Application of NtCrRLK47 gene in relieving continuous cropping obstacle of tobacco

Abstract

The invention discloses an application of an NtCrRLK47 gene in relieving continuous cropping obstacles of tobaccos. The serial number of the gene in a Chinese tobacco genome database is Ntab0776290.1. The invention further provides a method for relieving continuous cropping obstacles of tobacco, tobacco continuous cropping is performed in soil after the mutant material is planted by knocking out or interfering expression of the gene in tobacco or constructing a tobacco mutant strain with the tobacco NtCrRLK47 gene knocked out, the continuous cropping obstacles of the tobacco can be effectively relieved, the yield of the tobacco is increased, and the yield of the tobacco is increased. The method has an application prospect in the field of tobacco planting.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an application of a NtCrRLK47 gene in tobacco in alleviating tobacco continuous cropping obstacles. Background Art

[0002] Continuous cropping refers to the long-term cultivation of the same crop variety and closely related crops in the same field. With the continuous growth of the world's population, the demand for food and cash crops is also increasing. Due to the limited area of ​​arable land and the reduction in the amount of new arable land, continuous cropping has become the most common planting pattern in intensive and large-scale agriculture and horticultural production. Long-term continuous cropping will lead to a series of problems such as deterioration of soil physical and chemical properties, reduced soil enzyme activity, accumulation of autotoxic substances, changes in microbial communities, etc. Many crops, including potatoes, sophora flavescens and tobacco, will suffer from continuous cropping obstacles, which has a serious impact on agricultural production.

[0003] So far, the response to tobacco continuous cropping obstacles is mainly carried out from three aspects: biological, chemical, and agricultural measures. Biological control mainly uses the ability of long-term continuous tobacco to recruit self-toxic substance degrading microorganisms to alleviate continuous cropping obstacles by degrading self-toxic substances; the chemical aspect mainly develops functional organic fertilizers (FOFs) to alleviate tobacco continuous cropping obstacles by improving soil physical and chemical properties and bacterial community structure; agricultural measures include breeding and cultivation of disease-resistant varieties, reasonable crop rotation, supplementary fertilizers, and strengthening field management. Among them, breeding cultivars that can alleviate continuous cropping obstacles is of great significance. Breeding by using the differences in allelopathic effects between varieties to overcome self-toxic effects is expected to become an effective way to solve tobacco continuous cropping obstacles. Compared with other prevention and control methods, breeding cultivars that can alleviate continuous cropping obstacles not only has stable prevention and control effects and high adaptability to the environment, but also reduces the use of chemical fertilizers, improves soil physical and chemical properties, and improves economic benefits. It is an efficient, green and sustainable prevention and control measure.

[0004] CrRLK is a membrane receptor subfamily unique to plant cells that can sense internal and external signals, integrate metabolic, physiological and molecular processes, and regulate plant development. After being activated, it can produce a variety of physiological responses, such as physiological changes, formation or decomposition of protein complexes, metabolic reactions, modification of gene expression, and regulation of plant hormone activity. Therefore, it is of great significance to explore the CrRLK receptor family genes in the main tobacco that can effectively alleviate or eliminate the continuous cropping obstacles. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the NtCrRLK47 gene in tobacco in alleviating tobacco continuous cropping obstacles, and to provide a method for effectively alleviating tobacco continuous cropping obstacles.

[0006] In order to achieve the above-mentioned object, the present invention provides an application of the NtCrRLK47 gene in tobacco in alleviating tobacco continuous cropping obstacles, specifically, by sowing tobacco in soil planted with tobacco mutants in which the tobacco NtCrRLK47 gene has been knocked out, tobacco continuous cropping obstacles can be alleviated. The tobacco NtCrRLK47 gene can be used in the field of tobacco planting, including increasing the fresh weight, stem length and leaf area of ​​tobacco in tobacco continuous cropping, and has significant practical value. Among them, the tobacco NtCrRLK47 gene is numbered Ntab0776290.1 ​​in the Chinese Tobacco Genome Database.

[0007] The present invention also provides a method for alleviating tobacco continuous cropping obstacles, the specific steps are as follows:

[0008] (1) Constructing a knockout vector of the tobacco NtCrRLK47 gene and transforming it into Agrobacterium;

[0009] (2) constructing tobacco mutants with NtCrRLK47 gene knockout by Agrobacterium-mediated genetic transformation;

[0010] (3) After planting the obtained tobacco mutants in the soil, planting tobacco seedlings again can effectively alleviate the continuous cropping problems of tobacco, including increasing the fresh weight, stem length and leaf area of ​​tobacco in continuous cropping, which has significant practical value.

[0011] Preferably, the knockout vector is selected from a CRISPR editing vector, more preferably a p201N vector.

[0012] Preferably, in the construction of the above-mentioned knockout vector, the nucleotide sequence of the gRNA targeting the NtCrRLK47 gene is selected as shown in SEQ ID NO.10.

[0013] Preferably, the above-mentioned Agrobacterium is selected from LBA4404 Agrobacterium.

[0014] By constructing a gene knockout vector, a tobacco mutant with the tobacco NtCrRLK47 gene knocked out can be obtained after transformation with Agrobacterium.

[0015] The present invention has the following advantages:

[0016] The present invention discloses for the first time a new use of the tobacco NtCrRLK47 gene, and can effectively alleviate continuous cropping obstacles by knocking out the tobacco NtCrRLK47 gene.

[0017] The present invention also discloses a method for alleviating tobacco continuous cropping obstacles, which can effectively alleviate tobacco continuous cropping obstacles and increase tobacco yield by knocking out or interfering with the expression of the gene in tobacco, or constructing a tobacco mutant strain with tobacco NtCrRLK47 gene knocked out, and carrying out tobacco continuous cropping in soil after the mutant material is planted, thereby having application prospects in the field of tobacco planting. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of phylogenetic tree analysis of the AtCrRLK1L protein family in Arabidopsis and the NtCrRLK protein family in tobacco.

[0019] Figure 2 The figures are the phenotype comparison results of continuous cropping of tobacco CrRLK gene mutant materials in the present invention.

[0020] Figure 3 These are the test results of various indicators after continuous planting of tobacco CrRLK gene mutant materials in the present invention. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0022] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0023] Experimental Example 1 Identification of Tobacco CrRLK Family Genes

[0024] According to the Yunyan 87 genome in the Chinese tobacco genome database, the CrRLK1L genes in Arabidopsis, tomato, and potato reported in the literature were used as Qurey, and the genome of common tobacco Yunyan 87 was compared by Blastp. After pfam domain identification, genes containing both Malectin (PF12819.7, PF11721) and Pkinase (PF00069, PF07714) domains were selected, and they were preliminarily determined to be all CrRLK encoding genes in Yunyan 87 tobacco. The phylogenetic tree analysis was performed using the existing CrRLK1L family proteins in Arabidopsis and the full-length protein sequences of CrRLK obtained in tobacco. The results are as follows Figure 1 shown.

[0025] Depend on Figure 1It can be seen that some NtCrRLK genes have high homology with important CrRLK genes in Arabidopsis, such as NtCrRLK12 and NtCrRLK49, which have high homology with Arabidopsis FER, and NtCrRLK13, NtCrRLK19, NtCrRLK47, which have high homology with Arabidopsis THE1. Based on this, it is preliminarily determined that the family genes are all CrRLK encoding genes NtCrRLK01-NtCrRLK58 in Yunyan 87 tobacco, and their corresponding gene codes are shown in Table 1.

[0026] Table 1 Tobacco gene names and database numbers

[0027]

[0028] Experimental Example 2 Creation of tobacco CrRLK gene mutant materials and phenotypic identification after continuous cropping

[0029] 1. Creation of tobacco CrRLK gene mutant materials

[0030] According to the 58 NtCrRLK gene CDS sequences identified, they were analyzed to design editing gRNA. During the analysis, it was found that many NtCrRLK gene CDS sequences that were adjacent in evolutionary relationship had high similarity, and the same CRISPR target could be designed to construct a gene editing vector to achieve the purpose of knocking out two genes at the same time. In this way, a total of 20 NtCrRLK genes with high sequence similarity were selected, and a total of 10 gRNAs were designed for these 20 NtCrRLK genes, and CRISPR vectors were constructed.

[0031] Among them, the design of the target is mainly based on two websites: https: / / crispr.dbcls.jp / and https: / / cctop.cos.uni-heidelberg.de / . Finally, the analysis results of the two websites are combined to design gRNA. Guide RNA editing target sites were designed using the websites https: / / crispr.dbcls.jp / and https: / / cctop.cos.uni-heidelberg.de / . Two target sites were designed for each gene with an interval of about 100 bp, and they were constructed into the same CRISPR editing vector according to the Golden Gate method in the literature (Binder et al., 2014). First, the designed gRNA was introduced into the LI gRNA scaffold by site-directed mutagenesis. The gRNA was added to the gRNA-Fwd primer part. The LI gRNA scaffold was amplified by PCR reaction using constant gRNA-Rev primer and gRNA-Fwd primer with 5' end phosphorylation. After PCR amplification was confirmed by gel electrophoresis, the PCR fragments were connected, DpnI digested, Escherichia coli was transformed, and the plasmid was extracted and sent to the company for sequencing to verify the accuracy of gRNA integration. The obtained LI gRNA vector was then cloned into the secondary vector pUC57 gRNAshuttle by BsaⅠ enzyme digestion and T4 ligase ligation. Finally, the obtained secondary vector was transferred to the p201N vector containing 2x35S::Cas9-NosT by Gatway cloning. The plasmid was extracted after PCR positive detection and transformed into LBA4404 Agrobacterium after sequencing verification to complete the construction of the CRISPR editing vector for the highly expressed CrRLK mutant in tobacco roots.

[0032] Among them, the selected genes and the corresponding gRNA information are shown in Table 2, and the primer design for constructing the CRISPR vector is shown in Table 3.

[0033] Table 2 Design of NtCrRLK knockout vector

[0034]

[0035]

[0036] Table 3 Primer design for CRISPR vector construction

[0037]

[0038]

[0039] Using Agrobacterium-mediated genetic transformation technology, Yunyan 87 was used as the background material, and the constructed CRISPR editing vector was genetically transformed in tobacco. The transformed tobacco plants were positively identified by PCR and sequencing, and homozygous tobacco CrRLK mutant genetic materials were finally obtained after breeding and screening. Among them, a total of 10 mutant materials were created, containing 20 CrRLK genes, namely: Ntrlk17 / 27, Ntrlk21 / 41, Ntrlk45 / 56, Ntrlk23 / 29, Ntrlk24 / 30 / 33, Ntrlk54 / 55, Ntrlk35 / 39, Ntrlk36 / 57, Ntrlk44 / 48, Ntrlk47.

[0040] 2. Phenotypic identification of tobacco CrRLK gene mutant materials after continuous cropping

[0041] The phenotype of the tobacco CrRLK mutant obtained above was identified under indoor growth conditions. Under long-day growth room conditions at 24°C, the obtained tobacco CrRLK homozygous mutant materials were sown, using 9 cm diameter nutrient pots, adding 110 g of nutrient soil to each nutrient pot, and then sowing a certain amount of wild-type tobacco Yunyan 87 and mutant material seeds on the surface after moistening with water, with Yunyan 87 sown as the control, and 10 mutants sown as the treatment group, with 3 technical replicates set for each group. The nutrient pots were placed in a growth room for cultivation and watered regularly. After one week, the tobacco seedlings were transplanted, with one tobacco plant transplanted into each nutrient pot. After four weeks, the above-ground stems and leaves were removed, and the underground roots and culture soil were mixed evenly. One-week-old Yunyan 87 were transplanted into the mixed soil and cultured under growth room conditions for four weeks. The growth indicators of Yunyan 87, including plant fresh weight, leaf area, root length, etc., were observed and recorded. The growth indicators of Yunyan 87 grown in the soil of mutant and wild-type tobacco were compared, and the indoor phenotype of mutant tobacco continuous cropping was summarized and evaluated.

[0042] After treatment in each group, the growth phenotype of transplanted Yunyan 87 was Figure 2 As shown in A, the leaf phenotypes of Yunyan 87 in each group are compared. Figure 2 As shown in B. By analyzing the growth phenotype after continuous cropping, it can be seen that continuous cropping in the soil after the mutant material Ntrlk47 was planted has the most significant effect in alleviating the continuous cropping obstacle.

[0043] Among them, the results of fresh weight, shoot length, root length and leaf area of ​​transplanted Yunyan 87 after each treatment are shown in Table 1. Figure 3As shown in A, B, C, and D in the figure. It can be seen that compared with the control group, the fresh weight of Yunyan 87 grown in the soil after the mutant material Ntrlk47 was planted was 128.3% higher than the control, the stem length was 77.9% higher than the control, the root length was 31.3% higher than the control, and the leaf area was 89.6% higher than the control. The above results prove that the mutation of NtCrRLK47 can effectively alleviate the obstacle of continuous cropping of tobacco. The experiments were repeated three times and showed consistent experimental results.

[0044] In summary, the present invention provides an application of a tobacco NtCrRLK47 gene in alleviating tobacco continuous cropping disorders, by knocking out or interfering with the expression of the gene in tobacco, or constructing a tobacco mutant strain with the tobacco NtCrRLK47 gene knocked out, and continuously cropping tobacco in the soil after the mutant material is planted, the continuous cropping disorder of tobacco can be effectively alleviated, and the tobacco yield can be increased, which has application prospects in the field of tobacco planting.

[0045] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. An application of NtCrRLK47 gene in alleviating tobacco continuous cropping obstacles, characterized in that: By sowing tobacco in soil planted with tobacco mutants with NtCrRLK47 gene knockout, tobacco continuous cropping obstacles can be alleviated; wherein, the tobacco NtCrRLK47 gene is numbered Ntab0776290.1 ​​in the Chinese Tobacco Genome Database.

2. An application of NtCrRLK47 gene in tobacco planting, characterized in that: The application includes increasing the fresh weight, stem length and leaf area of ​​tobacco in continuous tobacco cropping; wherein the tobacco NtCrRLK47 gene is numbered Ntab0776290.1 ​​in the Chinese tobacco genome database.

3. A method for alleviating tobacco continuous cropping obstacles, characterized in that: Here are the steps: (1) constructing a knockout vector of the tobacco NtCrRLK47 gene and transferring it into Agrobacterium, wherein the tobacco NtCrRLK47 gene is numbered Ntab0776290.1 ​​in the Chinese tobacco genome database; (2) constructing tobacco mutants with NtCrRLK47 gene knockout by Agrobacterium-mediated genetic transformation; (3) After planting the tobacco mutant in the soil, planting tobacco seedlings again can alleviate the continuous cropping obstacle of tobacco.

4. The method according to claim 3, characterized in that The knockout vector is selected from a CRISPR editing vector.

5. The method according to claim 4, characterized in that The knockout vector is selected from p201N vector.

6. The method according to claim 4, characterized in that In the construction of the knockout vector, the nucleotide sequence of the gRNA targeting the NtCrRLK47 gene is selected as shown in SEQ ID NO.

10.

7. The method according to claim 3, characterized in that The Agrobacterium is selected from LBA4404 Agrobacterium.

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