GhAL5 gene and application thereof in improving high-temperature resistance breeding of crops

Abstract

The invention belongs to the technical field of plant genetic engineering, and relates to a GhAL5 gene and application thereof in cultivation of heat-resistant cotton and rice germplasm. Researches find that expression of the AL5 gene is up-regulated by using an agrobacterium tumefaciens-mediated genetic transformation technology, so that heat resistance of cotton and rice can be enhanced, plant anther fertility can be enhanced, and leaf damage degree can be reduced. The invention provides a new approach and method for improving the heat resistance of cotton and rice, and has wide agricultural application prospect and market value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to application of a GhAL5 gene in breeding cotton and rice germplasm with heat resistance. BACKGROUND

[0002] Cotton is an important strategic material for people's livelihood and raw material for cotton textile industry, and has important economic value. Studies have shown that the reproductive growth stage of cotton is more sensitive to high temperature than the vegetative growth stage, and continuous high temperature threatens to easily cause anther malformation, pollen abortion, etc., thereby causing cotton bolls to drop and yield to reduce. Cotton is a summer crop, and its reproductive growth stage is highly synchronized with summer high-temperature weather, and is more susceptible to high temperature. Under the environment of intensified global greenhouse effect, summer high-temperature weather will occur more frequently and continuously.

[0003] Rice is one of the most important food crops, and the harm of high temperature to the whole growth period of rice has obvious stage difference, which will affect yield and quality through abnormal growth and development and physiological metabolism disorder. It is of important practical significance to carry out research on heat resistance of cotton and rice.

[0004] Therefore, it is of great importance to explore the mechanism of high-temperature resistance of cotton and rice, find key heat-resistant genes, and create heat-resistant germplasm for breeding heat-resistant varieties. The existing methods for breeding heat-resistant varieties include traditional hybridization and phenotype screening breeding and molecular breeding. The traditional breeding method needs 6-8 years to stabilize the characteristics of the variety, and the phenotype identification under high-temperature stress is greatly affected by the environment, and the manual observation is highly subjective and has low throughput, so the efficiency is low. The emerging molecular breeding uses gene editing technology to construct transgenic crops, which has been widely applied due to its high-efficiency expression stability. However, at present, the research on heat-resistant genes of cotton and rice is still in its early stages, and the available gene resources for heat-resistant breeding are limited, which seriously affects the yield of cotton and rice under high-temperature stress. SUMMARY

[0005] The GhAL5 gene of Gossypium hirsutum provided in the application is a positive regulator of high-temperature resistance of cotton, which can improve the heat resistance of cotton and rice, and has wide utilization value. In the examples of the application, overexpression of the GhAL5 gene enhances the heat resistance of cotton seedlings and the pollen activity of cotton under high-temperature conditions. Heterologous overexpression of the GhAL5 gene in rice also enhances the heat resistance of rice. Knockout of the GhAL5 gene or interference with the expression of the GhAL5 gene by RNAi leads to reduced heat resistance of cotton mutants. As a key heat-resistant gene, GhAL5 can provide a theoretical basis for improving the heat resistance of cotton and other species, and is an important gene resource.

[0006] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions:

[0007] In one aspect, the application provides a GhAL5 gene, wherein the nucleotide sequence of the gene GhAL5 is shown as SEQ ID NO. 3.

[0008] In another aspect, the application provides a GhAL5 protein, characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 4.

[0009] Further, the application provides an application of the GhAL5 gene or the GhAL5 protein in improving high-temperature resistance breeding of crops.

[0010] Further, the application provides an application of the GhAL5 gene or the GhAL5 protein in improving high-temperature resistance breeding of crops, characterized in that the temperature of the high temperature is not higher than 45 ℃.

[0011] Further, the application provides an application of the GhAL5 gene or the GhAL5 protein in improving high-temperature resistance breeding of crops, characterized in that the method for improving high-temperature resistance breeding of crops comprises the following steps:

[0012] Further, the application provides an application of the GhAL5 gene or the GhAL5 protein in improving high-temperature resistance breeding of crops, characterized in that the method for improving high-temperature resistance breeding of crops comprises the following steps:

[0013] Further, the application provides an application of the GhAL5 gene or the GhAL5 protein in improving high-temperature resistance breeding of crops, characterized in that the method for improving high-temperature resistance breeding of crops comprises the following steps:

[0014] Further, the application provides an application of the GhAL5 gene or the GhAL5 protein in improving high-temperature resistance breeding of crops, characterized in that the method for improving high-temperature resistance breeding of crops comprises the following steps:

[0015] Further, the application provides an application of the GhAL5 gene or the GhAL5 protein in improving high-temperature resistance breeding of crops, characterized in that the method for improving high-temperature resistance breeding of crops comprises the following steps:

[0016] Further, the application provides an application of the GhAL5 gene or the GhAL5 protein in improving high-temperature resistance breeding of crops, characterized in that the method for improving high-temperature resistance breeding of crops comprises the following steps:

[0017] Further, according to the application of the isolated GhAL5 gene or the GhAL5 protein in improving the high-temperature resistance breeding of crops, the PCR amplification program comprises: PCR reaction conditions are: 95 ℃ pre-denaturation for 5 min; 95 ℃ denaturation for 30 s; 60 ℃ annealing for 30 s; 72 ℃ extension for 1 min, 30 cycles; 72 ℃ extension for 5 min.

[0018] Further, according to the application of the GhAL5 gene or the GhAL5 protein in improving the high-temperature resistance breeding of crops, the transformation method is selected from the group consisting of calcium phosphate co-precipitation method, Ti plasmid method, Ri plasmid method, virus vector method, gene gun method, microinjection method, electroporation method and Agrobacterium-mediated method.

[0019] Further, according to the application of the GhAL5 gene or the GhAL5 protein in improving the high-temperature resistance breeding of crops, the crops comprise cotton and rice. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 GhAL5 gene overexpression plasmid vector pK2GW7-GhAL5 map;

[0021] Figure 2 GhAL5 gene CRISPR-Cas9 knockout plasmid vector pRGEB32-7-GhAL5 map;

[0022] Figure 3 GhAL5 gene RNAi plasmid vector pHELLSGATE4-GhAL5 map;

[0023] Figure 4 GhAL5 gene overexpression transgenic cotton material PCR positive detection result gel map;

[0024] Figure 5 CRISPR-Cas9 knockout GhAL5 transgenic cotton material PCR positive detection result gel map;

[0025] Figure 6 GhAL5 gene RNAi transgenic cotton material PCR positive detection result gel map;

[0026] Figure 7 GhAL5 mutant GhAL5-ko sequence analysis map at the target site;

[0027] Figure 8 GhAL5 gene relative expression map in the anther of the transgenic cotton strain;

[0028] Figure 9Figure 1 is a protein expression map of gene GhAL5 in GhAL5 overexpression plants with MYC as a tag;

[0029] Figure 10 Figure 2 is a phenotype map of different genetic materials of GhAL5 gene at seedling stage under high temperature;

[0030] Figure 11 Figure 3 is a male fertility detection map of different genetic materials of GhAL5 gene under normal temperature (NT) and high temperature (HT);

[0031] Figure 12 Figure 4 is an expression level analysis map of gene GhAL5 in GhAL5 overexpression rice lines;

[0032] Figure 13 Figure 5 is a high temperature tolerance phenotype detection result map of GhAL5 overexpression rice. DETAILED DESCRIPTION

[0033] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with embodiments. The embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0034] According to the information contained in the present application, various changes can be easily made to the precise description of the present application by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present application is not limited to the defined processes, properties or components, as these embodiments and other descriptions are only illustrative of specific aspects of the present application. In fact, various changes to the embodiments of the present application that are obvious to those skilled in the art or related fields are encompassed within the scope of the appended claims.

[0035] In order to better understand the present application without limiting the scope of the present application, all numbers expressing quantities, percentages and other numerical values used in the present application are to be understood as being modified in all instances by the term "about". The term "about" has its ordinary meaning in the art, and is used to indicate an inherent variation of error for a device or method that is being used to determine a particular value, or to encompass values close to the stated value, for example within 10% of the stated value (or range of values). Therefore, unless otherwise specified, the numerical parameters listed in the specification and attached claims are approximations. Values can be changed depending on the desired properties of the ideal nature that is sought to be obtained.

[0036] In the field of cotton planting, the temperature that the daily maximum air temperature reaches or exceeds the suitable temperature for cotton growth by 3-5 ℃ or more is called high temperature. The optimum temperature for cotton from sowing to emergence is 25-30 ℃, the optimum temperature for cotton from emergence to squaring, squaring to flowering and flowering to boll opening is 30 ℃, and the high temperature is 35 ℃; the optimum temperature range for the sexual reproduction process of cotton is 28-30 ℃, and it is generally considered that the air temperature above 33 ℃ is high temperature for sexual reproduction. The optimum temperature for the growth and development of cotton bolls is 25-30 ℃, and the temperature range that generally causes cotton to be significantly damaged is above 35 ℃.

[0037] In addition, it should be noted that, unless otherwise defined, the scientific and technical terms used in the context of the present application shall have the meanings commonly understood by a person of ordinary skill in the art. In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0038] The overexpression vector or knockout vector or RNAi vector of the above-mentioned target gene GhAL5 can be introduced into the cotton genome by genetic transformation method, so that the gene is overexpressed or no longer expressed or the expression is reduced. It has been verified by experiments in the present application that overexpression of GhAL5 gene can enhance the high temperature tolerance of cotton and rice; knockout of GhAL5 gene and RNA interference of GhAL5 gene lead to the decrease of high temperature tolerance of cotton.

[0039] The gene related to the heat tolerance of cotton, the overexpression vector, the knockout vector and the RNAi vector and the application provided by the present application will be described in detail below in combination with examples.

[0040] Example 1 Cloning, expression pattern analysis and transgenic plant expression quantity analysis of GhAL5 gene

[0041] In the previous study, it was found that GhAL5 gene, a kind of Alfin-like transcription factor, regulates the three-dimensional genomic structure of anther of cotton lines under high temperature stress through chromatin loop dynamics and TAD boundary recombination, thereby regulating the high temperature response mechanism that differentiates the high temperature tolerance and high temperature sensitivity of cotton lines. Therefore, GhAL5 gene will be selected for research in the present study.

[0042] 1.1 Cotton tissue RNA extraction and cDNA acquisition.

[0043] The anther samples of Gossypium hirsutum Jin668 strain (patent number: 201510833618.0; Li et al., 2019) TS (tetrad stage), TDS (tapetum degradation stage), ADS (anther dehiscence stage) were quickly frozen in liquid nitrogen, and total RNA was extracted using a method containing hydrochloric acid guanidine. The RNA was reverse transcribed into cDNA (Vazyme #R412, HiScript IV 1st Strand cDNA Synthesis Kit (+gDNA wiper)). The cDNA synthesized at TS, TDS, and ADS stages was mixed at a volume ratio of 1:1:1, and the mixture was used as a template for amplification of the GhAL5 gene.

[0044] 1.2 Acquisition of GhAL5 target gene sequence.

[0045] The reference sequence of the gene (Ghir_D06G009180) was obtained from the Gossypium hirsutum genomic database, and specific primers BP-GhAL5-F (SEQ ID NO. 1) and BP-GhAL5-R (SEQ ID NO. 2) with attB sites required for BP reaction were designed according to the sequence to amplify the full-length gene.

[0046] SEQ ID NO. 1:

[0047] GGGGACAAGTTTGTACAAAAAAGCAGGCTAC ATGGACGGCGGTGCTTCCT

[0048] SEQ ID NO. 2:

[0049] GGGGACCACTTTGTACAAGAAAGCTGGGTC CAACAACCATCGTCAAGGC

[0050] The underlined part is the attB site

[0051] The mixed cDNA obtained in 1.1 was used as a template for PCR amplification of the target gene, and the PCR reaction conditions were as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s; 60 °C annealing for 30 s; 72 °C extension for 1 min, 30 cycles; 72 °C extension for 5 min, to obtain a PCR product with attB sites.

[0052] The PCR product is connected to the pDONRZeo (zeo+) vector by BP reaction, and the operation is as follows: PCR product: 50-100 ng, BP enzyme (BR enzyme is purchased from Invitrogen Company (Gateway™ BP Clonase™ II Enzyme Mix#), pDONRZeo (zeo+) plasmid: 100 ng, and the recombined vector pDONERZeo-GhAL5 is obtained after enzyme connection at room temperature for 4 h. The recombined plasmid is transferred into E. coli, and after single colony picking and sequencing, the plasmid is extracted and stored in a refrigerator at -20 ℃.

[0053] The nucleotide sequence of the GhAL5 gene is as follows:

[0054] SEQ ID NO. 3:

[0055]

[0056] The sequence of the GhAL5 protein is as follows:

[0057] SEQ ID NO. 4:

[0058] MDGGASYNPRTVEEVFRDFKGRRAAMIKALTTDVEEFYKQCDPEKENLCLYGYP SEQWEVNLPAEEVPPELPEPALGINFARDGMQEKDWLSLVAVHSDAWLLAVAFY FGARFGFDKADRKRLFNMINDLPTIFEVVTGATKKQTKEKSSVSNHSSNKSKS NSKRGSESQPKYSKAAASKDEVEDGMEDEDDEEHGETLCGACGENYAADEFW ICCDICEKWFHGKCVKITPARAEHIKQYKCPSCSNKRARP

[0059] The CDS sequence of the GhAL5 gene is as follows:

[0060] SEQ ID NO. 5:

[0061] ATGGACGGCGGTGCTTCCTATAACCCACGTACAGTCGAAGAAGTCTTTCGAGATTTCAAGGGTCGTCGAGCTGCCATGATTAAAGCCCTCACTACTGATGTGGAAGAGTTCTACAAGCAGTGCGATCCAGAAAAGGAGAATCTTTGCCTTTATGGATATCCCAGTGAGCAGTGGGAGGTGAATTTACCTGCTGAAGAGGTGCCTCCAGAGCTTCCGGAGCCTGCACTGGGTATCAACTTTGCCAGAGATGGGATGCAAGAAAAGGACTGGTTGTCTTTGGTTGCTGTACACAGTGACGCGTGGTTACTTGCTGTGGCTTTCTATTTTGGTGCTAGGTTTGGATTCGATAAAGCTGATAGGAAACGCCTTTTCAATATGATAAATGATCTTCCGACAATATTTGAAGTTGTGACAGGGGCAACTAAGAAACAGACAAAGGAGAAATCGTCGGTTTCAAATCATAGCAGCAACAAATCTAAATCAAACTCTAAGCGAGGTTCTGAATCTCAGCCCAAGTATTCGAAGGCAGCAGCATCAAAGGATGAGGTTGAAGATGGCATGGAAGACGAAGACGATGAGGAGCATGGAGAGACGTTATGTGGGGCTTGTGGAGAGAATTATGCCGCTGATGAATTCTGGATTTGCTGTGATATCTGTGAGAAATGGTTCCATGGAAAGTGTGTTAAGATTACACCAGCAAGGGCGGAGCATATTAAGCAGTACAAATGCCCATCTTGCAGCAACAAGAGAGCACGGCCTTGACGATGGTTGTTGGGGTGGCTGTTTTATGTGAATTCTGGCATTGTGGGACTGCTAACTGTCTAGTAGGGGATGTAGTGAAGTTCGATGTTAGTTAATGTGACGTTTTAATGTAGGAGATCTGTGTTTTGACTTTTGATATGTAGGTGGTGAACTGGCTGTTTAATTTGATTATAAAATAGGATAATTATAATCAGTTATTTCTGCCAT

[0062] Example 2 Transgenic vector construction of GhAL5 gene

[0063] 2.1 Super expression vector construction

[0064] The pDONERZeo-GhAL5 plasmid was extracted using the bacterial strain saved in Example 1.2, 1 μL of the pDONERZeo-GhAL5 plasmid was recombined into the plant expression vector pK2GW7 (the LR enzyme was purchased from Invitrogen Company (Gateway™ LR Clonase™ II Enzyme Mix #3106343), USA; pK2GW7 was from Ghent University, Belgium) using the LR reaction, and the LR reaction system was prepared in a 200 μL centrifuge tube at room temperature: ddH2O 2.5 μL, pDONERZeo-GhAL5 plasmid 1 μL (100 ng / μL), pK2GW7 plasmid 1 μL (100 ng / μL), and LR enzyme 0.5 μL (10 U / μL). The reaction system was placed at room temperature for 4 h, and then transformed into DH5α competent cells by heat shock method, and the remaining operations were the same as in Example 1.2. The positive clone obtained was the super expression vector for transformation, named pK2GW7-GhAL5. The recombinant plasmid was transformed into E. coli, and a single colony was picked and extracted for plasmid preservation after sequencing.

[0065] The super expression plasmid vector map of pK2GW7-GhAL5 is shown in Figure 1 The basic backbone of the recombinant vector is pK2GW7, and the fusion target gene GhAL5 is driven by the constitutive promoter 35S. The resistance of the recombinant vector in E. coli and Agrobacterium is spectinomycin (spe+), and the resistance in transgenic plants is kanamycin (kan+).

[0066] 2.2 Knockout vector construction

[0067] First, the conserved region of the GhAL5 gene in Gossypium hirsutum was selected, and online sgRNA design was performed using the online website (http: / / cbi.hzau.edu.cn / CRISPR2 / ). The sgRNA with low off-target rate (SEQ ID NO. 6) was selected for subsequent construction of gene knockout plasmid.

[0068] SEQ ID NO. 6: TTGCTGTACACAGTGACGCG TGG

[0069] The underlined part is the PAM sequence.

[0070] The sgRNA is connected to the expression vector by a multi-cistronic tRNA-gRNA to obtain a gene editing vector, and the gene editing vector carries a tRNA-gRNA fusion fragment (nucleotide sequence as shown in SEQ ID NO. 9) and a Cas9 coding gene. The gene editing vector is transfected into a host cell for gene editing.

[0071] The tRNA-gRNA fusion fragment is preferably constructed into a pRGEB32-GhU6.7NPT II vector containing a Cas9 coding gene to obtain a gene editing vector. The construction method of the expression vector pRGEB32-GhU6.9-NPT II is described in the literature "Wang P, Zhang J, Sun L, et al. High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR / Cas9 system [J]. Plant Biotechnology Journal, 2018, 16(1): 137-150" and the patent "CN108203714A A method for editing cotton genes (publication date: 2018-06-26)". The specific operation is as follows:

[0072] PGTR4 plasmid as a template, IFGhAL5 ko-F (SEQ ID NO. 7) and IFGhAL5 ko-R (SEQ ID NO. 8) as primers for PCR amplification. The PCR amplification system is: ddH2O 8.6 μL, IFGhAL5 ko-F and IFGhAL5 ko-R primers each 0.2 μL, 2×Taq Master Mix (Dye Plus) (Vazyme #P112) 10 μL, PGTR4 plasmid 1 μL. The PCR amplification program is: 95 ℃ 5 min; 95 ℃ 30 s; 60 ℃ 30 s; 72 ℃ 30 s, 30 cycles; 72 ℃ 5 min. The tRNA-gRNA product is amplified, and the amplification primer sequence and the tRNA-gRNA product are as shown in SEQ ID NO. 9:

[0073] SEQ ID NO. 7:

[0074] AGCATCAGATGGGCAAACAAAGCACCAGTGGTCTAG

[0075] SEQ ID NO. 8:

[0076] TTCTAGCTCTAAAACCGCGTCACTGTGTACAGCAATGCACCAGCCGGGAAT

[0077] SEQ ID NO. 9:

[0078] AGCATCAGATGGGCA aacaaagcaccagtggtctagtggtagaatagtaccctgccacggtacagacccgggttcgattcccggctggtgca ttgctgtacacagtgacgcg GTTTTAGAGCTAGAA

[0079] Capitalized underlined is sequence homologous to vector pRGEB32-GhU6.7-NPT II; lower case un- underlined is tRNA; lower case underlined is target.

[0080] PCR amplified products were stored in 4 ℃ refrigerator. Related research results of PGTR plasmid were published in the literature“Xie K, Minkenberg B, Yang Y. Boosting CRISPR / Cas9 multiplex editing capability with the endogenous tRNA-processing system[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(11):3570 3575.”.

[0081] The tRNA-gRNA amplification product was then connected to the pRGEB32-GhU6.9NPT II expression vector using an in-fusion seamless cloning reaction. The expression vector was digested by BsaI (purchased from Beijing NEB Company, item number: R3733L), and the digestion system was as follows: rCutSmart Buffer 2 μL, BsaI 0.5 μL, pRGEB32-GhU6.7-NPT2 vector 2 μg, ddH2O up to 20 μL, 37 °C overnight, and then the linearized pRGEB32-GhU6.9-NPT II expression vector and the tRNA-gRNA amplification product were connected by ClonExpress Entry Step Cloning Kit (purchased from Nanjing Novozyme Biotech Co., Ltd.) to obtain the editing vector of the GhAL5 gene; the connection system included: tRNA-gRNA fusion fragment 30 ng, linearized pRGEB32-GhU6.9-NPT II expression vector 300 ng, 2 CE Mix 5 μL; incubated at 50 °C for 10 min, and then placed on ice for 5 min to obtain the connected editing vector.

[0082] The connected editing vector was named pRGEB32-7-GhAL5, and the vector map is shown in Figure 2 The vector takes pRGEB32-GhU6.7-NPT2 as the skeleton, and fuses tRNA+target+gRNA. The recombinant vector has kanamycin resistance (kan+) in E. coli and Agrobacterium, and also has kanamycin resistance (kan+) in transgenic plants.

[0083] The In-Fusion connection product was transformed into E. coli competent DH5a by heat shock transformation method. After 37 °C culture for 12-16 h, a single colony was selected for PCR positive detection, and the detection primer was U6-7-F (SEQ ID NO. 16) and IFGhAL5 ko-R (SEQ ID NO. 8). The PCR reaction conditions were as follows: 95 °C for 5 min; 95 °C for 30 s; 60 °C for 30 s; 72 °C for 30 s, 30 cycles; 72 °C for 5 min. The positive single colony was expanded and cultured, and sequencing was performed. The correct single colony bacterial liquid was used to extract the plasmid, and the mutant plasmid pRGEB32-7-GhAL5 for knockout was obtained.

[0084] 2.3 RNAi vector construction

[0085] GhAL5 RNAi-F (SEQ ID NO. 10) and GhAL5 RNAi-R (SEQ ID NO. 11) were designed with GhAL5 gene as target. PCR amplification was performed with cDNA reverse transcribed in Example 1.1 as template, and the amplification conditions were as follows: 95 ℃ 5 min; 95 ℃ 30 s; 60 ℃ 30 s; 72 ℃ 30 s, 30 cycles; 72 ℃ 5 min. The PCR amplification product was stored in a 4 ℃ refrigerator. Then, TA cloning reaction was used to connect the PCR amplification product to pGEM-Teasy expression vector. The reaction system was as follows: 2.5 μL 2X Rapid Ligation Buffer, 0.5 μL T4 DNA Ligase, 1 μL pGEM-Teasy, 1 μL PCR amplification product, and the connection product was obtained by connecting at 4 ℃ overnight. The enzyme connection product was named pGEM-Teasy-GhAL, and the recombinant plasmid was transformed into E. coli. After single colony was picked and sequencing was correct, the plasmid was extracted and stored.

[0086] SEQ ID NO. 10: TAAGCGACGTTCTGAATCTCAGCC

[0087] SEQ ID NO. 11: CCAGTTCACCACCTACATATCAAAAGTC

[0088] The pHellsgate4 vector was subjected to enzyme cutting and linearization treatment (the map is shown in FIG. 4), and the enzyme cutting system was as follows: rCutSmartBuffer 2 μL, XhoI / XbaI 0.5 μL (cut respectively), pHellsgate4 vector 2 μg, ddH2O to 20 μL, 37 ℃ overnight. Figure 3

[0089] ​The extracted pGEM-Teasy-GhAL5 plasmid was used as a template for PCR reaction with BPGhAL5 RNAi-F (SEQ ID NO. 12) and BPGhAL5 RNAi-R (SEQ ID NO. 13) as primers, and after detection and purification, BP reaction was performed: ddH2O 1.5 μL, pHellsgate4 (Spe+) 1 μL, pGEM-Teasy-GhAL5 plasmid 2 μL, Gateway BP Clonase 0.5 μL. Incubate at room temperature for about 4 h, transform into E. coli DH5α, and incubate at 37 ℃ for 12-16 h, then pick single colonies for PCR positive detection with 35S-F (SEQ ID NO. 14) and GhAL5 RNAi-R (SEQ ID NO. 11) as primers. Expand and culture the positive single colonies, and sequence, and the plasmid extracted from the correct single colony is the plasmid pHellsgate4-GhAL5 for RNAi.

[0090] SEQ ID NO. 12:

[0091] GGGGACAAGTTTGTACAAAAAAGCAGGCTGC TAAGCGAGGTTCTGAATCTCAGCC

[0092] SEQ ID NO. 13:

[0093] GGGGACCACTTTGTACAAGAAAGCTGGGTG CCAGTTCACCACCTACATATCAAAAGTC

[0094] Underlined is the BP linker

[0095] SEQ ID NO. 14: GACGCACAATCCCACTATCC

[0096] Example 3. Transformation of Agrobacterium with recombinant vector plasmid

[0097] The recombinant plasmids pK2GW7-GhAL5, pRGEB32-7-GhAL5 and pHellsgate4-GhAL5 were separately heat-shocked to transform Agrobacterium GV3101, and the positive clones were screened by spectinomycin (pK2GW7-GhAL5 and pHellsgate4-GhAL5) and kanamycin (pRGEB32-7-GhAL5), and then identified by PCR, and finally, the forward primer 35S-F (SEQ ID NO. 14) and the reverse primer GhAL5 cds-R (SEQ ID NO. 15) or IFGhAL5 ko-R (SEQ ID NO. 8) or GhAL5 RNAi-R (SEQ ID NO. 11) were used for sequencing to verify the correct construction of the vector, and the sequencing work was entrusted to Tianyi Biological Company.

[0098] SEQ ID NO. 15: CAACAACCATCGTCAAGGC

[0099] Example 4 Genetic transformation and identification of GhAL5 gene

[0100] 4.1 Agrobacterium-mediated genetic transformation

[0101] The seeds of upland cotton strain Jin668 with full appearance and uniform size were selected, the seed coat was carefully removed with tweezers, and the seeds were sterilized with 0.1% mercuric chloride solution for 15 min, then washed with sterile water for 3 times. The seeds were poured into sterile seedling culture medium, and after dark culture at 30°C for one day, the seedlings were supported and continued to be cultured in the dark at 30°C for 4-5 days.

[0102] 10 μL of each of the recombinant plasmids pK2GW7-GhAL5 and pHellsgate4-GhAL5 was inoculated into 1 mL of LB medium containing 100 mg / L spectinomycin; 10 μL of pRGEB32-7-GhAL5 plasmid was inoculated into 1 mL of liquid LB medium containing 100 mg / L kanamycin, and cultured at 28°C for 16-24 h on a shaker. 20 μL of activated bacteria was inoculated into 20 mL of liquid LB medium containing 100 mg / L spectinomycin (pK2GW7-GhAL5 and pHellsgate4-GhAL5) and 100 mg / L kanamycin (pRGEB32-7-GhAL5), and cultured at 28°C for 16-24 h on a shaker. 1 mL of activated bacteria was centrifuged at 5000 rpm for 5 min to collect the bacteria, and the bacteria were resuspended in 20 mL of MGL medium, and 50 mg / mL of Acetosyringone (AS) solution was added, and cultured at 28°C for 30 min on a shaker for cotton hypocotyl infection.

[0103] The process of Agrobacterium infection of cotton hypocotyls is as follows:

[0104] The above constructed transformation vector is introduced into the host cell of Gossypium hirsutum Jin 668 by Agrobacterium-mediated transformation method. The specific transformation steps are as follows: (1) in the clean bench, the cotton Jin 668 hypocotyls cultured for 4-5 days are cut into 0.7 cm small pieces on the sterile filter paper with a sterile blade, and then poured into a sterile 100 mL triangular flask, and then the Agrobacterium bacteria liquid resuspended with MGL medium is used to infect the hypocotyls for 2-3 min. (2) Discard the bacteria liquid, pour the infected hypocotyls onto the sterile filter paper and blow to the surface to dry, then inoculate the hypocotyls into the co-culture medium, place in parallel, and carry out the first culture in the 20℃ incubator in the dark for 36-48 h, then continue to inoculate the hypocotyls after the first culture into the 2,4-D induction medium, and carry out the second culture in the light at 28℃ for 14 hours per day, subculture once every 1 month, until the embryogenic callus is obtained. (3) The embryogenic callus is inoculated into the differentiation medium to obtain cotyledon embryos; (4) the cotyledon embryos are inoculated into the rooting medium for rooting induction, until the transgenic plant seedlings are obtained.

[0105] The above culture medium is configured as follows:

[0106] The sterile seedling medium is configured as follows: MS 25 mL / L, glucose 15 g / L, pH=6.1-6.2, distilled water to 1 L, Phytagel 2.6 g / L.

[0107] The MGL medium is configured as follows: tryptone 5 g / L, sodium chloride 5 g / L, magnesium sulfate heptahydrate 0.1 g / L, KH2PO4 0.25 g / L, mannitol 5 g / L, glycine 1 g / L, distilled water to 1 L, pH=5.8.

[0108] The co-culture medium is configured as follows: MS 50 mL / L, trace elements 10 mL / L, iron salt 10 mL / L, myo-inositol 10 mL / L, ammonium nitrate 10 mL / L, L-Gly 1 mL / L, B5 1 mL / L, 2,4-D 1 mL / L, KT 0.2 mL / L, magnesium chloride hexahydrate 0.9-1.0 g, glucose 30 g, pH=5.9, distilled water to 1 L, Phytagel 2.6 g / L.

[0109] The 2,4-D induction medium is configured by adding 100 mg / L kanamycin and 100 mg / L cephalosporin to the co-culture medium.

[0110] The differentiation medium is configured as follows: MS 50 mL / L, trace elements 10 mL / L, iron salt 10 mL / L, myo-inositol 10 mL / L, potassium nitrate 10 mL / L, L-Gly 1 mL / L, B5 1 mL / L, IBA 1 mL / L, KT 0.3 mL / L, glutamine 1 g / L, asparagine 0.5 g / L, glucose 30 g / L, pH=6.1-6.2, distilled water to 1 L, Phytagel 2.6 g / L.

[0111] The rooting medium is configured as follows: MS 25 mL / L, trace elements 5 mL / L, iron salt 5 mL / L, myo-inositol 10 mL / L, L-Gly 1 mL / L, B5 1 mL / L, glucose 15 g / L, pH=5.90-5.95, distilled water to 1 L, Phytagel 2.6 g / L.

[0112] 4.2 Transgenic plant identification

[0113] Transgenic plant positive detection

[0114] After the above transformation process, transgenic T0 generation plants are obtained. The T0 generation is strictly selfed, and T1 generation seeds are harvested and planted in the experimental base of Huazhong Agricultural University in Wuhan, Hubei Province. When the T1 generation plants grow to the true leaf germination stage, the leaf blades are taken and the leaf genomic DNA is extracted by the CTAB method. The primer 35S-F (SEQ ID NO. 14) and GhAL5 cds-R (SEQ ID NO. 15) are used to detect the insertion of the cds sequence in the overexpression plants; the primer U6-7-F (SEQ ID NO. 16) and IFGhAL5 ko-R (SEQ ID NO. 8) are used to detect the insertion of tRNA and sgRNA in the gene knockout plants; the primer 35S-F (SEQ ID NO. 14) and GhAL5 RNAi-R (SEQ ID NO. 11) are used to detect the insertion of T-DNA in the RNAi transgenic plants (T-DNA is a specific DNA fragment on the Ti plasmid of Agrobacterium, and the presence or absence of this fragment directly reflects whether the transformation is successful).

[0115] SEQ ID NO. 16: TGTGCCACTCCAAAGACATCAG

[0116] The PCR conditions are as follows: 95°C for 5 min; 95°C for 30 s; 60°C for 30 s; 72°C for 1 min, 30 cycles; 72°C for 5 min.

[0117] The transgenic plant positive detection results are as follows: Figure 4 、 Figure 5 and Figure 6Verification shows that the overexpression, knockout and RNAi transgenic materials are successfully constructed, wherein Figure 4 Gel map of PCR positive detection results of pK2GW7-GhAL5 transgenic cotton material for overexpression of GhAL5 gene; lane M represents Marker electrophoresis results, lanes 1-8 represent transgenic plant samples, wild type (WT) represents upland cotton Jin668, and negative (NC) represents PCR template as ultrapure water.

[0118] Figure 5 Gel map of PCR positive detection results of pRGEB32-7-GhAL5 transgenic material for CRISPR-Cas9 knockout of GhAL5 gene; lane M represents Marker electrophoresis results, lanes 1-9 represent transgenic plant samples, wild type (WT) represents upland cotton Jin668, and negative (NC) represents PCR template as ultrapure water; transgenic positive material PCR can amplify the corresponding band (956bp), and the wild type has no band.

[0119] Figure 6 Gel map of PCR positive detection results of pHellsgate4-GhAL5 transgenic material for RNAi of GhAL5 gene; lane M represents Marker electrophoresis results, lanes 1-8 represent transgenic plant samples, wild type (WT) represents upland cotton Jin668, and negative (NC) represents PCR template as ultrapure water, transgenic material PCR can amplify the corresponding band, and the wild type has no band.

[0120] Transgenic plant pure line detection:

[0121] The T1 generation seeds obtained by self-crossing are clipped to remove the seed coat, disinfected with 0.1% mercuric chloride solution for 15 min, and then washed repeatedly with sterile water for 3 times. The seeds are placed on the surface of sterile seedling medium containing 100 mg / L kanamycin, and then cultured in the dark at 30°C for one day, and then the seedlings are supported and cultured in the dark at 30°C for 4-5 days. Whether the transgenic seedlings grow normally with lateral roots is observed, and if the lateral roots grow, it indicates that the positive transgenic plant. The positive plants are watered and potted for 14-30 days, and then transplanted to the field. Then, each generation is self-crossed for seed saving, until the resistant separation does not occur, and then the next step of phenotype and function identification is carried out.

[0122] Example 5 GhAL5 gene CRISPR transgenic plant editing efficiency detection

[0123] The transgenic line plants obtained by tissue culture of Example 4 were subjected to mutation type identification induced by CRISPR / Cas9 system using Hi-TOM platform. The specific implementation process is as follows: leaf genomic DNA extracted by CTAB method (HiPure HP Plant DNA Kit # Magen, D3187); first round PCR was performed using Hi-TOM detection primers (SEQ ID NO. 17 and SEQ ID NO. 18), and then primers 2P-F-6 (SEQ ID NO. 19) and 2P-R-6 (SEQ ID NO. 20) were added with labels (Barcode) respectively for the second round of PCR amplification to correspond to different single plants, and the PCR products were mixed in equal amounts and purified. The purified PCR products were subjected to second generation sequencing, and the sequencing results of independent single plants were obtained according to the Barcode labeled primers. The results with higher reads were aligned with the reference genome sequence to obtain the complete single plant target gene mutation site. Finally, the CRISPR transgenic line GhAL5-ko with larger differences in phenotype from the wild type was obtained for subsequent experiments.

[0124] SEQ ID NO. 17: ggagtgagtacggtgtgctgc TTATCACGATTTTAGTCGT

[0125] SEQ ID NO. 18: gagttggatgctgg ATGGAAACCTAGCACCAAAATAGAAAGC

[0126] Underlined to identify HI-TOM linker

[0127] SEQ ID NO. 19:

[0128] AATGATACGGCGACCACCGAGATCTACACTACCGAGAACACTCTTTCCCTACACGACGCTCTT

[0129] SEQ ID NO. 20:

[0130] CAAGCAGAAGACGGCATACGAGATAGACAGCTGTGACTGGAGTTCAGACGTGTGCTCTT

[0131] The amplification system is: ddH2O 8.6 μL, and primers 0.2 μL each, 2 × Taq Master Mix (Dye Plus) (Vazyme # P112) 10 μL, and HI-TOM PCR first round product as template.

[0132] PCR conditions: 95 ℃ 5 min; 95 ℃ 30 s; 60 ℃ 30 s; 72 ℃ 30 s, 30 cycles; 72 ℃ 5 min.

[0133] PCR products were mixed and purified in equal amounts and then sent for testing. The editing efficiency test results are shown in Table 1. Figure 7 Table 1: Editing efficiency test results

[0134] Example 6: Analysis of the relative expression amount of GhAL5 in wild type, overexpression and RNAi plants

[0135] 6.1 RNA was extracted from anthers of wild type (WT), 5 overexpression plants and 5 RNAi plants and reverse transcribed into cDNA (the method was the same as in Example 1). The cDNA was diluted 100 times with ddH2O as a template. GhUB7 (GenBank: DQ116441) was used as an internal reference. The relative expression amount of GhAL5 gene was determined by qPCR experiment. The primers used were SEQ ID NO. 21 and SEQ ID NO. 22. The kit used was ChamQ Blue Universal SYBR qPCR Master Mix, Vazyme #Q312. The results are shown in Table 2. Figure 8 Table 2: Relative expression amount of GhAL5 in wild type, overexpression and RNAi plants

[0136] SEQ ID NO. 21: TGCTGTGGCTTTCTATTTTGGTG

[0137] SEQ ID NO. 22: TGCTGCTATGATTTGAAACCGAC

[0138] 6.2 Protein extraction and Western blot analysis of GhAL5 overexpression lines

[0139] Total protein was extracted from anthers of GhAL5 overexpression lines and wild type cotton using a phenol-based method. Western blot was used to test the expression of MYC-GhAL5 in overexpression plants. The primary antibody was: Abmart Mouse anti Myc-Tag mAb, item number: AE010. The secondary antibody was: Abmart HRP-conjugated Goat anti-Mouse IgG (H+L), item number: AS003. The results are shown in Table 3. Figure 9 Table 3: Western blot analysis of GhAL5 overexpression lines

[0140] 6.3 Phenotype analysis of different genetic materials of GhAL5 under high temperature in seedling stage.

[0141] For different genetic materials of GhAL5, overexpression (GhAL5-OE1 and GhAL5-OE7), RNAi plants (GhAL5-Ri2 and GhAL5-Ri7), and knockout lines (GhAL5-KO), high temperature treatment was performed in seedling stage. Four-leaf-stage cotton seedlings were placed at 42°C and 48°C for 48 h, and then recovered under normal conditions for seven days before phenotype recording. The results are shown in Figure 10 Compared with wild type (WT) (Fig. 6A), GhAL5-OE plants showed significantly enhanced heat tolerance (Fig. 6B). The leaf phenotype after high temperature treatment is shown in Fig. 6C. Compared with wild type, the leaves of GhAL5-OE plants were less damaged by heat. Figure 10 Figure 10 Figure 10

[0142] 6.4 Male fertility detection of different genetic materials of GhAL5 under normal temperature (NT) and high temperature (HT).

[0143] The anther fertility of wild type (WT), overexpression (GhAL5-OE1 and GhAL5-OE7), RNAi plants (GhAL5-Ri2 and GhAL5-Ri7), and knockout lines (GhAL5-KO) under normal temperature and high temperature was analyzed.

[0144] The process from pollen maturation to release is as follows: microspores undergo mitosis to form mature pollen grains containing nutritive cells and reproductive cells, and the outer and inner walls develop completely, accumulate nutrients, and then enter a dormant state; at the same time, the tapetum and middle layer of the anther wall gradually disintegrate, the inner wall of the anther locule differentiates into a fibrous layer, and the anther apex or side forms a predetermined split. If high temperature is encountered, it will destroy the internal structure and stability of nutrients of pollen grains, leading to a decrease in pollen viability or even abortion; at the same time, high temperature will accelerate abnormal water loss of the anther, causing uneven shrinkage of the fibrous layer, which may cause premature rupture of the split or incomplete cracking, and some pollen grains are released or trapped in the anther before maturation. Anther fertility can be characterized from two aspects of morphology and cytology:

[0145] Morphology: fertile anthers are plump and light yellow in color, and crack smoothly; sterile anthers are small and shriveled, with abnormal color (white or brown), and often do not crack or crack incompletely;

[0146] Cytology: TTC staining is used, and red pollen observed under a microscope after TTC staining is considered as fertile pollen, and sterile pollen is not stained. The proportion of fertile pollen is determined by visual observation.

[0147] ​​​TTC (2, 3, 5-triphenyltetrazolium chloride) dyeing formula: TTC powder 4 g / L (source leaf biological CAS#298-96-4), di-potassium hydrogen phosphate trihydrate 14.03 g / L (Shanghai test 10017518), potassium dihydrogen phosphate 5.32 g / L (Shanghai test 10017618).

[0148] The plants at the flowering stage were selected. When the daily maximum temperature was 28-35 °C and the night temperature was lower than 27 °C, the samples were taken as normal temperature samples. When the daily maximum temperature was higher than 35 °C and the night temperature was higher than 28 °C, it was considered as high temperature stress. After more than 7 consecutive days, the samples were taken as HT treatment samples. The flowers of NT and HT single plants were taken, and after removing the bract leaves and sepals, they were photographed, and the anthers were clamped into a 2 mL centrifuge tube containing 1 mL TTC (4 g / L) for dark reaction for 20 min. The pollen staining was observed under a upright microscope.

[0149] The results are shown in Figure 11 , wherein NT represents normal temperature, and HT represents high temperature. Figures A-L are the anther photographs of wild type, overexpression plants OE1, overexpression plants OE2, knockdown plants Ri2, knockdown plants Ri7, and knockout plants, and the scale bar is 1 cm. Figures a-l are the pollen TTC staining photographs of wild type, overexpression plants OE1, overexpression plants OE2, knockdown plants Ri2, knockdown plants Ri7, and knockout plants, and the scale bar is 200 μm. It can be seen that: the GhAL5 overexpression lines show enhanced heat tolerance, and the pollen viability of GhAL5-OE cotton remains at 78-83% under high temperature treatment, while severe sterility of pollen is observed in the wild type control, and the pollen viability is 5%-28%. The RNAi lines and knockout mutants show reduced heat tolerance, and the GhAL5 knockout mutant (GhAL5-ko) is more sensitive to heat, and the pollen viability is significantly reduced to 13.03%.

[0150] 6.5 Heat tolerance analysis of rice plants heterologously overexpressing GhAL5 gene

[0151] The rice japonica variety Zhonghua No. 11 (ZH11) line heterologously overexpressing GhAL5 gene (rice transgenic tissue culture work was commissioned to Wuhan Tianwen Biological Technology Co., Ltd.) was taken, and rice plants GhAL5OE10 / OE27 with significantly improved expression were obtained, as shown in Figure 12 . The rice plants GhAL5OE10 / OE27 and Zhonghua No. 11 wild type were subjected to 48 h of HT treatment at 42 °C and 48 °C, followed by recovery to normal conditions for seven days, and phenotype observation was performed (scale = 4 cm). Overexpression of GhAL5 can significantly enhance the heat tolerance of rice plants, and the results are shown in Figure 13 .

Claims

1. A GhAL5 gene, characterized in that, The nucleotide sequence of the gene GhAL5 is shown in SEQ ID NO.

3.

2. A GhAL5 protein, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

4.

3. The application of the GhAL5 gene according to claim 1 or the GhAL5 protein according to claim 2 in breeding to improve the high-temperature resistance of crops.

4. The application of the GhAL5 gene or GhAL5 protein according to claim 3 in breeding to improve the high-temperature resistance of crops is characterized by, The temperature mentioned above does not exceed 45°C.

5. The application of the GhAL5 gene or GhAL5 protein according to claim 3 in breeding to improve the high-temperature resistance of crops, characterized in that, The method for improving the high-temperature resistance of crops includes the following steps: A GhAL5 gene overexpression vector was constructed, transformed into crops, and transgenic crops with increased GhAL5 gene expression were obtained.

6. The application of the GhAL5 gene or GhAL5 protein according to claim 5 in breeding to improve the high-temperature resistance of crops, characterized in that, The construction of the GhAL5 gene overexpression vector includes the following steps: Using cotton genomic DNA as a template, the GhAL5 gene was amplified by PCR using the primer pair shown in SEQ ID NO.1-2. The amplified GhAL5 gene was then ligated into the plant expression vector pK2GW7 via a Gateway reaction to obtain the GhAL gene overexpression vector.

7. The application of the GhAL5 gene or GhAL5 protein according to claim 6 in breeding to improve the high-temperature resistance of crops, characterized in that, The Gateway reaction includes the following steps: The amplified GhAL5 gene was ligated into the pDONRZeo vector via the BP reaction, and then the GhAL5 gene in the pDONRZeo vector was ligated into the plant expression vector pK2GW7 via the LR reaction to obtain the GhAL5 gene overexpression vector.

8. The application of the GhAL5 gene or protein as described in claim 6 in breeding to improve the high-temperature resistance of crops, characterized in that, The PCR amplification procedure includes the following PCR reaction conditions: 95 ℃ pre-denaturation for 5 min; 95 ℃ denaturation for 30 s; 60 ℃ annealing for 30 s; 72 ℃ extension for 1 min, 30 cycles; 72 ℃ extension for 5 min.

9. The application of the GhAL5 gene or GhAL5 protein according to claim 5 in breeding to improve the high-temperature resistance of crops, characterized in that, The transformation method is selected from calcium phosphate coprecipitation, Ti plasmid method, Ri plasmid method, viral vector method, gene gun method, microinjection method, electroporation method, or Agrobacterium-mediated transformation.

10. The application of the GhAL5 gene or GhAL5 protein according to claim 5 in breeding to improve the high-temperature resistance of crops, characterized in that, The crops mentioned include cotton and rice.

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