CN116083457A - Application of abnormal cotton leucine-rich repeat receptor kinase gene - Google Patents

Abstract

The invention discloses an application of abnormal cotton (Gossypium anomalum) leucine-rich repetitive receptor kinase gene, which consists of 3885 bases, wherein the 1 st base at the 5' end is a transcription initiation site, and the 3885 st base is a transcription termination site. Expression of this gene can cause the abnormal cotton single piece introgression line CSSL11-9 to develop a lethal phenotype. Silencing the gene expression in abnormal cotton single piece introgression line CSSL11-9 inhibited the lethal phenotype and single piece introgression line CSSL11-9 survived normally. In addition, the gene can also endow cotton with verticillium wilt and fusarium wilt resistance, and CSSL11-9 plants with silenced gene have obviously reduced resistance to cotton verticillium wilt and fusarium wilt, thereby providing important gene resources for cotton verticillium wilt and fusarium wilt resistance breeding.

Description

Application of Abnormal Cotton Leucine Rich Repeat Receptor Kinase Gene

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of abnormal cotton leucine-rich repeat receptor kinase gene.

Background technique

As early as the beginning of the 20th century, people realized that the use of wild resources can compensate for the superior alleles lost in modern breeding, but interspecific reproductive isolation limits the use of wild resources in breeding. Hybrid lethality or hybrid weakness and hybrid breakdown are the two main types of reproductive isolation after fertilization. Among them, hybrid lethality or hybrid disadvantage occurs in the F ₁ generation, showing plant death, sterility, dwarfism, and reduced vitality; while some interspecific hybrids can produce normal hybrid F ₁ , but the plants of the F ₂ generation often show disadvantages Or lethal, a phenomenon known as hybrid recession. Elucidating the mechanism behind reproductive isolation can break reproductive barriers for traditional crop breeding, promote the exchange of genomes between species, and will help promote the application of excellent traits in wild species in modern breeding.

et al.，2017；Yoneya et al.，2021)相关基因在不同物种中进行了基因定位，部分基因已经被图位克隆。Alcazar等(2010)发现编码一种类受体激酶SRF3(Strubbelig receptor family 3)基因的等位变异决定了拟南芥种间的遗传不亲和性。拟南芥中编码富含亮氨酸重复序列的NLR蛋白DM1(DANGEROUS MIX 1)和DM2d(Bomblies etal.，2007；Chae et al.，2014)以及DM10和DM11(Barragan et al.，2021)的上位性互作造成了F₁杂种致死。Chen等(2013)证实Hwil(Hybrid weakness 1)基因座上的两个富含亮氨酸重复序列类受体激酶(LRR-RLK)基因和Hwi2位点编码的一种分泌型的类枯草杆菌蛋白酶(Sublitisin-likeproteases)决定了水稻种间杂种劣势。Deng等(2019)证实了富亮氨酸重复序列(CC-NLR)基因是Le₄位点的编码基因，决定了海岛棉和陆地棉的种间杂交致死。Si等(2021)发现小麦中编码CC-NLR蛋白的基因Ne2诱导了小麦的杂交致死。上述研究表明，这些成对有害的上位性互作通常为编码受体激酶(Receptor kinase，RK)或NLR免疫受体家族的成员。The genetic pattern of hybrid lethality fits the Bateson-Dobzhansky-Muller interspecies hybrid incompatibility model. Hybrid lethality includes two types, hybrid lethality and hybrid lethality. The hybrid lethal phenotype occurs in the F ₁ generation and is controlled by one or two pairs of dominant genes; while the hybrid decline occurs in the F ₂ generation and is controlled by one or two pairs of recessive genes. Hybrids have been lethal in existing studies (Zuellig et al., 2018; Jia et al., 2021; Li et al., 2021; Si et al., 2021a; Tezuka et al., 2021; Zhang et al., 2022) and hybrid recession (Yamamoto et al., 2007; Jiang et al., 2008;

et al., 2017; Yoneya et al., 2021) related genes have been mapped in different species, and some genes have been cloned map-wise. Alcazar et al. (2010) found that the allelic variation of the gene encoding a receptor-like kinase SRF3 (Strubbelig receptor family 3) determines the genetic incompatibility between Arabidopsis species. Arabidopsis encoding leucine-rich repeat NLR proteins DM1 (DANGEROUS MIX 1) and DM2d (Bomblies et al., 2007; Chae et al., 2014) and DM10 and DM11 (Barragan et al., 2021) The epistasis interaction caused the lethality of the _F1 hybrids. Chen et al. (2013) confirmed that two leucine-rich repeat receptor-like kinase (LRR-RLK) genes on the Hwil (Hybrid weakness 1) locus and a secreted subtilisin-like protease encoded by the Hwi2 locus (Sublitisin-like proteases) determines the interspecific heterosis in rice. Deng et al. (2019) confirmed that the leucine-rich repeat sequence (CC-NLR) gene is the gene encoding the Le ₄ locus, which determines the lethality of interspecific hybridization between sea island cotton and upland cotton. Si et al. (2021) found that Ne2, a gene encoding CC-NLR protein in wheat, induced hybrid lethality in wheat. The above studies have shown that these pairwise deleterious epistatic interactions usually encode receptor kinases (Receptor kinase, RK) or members of the NLR immune receptor family.

Hybridization combines genetic material from different genomes. Genetic interactions in hybrids can produce beneficial phenotypes known as heterosis. Instead, such interactions can result in deleterious phenotypes such as hybrid lethality. This "genetic incompatibility" was first observed in common beans, with seedlings showing symptoms resembling those of soybean mosaic virus infection (Burkholder et al., 1926). Kostoff et al. (1930) found that necrotic tobacco hybrid seedlings exhibited similar symptoms to pathogen responses, and for the first time suggested that genetic incompatibility involved the plant immune system. With the rapid development of biochemistry and molecular biology, it has been confirmed that hybrid lethality is the activation of autoimmunity in the absence of external pathogen infection to cause immune system disorders (Li et al., 2021). Hybrid lethality is typically characterized by the process of PCD (Wan et al., 2021). The activation of the autoimmune system results in increased plant defenses against foreign pathogens. Bomblies et al. (2007) found that the symptoms of Arabidopsis hybrid necrosis were similar to hypersensitivity reactions, many genes involved in defense were transcriptionally induced, and the dead plants showed strong downy mildew resistance. Chen et al. (2014) found that rice plants with a hybrid disadvantageous phenotype showed enhanced resistance to bacterial blight. Therefore, it is speculated that there may be a functional correlation between hybrid lethality-related genes and disease resistance traits (xiao et al., 2021).

LRR-RLK senses extracellular signals from the N-terminal extracellular domain and transmits the information to the C-terminal intracellular kinase domain through the transmembrane region, causing it to undergo phosphorylation or dephosphorylation reactions, turning on or off downstream target proteins ( Lehti-shiu et al., 2012). The remarkable feature of the extracellular region of LRR-RLK is that it contains LRRs domains arranged in tandem, which consist of 20-30 repeated amino acids. The tertiary structure of LRR forms a new α-helix/β-sheet and participates in the interaction between proteins. This mechanism of action is the basis of the cellular molecular recognition process. The diversity of extracellular LRRs enables LRR-RLKs to specifically recognize different ligand molecules, thereby correctly transmitting different extracellular signals. LRR-RLK members play a very important role in regulating plant disease resistance (Wang et al., 2018). LRR-RLK responds to a variety of pathogen-derived molecules found in the extracellular space, and the plant's own immune system usually uses PAMPs-triggered immunity (PTI) to initiate a defense response to achieve basic resistance to pathogens . Due to the complexity of plant signal transduction pathways and the complementary functions between different proteins, there are not many reports on the ligand molecules of LRR-RLK interaction and complex signaling pathways. Arabidopsis BAK1 (BRI1associated receptor kinase 1) typical LRR receptor-like kinase. BAK1 recognizes pattern recognition receptors on the cell membrane surface, and phosphorylates immune signals to trigger PTI through the MAPK cascade (Wu et al., 2020).

Contents of the invention

The purpose of the present invention is to provide an abnormal cotton leucine-rich repeat receptor kinase gene (GoanoHBD1), which can cause the lethal phenotype of upland cotton plants and can endow cotton with resistance to Verticillium wilt and Fusarium wilt.

To achieve the above object, the present invention takes the following technical solutions:

An abnormal cotton leucine-rich repeat receptor kinase gene (GoanoHBD1), the nucleotide sequence of which is shown in SEQ ID NO.1. The abnormal cotton leucine-rich repeat receptor kinase gene (GoanoHBD1) comes from the wild cotton species of the cotton genus.

The amino acid sequence of the protein encoded by the abnormal cotton leucine-rich repeat receptor kinase gene (GoanoHBD1) is shown in SEQ ID No.2.

Among them, the SEQ ID NO.1 in the sequence consists of 3885 bases, the first base from the 5' end is the transcription initiation site, and the 3885th base is the transcription termination site, and the complete coding frame is 3885 bases, encoding 1294 amino acids, with a molecular weight of 323.48KD and an isoelectric point of 4.58.

The method for obtaining the sequence of the abnormal cotton leucine-rich repeat receptor kinase gene (GoanoHBD1) above is as follows: extract the RNA from the leaf tissue of the wild cotton species of the genus Gossypis, reverse transcribe the obtained cDNA as a template, and use F: TCCGTTTATCAATCTGTTCC, R: ACATTACCACCAATCAAGCA primers Yes, the amplified sequence of the abnormal cotton leucine-rich repeat receptor kinase gene (GoanoHBD1).

The present invention relates to a leucine-rich repeat receptor kinase gene (GoanoHBD1), which is induced and expressed by temperature. When the ambient temperature was higher than 26°C, the expression level of GoanoHBD1 increased significantly. The up-regulated expression of this gene can cause the lethal phenotype of the abnormal cotton single-segment introgression line CSSL11-9. Silencing the leucine-rich repeat receptor kinase gene (GoanoHBD1), the single-segment introgression line CSSL11-9 plants can survive normally, and the GoanoHBD1-silencing CSSL11-9 plants have significantly reduced resistance to cotton verticillium wilt and Fusarium wilt .

Utilizing the characteristics of the leucine-rich repeat receptor kinase gene mentioned above, the lethal phenotype of the abnormal cotton single-segment introgression line CSSL11-9 can be inhibited by silencing the abnormal cotton leucine-rich repeat receptor kinase gene GoanoHBD1 .

Cotton plants sensitive to Verticillium wilt and Fusarium wilt can be obtained by silencing the abnormal cotton leucine-rich repeat receptor kinase gene GoanoHBD1, which can be used to prove the abnormal cotton leucine-rich repeat receptor by reverse proof Kinase genes can improve the resistance of cotton to Verticillium wilt and Fusarium wilt. Therefore, the present invention also protects the application of the abnormal cotton leucine-rich repeat receptor kinase gene GoanoHBD1 in endowing cotton with resistance to Verticillium wilt and Fusarium wilt, specifically, it can be operated in a transgenic manner. More specifically, it can be introduced into the target plant through the recombinant expression vector. In the method, the recombinant expression vector can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conduction, Agrobacterium-mediated, and The transformed plant tissue is grown into plants.

Therefore, the present invention also protects a method of plant breeding, said method being the following (1) or (2):

(1) by increasing the activity of the GoanoHBD1 protein in the target plant, obtain plants with stronger resistance to Verticillium wilt and Fusarium wilt than the target plant;

(2) by promoting the expression of the GoanoHBD1 gene in the target plant, obtain plants with stronger resistance to Verticillium wilt and Fusarium wilt than the target plant;

The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.2; preferably, the target plant is cotton.

Wherein, the implementation of "promoting the expression of the S1BBX31 gene in the target plant" can be as follows (1) or (2) or (3):

(1) introducing the SlBBX31 gene into the target plant;

(2) introducing strong promoters and/or enhancers;

(3) Other common methods in this field.

The polypeptide comprising the amino acid sequence or at least part of the sequence provided by the present invention may still have biological activity or even have new biological activity after certain amino acids are removed or substituted.

The protein encoded by the nucleotide sequence provided by the present invention and the nucleotide sequence and protein functionally identical or similar to the leucine-rich repeat receptor kinase gene (GoanoHBD1) can be synthesized.

The gene comprising the nucleotide sequence provided by the present invention or at least part of the nucleotide sequence can be constructed by genetic recombination to construct a recombinant plasmid to obtain a new biosynthetic pathway, and can also be inserted, replaced, deleted or inactivated to obtain a new biosynthetic way.

The non-ribosomal peptide synthetase provided by the invention may be derived from one or more non-ribosomal peptide synthetase domains, modules or genes by deletion, insertion or inactivation from the same or different non-ribosomal peptide synthetase systems And produce new polypeptide compounds.

Fragments or genes comprising the nucleotide sequence or at least part of the nucleotide sequence provided by the present invention can be used to construct a non-ribosomal peptide synthetase library or a non-ribosomal peptide synthetase derived library or combined library.

This gene can also be used in genetic engineering, protein expression, enzyme-catalyzed reactions, etc., and can also be used to find and discover compounds or genes used in medicine, industry or agriculture to expand the leucine-rich repeat receptor kinase gene (GoanoHBD1) The range of sources has a high application prospect.

In the present invention, there is no particular limitation on the plants applicable to the present invention, as long as they are suitable for gene transformation operations, such as various crops, floral plants, or forestry plants. Said plant can be, for example (not limited to): dicotyledonous plant, monocotyledonous plant or gymnosperm plant.

As a preferred manner, the "plant" includes but is not limited to: cotton, and any gene that has this gene or is homologous to it is applicable.

The "plant" mentioned in the present invention includes the whole plant, its parent and progeny plants and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues and organs. Different parts have our target gene or nucleic acid. The "plant" mentioned here also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, where each of the aforementioned objects contains the gene/nucleic acid of interest .

The invention includes any plant cell, or any plant obtained or obtainable by a method therein, and all plant parts and propagules thereof. This patent also covers transfected cells, tissues, organs or whole plants obtained by any of the aforementioned methods. The only requirement is that the offspring exhibit the same genotypic or phenotypic characteristics, and the offspring obtained using the method of this patent have the same characteristics.

The invention also extends to harvestable parts of plants as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. It further relates to other derivatives of the harvested plants, such as dry granules or powders, oils, fats and fatty acids, starches or proteins. The invention also relates to foods or food additives obtained from related plants.

The present invention has the following advantages:

(1) The leucine-rich repeat receptor kinase gene (GoanoHBD1) obtained in the present invention can cause a lethal phenotype in upland cotton.

(2) The leucine-rich repeat receptor kinase gene (GoanoHBD1) obtained in the present invention is a brand-new gene that can endow plants with resistance to Verticillium wilt and Fusarium wilt, and the function of this gene in plants has not been reported yet. The leucine-rich repeat receptor kinase gene (GoanoHBD1) obtained in the present invention can further analyze the molecular mechanism of cotton resistance to Verticillium wilt and Fusarium wilt. Whether the leucine-rich repeat receptor kinase gene (GoanoHBD1) is involved in the signaling pathway related to disease resistance, what are its upstream and downstream genes, and what are the proteins that interact with it, so further research on this gene can be found in Theoretically expand the understanding of the mechanism of cotton resistance to Verticillium wilt and Fusarium wilt.

(3) Plants resistant to Verticillium wilt and Fusarium wilt can be obtained by transgenic means, specifically, the GoanoHBD1 gene can be introduced into the target plant to obtain a transgenic plant, and the plant's resistance to Verticillium wilt and Fusarium wilt is higher than that of the target plant , to provide a new way for plant breeding against Verticillium wilt and Fusarium wilt.

Description of drawings

Figure 1 shows the use of virus-mediated gene silencing (VIGS) to silence the leucine-rich repeat receptor kinase gene (GoanoHBD1) in the abnormal cotton single-segment introgression line CSSL11-9, and to silence the leucine-rich repeat receptor kinase gene (GoanoHBD1) single-segment introgression line CSSL11-9 plants can survive normally. a: qRT-PCR detection of GoanoHBD1 gene expression in VIGS silenced plants; b: Phenotype of GoanoHBD1 gene VIGS silenced plants.

Figure 2 shows the expression of the leucine-rich repeat receptor kinase gene (GoanoHBD1) induced by different temperatures.

Figure 3 shows the use of VIGS to silence the leucine-rich repeat receptor kinase gene (GoanoHBD1) in the abnormal cotton single-segment introgression line CSSL11-9, and to silence the leucine-rich repeat receptor kinase gene (GoanoHBD1) in the single-segment introgression line CSSL11-9 showed significantly reduced resistance to Verticillium wilt. a: Resistance analysis of positive control pTRV2::GhCLA1, negative control pTRV2::00 injected with empty vector, injected pTRV2::GoanoHBD1, single-segment introgression line and Su 8289 seedlings against Verticillium dahliae; b: qRT-PCR detection of VIGS silenced plant GoanoHBD1 Gene expression; c: Statistics of disease grade index of cotton seedlings 28 days after inoculation with Verticillium dahliae; standard deviation was calculated for 3 repetitions, **: P<0.01.

Figure 4 shows the use of VIGS to silence the leucine-rich repeat receptor kinase gene (GoanoHBD1) in the abnormal cotton single-segment introgression line CSSL11-9, and to silence the leucine-rich repeat receptor kinase gene (GoanoHBD1) in the single-segment introgression line CSSL11-9 showed significantly reduced resistance to Fusarium wilt. a: Resistance analysis of single-segment introgression lines CSSL11-9, Su8289, empty vector negative control pTRV2::00 and pTRV2::GoanoHBD1 seedlings against Fusarium wilt; b: qRT-PCR detection of GoanoHBD1 gene expression in VIGS silenced plants; c: Statistics on the disease grade index of cotton seedlings inoculated with Fusarium wilt; the standard deviation was calculated for three repetitions, **: P<0.01.

Detailed ways

The present invention will be described in detail through specific examples below. These embodiments are provided for a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

"Includes" or "comprises" mentioned throughout the specification and claims is an open term, so it should be interpreted as "including but not limited to". The subsequent description in the specification is a preferred implementation mode for implementing the present invention, but the description is for the purpose of the general principles of the specification, and is not intended to limit the scope of the present invention. The scope of protection of the present invention should be defined by the appended claims.

1. Test material

The upland cotton Su8289 and abnormal cotton used in this experiment were imported from Jiangsu Academy of Agricultural Sciences. The abnormal cotton single-segment introgression line CSSL11-9 described in the present invention is disclosed in the invention patent with publication number CN 111763757 A, titled "Abnormal cotton chromosome fragments capable of causing lethal upland cotton and their molecular markers and applications".

2. Test method

2.1 Acquisition of the leucine-rich repeat receptor kinase gene (GoanoHBD1)

Using 230 pairs of SSR primers developed by our research group to uniformly cover the abnormal cotton chromosome group, the genome-wide foreground and background identification of the single-segment introgression line CSSL11-9 was carried out. The amplification products of only 6 pairs of SSR molecular marker primers were different between the single-segment introgression line CSSL11-9 and the recurrent parent Su8289. Polymorphic markers NAU5192, A11_175, JAAS3191, A11_243, JAAS3310, A11_193 were screened (the primers are in the invention patent with the publication number CN 111763757A, the title of the invention is "abnormal cotton chromosome fragments that can cause upland cotton lethality and its molecular markers and applications" published in). Nanjing Qingke Biotechnology Co., Ltd. completed the synthesis of all primers.

Using Su8289 as the female parent and CSSL11-9 as the male parent, a _F2 population containing 2337 individuals was constructed, and the SSR primers from abnormal cotton (Zhai et al., 2015) were further selected for the abnormal cotton chromosome fragment A11-9 The interval was encrypted, and finally the key genes derived from the lethal traits of abnormal cotton were finely mapped between JAAS3191-JAAS3050, covering 63.87kb of the upland cotton genome, with a total of 8 functionally encoded genes (Table 1).

Table 1 Genes and annotations of the 8 functional codes in the candidate interval

In order to identify the key genes derived from the lethal traits of abnormal cotton, we silenced 8 candidate genes in the candidate interval by virus-mediated gene silencing (VIGS) method. The vectors used in VIGS were pTRV1 and pTRV2, respectively, and the cotton albino gene (GhCLA1) was used as a control. The primers of the VIGS vectors were designed for the 8 candidate gene sequences, and the amplified fragments were cloned and sequenced to verify that they were correct, and then connected to the TRV2 gene silencing vector in the TRV system. Pick positive pTRV1, pTRV2 (negative control) and pTRV2::GhCLA1 (positive control) and a single colony of Agrobacterium GV3101 containing the candidate gene pTRV2 plasmid, and culture it until the OD600 of the bacterial solution is about 0.5. Collect bacterial cells by centrifugation at 4,000rpm for 10 minutes at room temperature, resuspend with an appropriate volume of resuspension (10mM MgCl ₂ , 10mM MES and 200μM hexanoylsyringone) to a final concentration of 2.0, and let the resuspension stand at room temperature for 3h , Mix the recovery solutions of TRV1 and TRV2 at a ratio of 1:1 by volume. The Agrobacterium inoculation experiment was carried out after the two cotyledons of the cotton seedlings were fully unfolded. The Agrobacterium mixture containing pTRV1 and pTRV2::Goano_ORF (1:1) was injected into normal growing Su8289 and CSSL11-9 plants at the one-leaf stage by leaf needle infiltration method. The inoculated cotton seedlings were cultured in an environment with an ambient temperature of 23° C. and a 16h/8h light/dark cycle. Silenced pTRV2::00 and pTRV2::GhCLA1 were used as negative control and positive control, respectively. Two weeks later, the new leaves of the plants injected with pTRV2::GhCLA1 were completely albino, and the RNA in the leaves of the plants inoculated with the target gene was extracted, and qRT-PCR was used to detect whether the target gene was still expressed (Fig. 1a). The expression levels of all gene silenced plants were extremely significantly lower than those of the empty control, indicating that the gene was silenced by effective VIGS. At the same time, the culture environment temperature was changed to 27°C, and the photoperiod was unchanged. CSSL11-9, pTRV2::Goano_ORF1, pTRV2::Goano_ORF2, pTRV2::Goano_ORF4, pTRV2::Goano_ORF5, pTRV2::Goano_ORF6, pTRV2::Goano_ORF7 and pTRV2::Goano_ORF8 silenced CSSL11-9 plants were found after one week Both began to show lethal phenotypes, but only the plants in which the Goano_ORF3 gene was silenced in the CSSL11-9 plants survived normally (Fig. 1b), and Goano_ORF3 was preliminarily determined to be the key gene derived from the lethal traits of abnormal cotton. We named the Goano_ORF3 gene GoanoHBD1 (Hybrid breakdown).

2.2 Expression analysis of leucine-rich repeat receptor kinase gene (GoanoHBD1)

We planted single-segment introgression line CSSL11-9 plants in a constant temperature incubator at 23°C. When the plants grew to the two-leaf stage, they were transferred to constant temperature incubators at 30°C, 26°C, and 23°C respectively, and the sampling time was 0h and 24h. , 72h and 120h, the top leaves of plant CSSL11-9 were taken, and we analyzed the expression of GoanoHBD1 induced by different temperatures. Take 1 μg of total RNA and operate according to the instructions of the TAKARA reverse transcription kit. Synthesis of first-strand cDNA. After the reverse transcription product was diluted 10 times, 1 μL was taken for qRT-PCR. The cotton actin gene (GenBank: AY305723.1) was used as an internal reference, and the primer sequence was F: ATCCTCCGTCTTGACCTTG, R: TGTCCGTCAGGCAACTCAT. The real-time fluorescent quantitative PCR was operated by ABI QuantStudio5 fluorescent quantitative PCR instrument (ABI, USA), and the method was TB Green I dye method. The reaction system was 20 μl, cDNA, 1 μl; Primer F (10 μM), 1 μl; Primer R (10 μM), 1 μl; TB green Imix, 10 μl, and rehydrated to 20 μl. The program is: 95°C, 10min; 95°C, 10s, annealing: 58°C, 20s; 72°C, 30s; 40 Cycles; 72°C, 10min, and finally run the dissolution program. Relative expression of target gene=2- ^ΔΔΔCT , ΔΔΔCT=[(Ct _{target gene} -Ct _{internal reference gene} ) _{treatment at specified time-} (Ct _{target gene} -Ct _{internal reference gene} ) _0h ] _VD -[(Ct _{target gene} -Ct _{internal reference gene} ) _{Treatment at specified time-} (Ct _{target gene} -Ct _{internal reference gene} ) _0h ] _CK . The primers used to detect the expression of GoanoHBD1 gene are F: GTACGATCTTGAGATTGATT, R: TATGGCGGTAAATGTTTC.

The results showed that in the single-segment introgression line CSSL11-9, the expression of GoanoHBD1 was induced by temperature, and the expression of GoanoHBD1 increased significantly when the ambient temperature was greater than or equal to 26°C. The expression level gradually increased with the prolongation of the induction time, and the expression level reached the highest when the lethal phenotype appeared in the top leaves (Fig. 2).

2.3 Sequence analysis of the leucine-rich repeat receptor kinase gene (GoanoHBD1)

According to the genome sequence of GoanoHBD1 gene, specific primers were designed to extract the RNA of abnormal cotton leaves, and the cDNA obtained by reverse transcription was used as a template to amplify the open reading frame of GoanoHBD1 gene in abnormal cotton. The results showed that the ORF length of the GoanoHBD1 gene was 3885bp (SEQ ID NO.1), the gene encoded 1294 amino acids (SEQ ID NO.2), 323.48KD, and the isoelectric point was 4.58.

2.4 Analysis of Verticillium wilt resistance of the leucine-rich repeat receptor kinase gene (GoanoHBD1)

The Verticillium dahliae bacterium used in the present invention is V991, which can be introduced by scientific research institutes and cultivated by the following methods. 20g of sucrose, 1000ml of distilled water) surface, transferred to the liquid potato medium (200g of potatoes, 20g of sucrose, 1000ml of distilled water) after two weeks, shake culture at room temperature for 5 days, filter the cultured bacteria solution, and measure the concentration of bacteria spores by hemocytometer , Dilute the concentration to 5×10 ⁷ spores/ml. The inoculation method is as follows: develvet the test seeds with concentrated sulfuric acid, dry them, sow them in a paper cup with a volume of 460ml, 3 seeds in a cup, thin out the seedlings after emergence, and keep 1 plant in each cup. When the cotton plant grows to two leaves and one heart, the bottom of the paper cup is torn off to achieve the purpose of root damage. Each material was inoculated with the conidia suspension of Verticillium dahliae at a concentration of 5×10 ⁷ spores/ml, 30ml per nutrient bowl.

The investigation of Verticillium wilt incidence level of the present invention is carried out according to the following standards, level 0: healthy plants, no diseased leaves, normal growth; level 1: less than 1/4 of the leaves of the plants are sick, turning yellow and wilting; level 2: 1/4 of the plants are sick. More than one and less than one-half of the leaves are diseased, yellowing and wilting; Grade 3: more than one-half to less than three-quarters of the leaves are diseased, yellowing and wilting; Grade 4: more than three-quarters of the leaves are diseased, or Plants die. The disease index (DI) of each strain was calculated according to the survey results. DI=[∑(Ni×i)/(N×4)]×100; i=0～4, Ni=number of plants at each level.

In the present invention, we analyzed the resistance of the GoanoHBD1 gene to Verticillium wilt of cotton using the following method.

Using a virus-mediated gene silencing method, the resistance of cotton to Verticillium wilt was verified after GoanoHBD1 gene silencing (Fig. 3a). The virus-mediated gene silencing method is the same as 2.1. When the cotton seedlings injected with TRV2::GhCLA1 showed albino phenotype, the expression of GoanoHBD1 gene was detected in cotton (Fig. 3b), and the cotton actin gene (GenBank: AY305723.1) was used as an internal reference. At the same time, Verticillium dahliae was inoculated, and the inoculated cotton seedlings were cultured at an ambient temperature of 25°C and a 12h/12h light/dark cycle environment, and the disease resistance of the silenced cotton was investigated.

The results showed that 28 days after inoculation with Verticillium dahliae, the disease index of CSSL11-9 plants after silencing the GoanoHBD1 gene was 65.35%, which was significantly higher than that of CSSL11-9 plants without injection (CK, 28.13%) and CSSL11-9 plants injected with empty vector ( The pTRV2::00, 29.03%) control (Fig. 3c) shows that the GoanoHBD1 gene plays an important role in regulating cotton Verticillium wilt resistance.

2.5 Fusarium wilt resistance analysis of the leucine-rich repeat receptor kinase gene (GoanoHBD1)

The fusarium wilt bacterial strain used in the present invention is No. 7 bacterial strain. Drop No. 7 bacterial strain of Fusarium wilt on the surface of solid potato medium (200g of potatoes, 17g of agar, 20g of sucrose, 1000ml of distilled water), and the plaque is about 0.5cm; dry and seal and place it in a 25°C constant temperature incubator to avoid light for cultivation; 5 After 7 days, transfer the bacterial block of Fusarium wilt bacteria to liquid potato medium (200g of potatoes, 20g of sucrose, 1000ml of distilled water) for cultivation, use gauze to filter the bacterial liquid after 7 days, use a hemocytometer to count the spore concentration of Fusarium wilt under a microscope, and dilute the concentration to 5×10 ⁷ spores/ml. The inoculation method is as follows: Put the bacteria block of Fusarium wilt into the liquid potato culture medium for cultivation, and use gauze to filter the bacterial liquid after 7 days; break up the Pintotop cultivation medium and crush it until the medium does not form agglomerates and the particle size is uniform. ; Pour the concentration of 5 × 10 ⁷ spores/ml Fusarium wilt bacterial liquid into the crushed matrix, knead it several times, and fully mix the germs and the matrix; put the inoculated matrix into a 50-hole tray, and put more The trays filled with substrate are stacked together and pressure is applied from above to form a seeding hole in each hole of the tray. Seeds were treated with concentrated sulfuric acid and dried, then sowed, 3 seeds per hole, covered with soil, watered, and covered with film. Place in an environment with an ambient temperature of 25°C and a 12h/12h light/dark cycle. After emergence, remove the film in time, thin the seedlings, and keep 1 plant in each hole.

Fusarium wilt disease progression investigation of the present invention is carried out according to following standard: 0 grade, cotton plant is healthy, grows normally, no diseased leaf; Above, less than one-half of the leaves show symptoms of disease; Grade 3, more than one-half of the leaves of the cotton plants show symptoms of disease, but not dead; Grade 4, the cotton plants are dead. According to the disease level of a single plant, the disease resistance index and mortality of different varieties were calculated to evaluate the resistance level of cotton to Fusarium wilt. Disease resistance index (DI)=[∑(Ni×i)/(N×4)]×100; i=0～4, Ni=number of plants at each level. Mortality rate = single dead plant × 100%/number of all investigated plants.

In the present invention, we analyzed the resistance of GoanoHBD1 gene to cotton wilt using the following method.

Using a virus-mediated gene silencing method, the resistance of cotton to Fusarium wilt after GoanoHBD1 gene silencing was verified (Fig. 4a). The virus-mediated gene silencing method is the same as 2.1. When the cotton seedlings injected with TRV2::GhCLA1 showed albino phenotype, the expression of GoanoHBD1 gene was detected in cotton (Fig. 4b), and the cotton actin gene (GenBank: AY305723.1) was used as an internal reference.

The results showed that the disease index of CSSL11-9 plants after silencing the GoanoHBD1 gene was 51.67%, which was significantly higher than that of CSSL11-9 plants without injection (CK, 18.58%) and CSSL11-9 plants injected with empty vector (pTRV2::00, 18.83%) ) (Fig. 4c), the GoanoHBD1 gene plays an important role in regulating the resistance of cotton Fusarium wilt.

Although the present invention has been described in detail with general descriptions and specific examples above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (6)

1. The function of the abnormal cotton leucine-rich repeat receptor kinase gene GoanoHBD1 in inducing the lethal phenotype of cotton plants is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. The application of the abnormal cotton leucine-rich repeat receptor kinase gene GoanoHBD1 in imparting verticillium wilt and fusarium wilt resistance to cotton is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

3. A plant breeding method characterized in that the method is (1) or (2) below:

(1) The plant with verticillium resistance and fusarium wilt resistance stronger than the target plant is obtained by increasing the activity of the GoanoHBD1 protein in the target plant;

(2) The plant with verticillium resistance and wilt resistance stronger than the target plant is obtained by promoting the expression of the GoanoHBD1 gene in the target plant;

the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

4. A method according to claim 3, wherein the plant is cotton.

5. The application of the silent abnormal cotton leucine-rich repeat receptor kinase gene GoanoHBD1 in obtaining cotton plants sensitive to verticillium wilt and fusarium wilt is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

6. The application of the silent abnormal cotton leucine-rich repeat receptor kinase gene GoanoHBD1 in inhibiting the occurrence of lethal phenotype of abnormal cotton single-piece introgression line CSSL11-9 is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

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