in SEQ ID NO.1; (2) An amino acid sequence derived from (1) with equivalent function, wherein one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO.

1.

2. Cotton encoding the protein of claim 1 GhCYP71A1 Gene.

3. The gene according to claim 2, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

2.

4. A biological material containing the gene according to claim 2 or 3, characterized in that: The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineered bacteria or non-renewable plant cells or tissues.

5. Use of the protein according to claim 1, the gene according to claim 2 or 3, or the biological material according to claim 4 in regulating the cold tolerance of cotton.

6. The use according to claim 5, characterized in that: The regulation is negative regulation; preferably, by inhibiting or eliminating the expression or activity of the protein or gene, the cold tolerance of cotton is improved, the root growth of cotton is promoted, the survival rate of cotton is improved, or the accumulation of organic matter in cotton is promoted.

7. Use of the protein according to claim 1, the gene according to claim 2 or 3, or the biological material according to claim 4 in the preparation of transgenic cotton.

8. Use of the protein according to claim 1, the gene according to claim 2 or 3, or the biological material according to claim 4 in cotton variety improvement.

9. A method for improving the cold resistance of cotton, characterized in that: include: Using genetic engineering to inhibit or eliminate The expression level or activity of the protein according to claim 1 or the gene according to claim 2 or 3.

10. The method according to claim 9, characterized in that The genetic engineering approach includes gene silencing technology.

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