Application of tea tree glucan endoglycosyltransferase-related proteins in rooting of cuttings

Abstract

The invention discloses application of tea tree glucan endoglycosyltransferase-related proteins in promoting tea tree cutting rooting, comprising truncated proteins of proteins TEA021045, TEA021057, TEA031639 and Novel.313, wherein the amino acid sequences of the truncated proteins are shown in SEQ ID NO.1‑4, and the four truncated proteins are expressed and purified by Pichia pastoris and applied to tea tree cutting. The results show that exogenous application of the four truncated proteins can significantly improve the rooting rate of tea tree cuttings, thereby effectively solving the problem of tea tree rooting difficulty in cutting seedling cultivation.

Description

Technical Field

[0001] The invention belongs to the biological field, and in particular relates to the application of tea tree glucan endoglycosyltransferase-related protein in tea tree cutting rooting. Background Art

[0002] Plant cells are surrounded by polysaccharide-rich cell walls, which determine the cell's morphology and play an important role in the development process. The polysaccharides in plant cell walls can be divided into cellulose, hemicellulose and pectin. These components are "cut and pasted" under the catalysis of specific enzymes, thereby completing the cell wall remodeling during plant growth and development. The most important enzyme involved in cell wall modification is transglucanase, which belongs to the glycoside hydrolase family 16. Among them, the most intensively studied is xyloglucan endoglycosyltransferase / hydrolase (XTHs), which is a cell wall loosening enzyme, including xyloglucan endoglycosyltransferase (XETs) and xyloglucan endoglycosylhydrolase (XEHs).

[0003] XTH plays a role in plant development and response to adverse stress. The transcription product of Arabidopsis AtXTH9 accumulates in the plant stem tip, and the internodes of the plant are significantly shortened after mutation. AtXTH27 plays an important role in the formation of Arabidopsis rosette leaves and the development of vascular tissue. Miedes et al. showed that the activity of XET increases during the growth of tomato fruit and decreases during the ripening process, indicating that SiXTH5 and SiXTH8 can maintain the stability of cell wall components during fruit development. XTH also has an important function in responding to adverse stress. Cho et al. overexpressed the pepper CaXTH3 gene in Arabidopsis, which increased the number of mesophyll cells and significantly enhanced the plant's tolerance to water stress. However, the role of XTH in the development of adventitious roots in plants has not been reported.

[0004] Tea tree (Camellia sinensis [L.] O. Kuntze) is a perennial woody plant originating from China and widely planted in mountainous areas of China as an economic crop. Cuttings are the main asexual propagation method for tea trees, which are easy to operate, have a high propagation coefficient, and are easy to operate mechanically. However, due to the low survival rate and rooting rate of some difficult-to-root varieties and ancient tree resources, it has brought great limitations to the promotion of tea tree varieties and resource conservation.

[0005] Tea tree rooting is the key to the success of tea tree cutting seedling cultivation. To improve the rooting rate of tea tree cuttings, the commonly used strategies in production are to control the seedling environment (CN104641920A and CN104541837A) and use root-promoting substances (CN104012288A); if the cultivation environment is controlled, a large amount of human resources and cultivation facilities are required, and the one-time investment is large. Due to the different geographical locations of China's tea tree seedling cultivation areas, the control environment technology of one cultivation area may not be suitable for other tea tree seedling cultivation areas; auxin substances such as ABT1 are commonly used in plant cutting rooting and tea tree seedling cultivation. If root-promoting substances are used to treat cuttings or mother trees, the type, use method and concentration of rooting substances will affect the rooting of tea tree cuttings. The exploration process is cumbersome and time-consuming and labor-intensive in production practice. Therefore, it is very meaningful to improve the rooting rate of tea tree cuttings without relying on the auxin approach for the preservation of rare tea resources. Summary of the invention

[0006] In order to solve the problem of difficulty in rooting cuttings of tea tree varieties that are difficult to root and ancient tree resources, the present invention provides the use of xyloglucan endoglycosyltransferase-related proteins in promoting the rooting of tea tree cuttings, which significantly improves the rooting rate of tea tree cuttings.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The truncated proteins related to the rooting of tea tree cuttings include truncated proteins of TEA021045, TEA021057, TEA031639 and Novel.313. The sequence of the truncated protein of TEA021045 is shown in SEQ ID NO.1, the sequence of the truncated protein of TEA021057 is shown in SEQ ID NO.2, the sequence of the truncated protein of TEA031639 is shown in SEQ ID NO.3, and the sequence of the truncated protein of Novel.313 is shown in SEQ ID NO.4.

[0009] Biomaterial, wherein the biomaterial is any one of the following A1)-A3):

[0010] A1) nucleic acid sequences encoding the above-mentioned truncated proteins of TEA021045, TEA021057, TEA031639 and Novel.313; the gene sequence encoding the truncated protein of TEA021045 is shown in SEQ ID NO.5, the gene sequence encoding the truncated protein of TEA021057 is shown in SEQ ID NO.6, the gene sequence encoding the truncated protein of TEA031639 is shown in SEQ ID NO.7, and the gene sequence encoding the truncated protein of Novel.313 is shown in SEQ ID NO.8.

[0011] A2) an expression cassette containing the nucleic acid sequence described in A1);

[0012] A3) A recombinant vector or recombinant microorganism containing the nucleic acid sequence described in A1).

[0013] Application of tea tree glucan endoglycosyltransferase-related proteins in promoting the rooting of tea tree cuttings: The truncated proteins of TEA021045, TEA021057, TEA031639 and Novel.313 were expressed and purified, and then used to treat tea tree cuttings (Echa No. 10 and Yingshuang) and the rooting ratios of the cuttings were counted. The results showed that all four truncated proteins could significantly improve the rooting rate of tea tree cuttings.

[0014] Application of crude extract of xyloglucan endoglycosyltransferase in promoting rooting of tea tree cuttings: In a specific embodiment of the present invention, crude extract XET of tea tree is extracted, and the rooting rates of tea tree cuttings treated with crude extracts of IBA, XET and both at the same time are compared. The results show that crude extract XET has the same effect of promoting rooting as IBA, and crude extract XET can synergize with IBA to promote rooting of tea tree cuttings, and the rooting effect is significantly higher than that of using either alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The changes in the content of abscisic acid during the cutting process of Echa No. 10 and Yingshuang. A. Changes in ABA content at the base of Echa No. 10 cuttings, B. Changes in ABA content in the leaves of Echa No. 10 cuttings, C. Changes in ABA content at the base of Yingshuang cuttings, D. Changes in ABA content in the leaves of Yingshuang cuttings. The ordinate is the endogenous ABA content (ng / g), and the abscissa is the time after cutting (month).

[0016] Figure 2 The figure shows the change of rooting rate after exogenous abscisic acid treatment on the base of tea cuttings. CK is the control group, ABA is the treatment with abscisic acid for 8 hours, EC10 and YS are the abbreviations of Echa No. 10 and Yingshuang, respectively, * indicates significant difference (P < 0.05), exogenous abscisic acid treatment on tea cuttings can significantly increase the rooting rate of cuttings.

[0017] Figure 3 The rooting rate of tea cuttings was changed after the base of the cuttings was treated with abscisic acid synthesis inhibitor. CK was the control group, Na2Wo4 was the sodium tungstate immersion treatment for 8 hours, EC10 and YS were the abbreviations of Echa No. 10 and Yingshuang, respectively, and * indicated significant difference (P < 0.05).

[0018] Figure 4 This is the result of GO enrichment analysis of E-cha No. 10.

[0019] Figure 5 This is the result of GO enrichment analysis of Yingshuang.

[0020] Figure 6The results of TPM analysis, FPKM analysis and qRT-PCR analysis are shown in Figure 2. Four candidate genes, TEA021045, TEA021057, TEA031639 and Novel.313, were identified through transcriptome analysis.

[0021] Figure 7 The effect of exogenous abscisic acid treatment on XET content and enzyme activity at the base of tea tree cuttings. A. Changes in XET protein content at the base of cuttings after exogenous abscisic acid treatment, B. Changes in XET enzyme activity at the base of cuttings after exogenous abscisic acid treatment. CK is the control group, TR is the exogenous abscisic acid treatment group, EC10 and YS are the abbreviations of Echa No. 10 and Yingshuang, respectively, and * indicates significant difference (P < 0.05).

[0022] Figure 8 The effect of purified XET-related protein treatment on the rooting rate of tea cuttings at the base was investigated. T1, T2, T3 and T4 were purified proteins (full length) of TEA021045, TEA021057, TEA031639 and Novel.313, respectively, and CK was the control group; EC10 and YS were the abbreviations of Echa No. 10 and Yingshuang, respectively. DUCAN was used for the significance test, and different lowercase letters indicated significant differences (P < 0.05).

[0023] Fig. 9 The effect of XET crude enzyme solution, IBA and XET crude enzyme solution + IBA treatment on the rooting rate of tea tree cuttings base. CK is the control group, XET is XET crude enzyme solution treatment of cuttings base for 8 hours, IBA is indolebutyric acid treatment of cuttings base for 8 hours, XET + IBA is XET crude enzyme solution and indolebutyric acid co-treatment of cuttings base for 8 hours.

[0024] Fig.10 The experimental design of protein truncation and the effect of truncated protein on rooting rate after the base of cuttings was treated. AD is the experimental design of protein truncation, E is the tea cuttings treated with truncated protein of TEA021045, F is the tea cuttings treated with truncated protein of TEA021057, G is the tea cuttings treated with truncated protein of TEA031639, and H is the tea cuttings treated with truncated protein of Novel.313. DETAILED DESCRIPTION

[0025] Example 1: Expression and purification of tea tree XETs promotes rooting of tea tree cuttings

[0026] The previous research of the inventor's research group found that ABA can promote the rooting of tea tree cuttings. In order to clarify the function of ABA in tea tree cuttings, we determined the ABA content in two tea varieties (Echa No. 10 and Yingshuang, the same below) ( Figure 1) and exogenously used ABA and its synthesis inhibitor Na2Wo4 to treat tea tree cuttings. The results showed that exogenous ABA treatment of the base of tea tree cuttings could significantly increase the rooting rate of tea tree cuttings ( Figure 2 and 3 ).

[0027] In order to reveal the transcriptional regulation of ABA-induced rooting of tea cuttings, we extracted RNA from the base of tea cuttings treated with ABA for 8 hours for transcriptome sequencing, and set the solvent (1% ethanol) treatment for 8 hours as a control. After obtaining clean reads, we performed DEGs analysis, Venn analysis and cluster analysis, and then DEGs were used for GO enrichment analysis, TPM analysis and FPKM analysis. The results showed that after ABA treatment, genes related to cell wall activity at the base of tea cuttings were activated in large numbers, and 21 XET-related genes were upregulated in Echa No. 10, and most of these genes were also upregulated in Yingshuang. The transcriptome data were verified by qRT-PCR, and the results were basically consistent with the transcriptome data ( Figure 4 , 5 , 6). These results suggest that ABA may promote the rooting of tea cuttings by activating the XET pathway. In order to verify whether the XET protein level and enzyme activity were affected by exogenous abscisic acid, we conducted an ELISA experiment. The results showed that the protein content and enzyme activity of XET increased significantly after ABA treatment, indicating that ABA affects the rooting of tea cuttings by mediating the activity and content of XET ( Figure 7 The above results indicate that XET may have an important function in the tea tree cutting process. In order to verify the specific function of this protein, we selected the four genes (TEA021045, TEA021057, TEA031639 and Novel.313) that were most significantly upregulated in the transcriptome data for cloning. After cloning, we performed eukaryotic expression purification and exogenous treatment of tea tree cuttings for verification.

[0028] 1. Gene cloning

[0029] The cDNA sequences of TEA021045, TEA021057, TEA031639 and Novel.313 were cloned by 3'-RACE and 5'-RACE techniques (rapid-amplification of cDNA ends). The RNA of Echa No. 10 was extracted using the Beijing Huayueyang Biopolysaccharide and Polyphenol Plant RNA Extraction Kit, and the templates for 3'-RACE and 5'-RACE were obtained by reverse transcription. The PCR amplification product was purified and recovered, connected to the pJET1.2 vector, and transformed into the Escherichia coli strain DH5α. Positive colonies were screened by ampicillin resistance, and an appropriate number of monoclonal colonies were picked for DNA sequencing after overnight incubation at 37°C. The primer sequences are shown in Table 1. In order to express TEA021045, TEA021057, TEA031639 and Novel.313 fusion proteins in the Pichia pastoris pPICZαA vector, we amplified their cDNAs using Phusion polymerase and HTG-specific primers from New England Biolabs, which added a 5'Eco RI restriction site upstream and replaced the stop codons of TEA021045, TEA021057, TEA031639 and Novel.313 with an Xba I site to encode their N-terminal sequences. The PCR products were purified and recovered and ligated to the pJET1.2 vector. After transformation, the positive transformants were plated and picked. Then, the TEA021045, TEA021057, TEA031639 and Novel.313 gene sequences were excised from the recombinant vector by complete digestion with Xba I and partial digestion with Eco RI, ligated into the pPICZαA vector after gel recovery, and transformed into the Escherichia coli strain DH5α to obtain positive transformants.

[0030] Table 1: Primer sequence list

[0031]

[0032] 2. Pichia pastoris eukaryotic expression

[0033] E. coli carrying the recombinant pPICZαA plasmid were screened using 40 μg / ml zeocin (InVitrogen) on low-salt (65 mM sodium chloride) LB medium at pH 7.5. The plasmid was linearized by Sac I restriction endonuclease digestion and transferred into Pichia pastoris strain SMD1168H by electroporation, screened on low-salt LB plates containing 100 mg / l zeocin, positive colonies confirmed by DNA sequencing, and cultured overnight in low-salt LB medium supplemented with 100 mg / l zeocin, 1% (w / v) glycerol, and 0.4 mg / l biotin. After the culture was completed, the cells were recovered by centrifugation and resuspended in expression medium (low-salt LB medium, containing 100 mg / l zeocin, 1% (v / v) methanol, and 0.4 mg / l biotin).

[0034] The C-termini of recombinant TEA021045, TEA021057, TEA031639 and Novel.313 carry a 6×His tag, which can be used to 2+ A saturated chelating agarose column was used for affinity chromatography for purification. The protein expressed by Pichia pastoris was bound to the column in buffer A (20mM Na2HPO4, 0.75M NaCl, 0.05% Triton X-100, pH 7.0) with the addition of protease inhibitors, and washed with 50mM imidazole solution after binding. The protein was then transferred to buffer B (20mM citrate, pH 6.3, containing 0.05% Triton X-100 but without NaCl) containing 0.5% (w / v) chlorobutanol and protease inhibitors using a PD-10 desalting column (GE Healthcare). The protein was then eluted in the following order: 5ml buffer A, 3ml buffer B, and finally buffer C (5mM citrate and 5mM formate); the purified protein was collected and used in the following step "6, cutting".

[0035] 3. Collection of cuttings

[0036] From the mother tea plants in the mother garden, we collected disease- and insect-free semi-lignified branches as materials. The varieties were Echa No. 10 and Yingshuang. After collection, they were placed in a greenhouse and covered with weed-proof cloth for standby use. The temperature did not exceed 40°C, and the branches could be stored for no more than 2 days.

[0037] 4. Preparation of cutting medium

[0038] The matrix is ​​a mixture of peat soil, perlite and sand in a volume ratio of 2:1:1. The evenly mixed matrix is ​​placed in a seedling tray for later use. The matrix is ​​sprayed with methyl thiophanate (700 times liquid prepared with 70% powder) until it is moistened, disinfected with methyl thiophanate, and then washed once with large amounts of water.

[0039] 5. Cuttings processing

[0040] The collected branches are trimmed into stem ends with buds containing a growth point, half a leaf, and a length of 2-5 cm. The trimmed cuttings are soaked and disinfected with carbendazim (the concentration of carbendazim solution is 1000 times the liquid prepared with 50% powder). After disinfection, they are washed once with clean water for use; the cut end of the cuttings is parallel to the direction of the leaves, and the upper cut end is higher than the growth point.

[0041] 6. Cuttings

[0042] The base of the treated cuttings was immersed in three 2 mg / ml XET purified protein solutions (TEA021045, TEA021057, TEA031639 and Novel.313) for 8 h, and the stem segments were inserted into the matrix perpendicular to the matrix surface to ensure that the leaf buds were above the matrix. The matrix was then pressed tightly and rooting water was poured in to make the cuttings contact with the matrix.

[0043] 7. Seedling management

[0044] First, 30 days after cutting is the period when the base of the tea tree cuttings swells, the relative moisture content of the seedling medium is controlled at 80-90%, and the relative humidity of the air is maintained at 80-90%; second, temperature control: 30℃ is the temperature for rooting tea tree cuttings, and water management should be strengthened when the temperature is higher than 35℃; third, light management: use a shade net with a shading rate of 70% for shade management, and gradually increase the light 40 days after cutting; fourth, pest and disease control: during the seedling period, tea aphids, tea green leafhoppers, and anthracnose pests should be controlled.

[0045] The purified protein was used to treat tea tree cuttings, and the rooting rate of the cuttings was counted. The results showed that ( Figure 8 ), all four purified proteins could significantly improve the rooting rate of tea tree cuttings, among which TEA031639 and Novel.313 had the best effects in the two tested varieties.

[0046] Example 2: XET crude extract promotes rooting of tea tree cuttings

[0047] During the rooting process of tea tree cuttings, auxin-type plant hormones, such as indolebutyric acid and naphthylacetic acid, are the main factors for tea tree rooting and have been widely used in production practice. Our previous studies have shown that XET can soften cell wall components, so XET may have the potential to synergize with other plant hormones to improve the rooting rate of tea tree cuttings. To verify this hypothesis, tea tree cuttings were treated with IBA, XET crude extracts, and both at the same time, and the rooting rate was counted.

[0048] The preparation method of XET crude extract is as follows: 1.5g plant stem tip fresh sample, add 8ml extract (10mmol calcium chloride, 300mmol sodium succinate, 20mmol ascorbic acid, 15% glycerol (v / v), 3% PVPP (w / v)) and appropriate amount of quartz sand ice bath grinding into homogenate, after standing on ice for 2h, filter with magic filter cloth, centrifuge at 4℃, 12000×g for 5 minutes, the supernatant is XET crude enzyme solution, after packaging, store in ultra-low temperature refrigerator and use for subsequent experiments. The enzyme solution concentration is quantified by BCA. Its use in cutting test method is the same as in Example 1, XET crude enzyme solution concentration is 2mg / L, IBA concentration is 100mg / L.

[0049] The results show that ( Fig. 9 ), XET and IBA alone can significantly improve the rooting rate of tea tree cuttings, among which IBA has a better effect, but there is no significant difference between the two treatments; further, the effect of promoting rooting is best when the two are used simultaneously, and is significantly higher than when either is used alone; these results indicate that XET does not rely on conventional auxin pathways to promote the rooting of tea tree cuttings, and has the effect of synergizing with other plant hormones to improve the rooting rate of tea tree cuttings.

[0050] Example 3: The N-terminus of XETs has a strong ability to promote rooting of cuttings

[0051] According to the predicted protein structures of TEA021045, TEA021057, TEA031639 and Novel.313, this study Fig.10As shown in AD, a protein truncation experiment was designed, and the truncation primers are shown in Table 2. The following is an example of TEA021045 gene truncation, and the truncation of other genes is similar to TEA021045; primers TEA021045 / NTD-F and TEA021045 / NTD-R amplify the N-terminal 1-220 amino acids of TEA021045, and the EcoRI restriction site is added to the 5' end of the upstream primer, and the KpnI restriction site and stop codon are added to the 5' end of the downstream primer. Primers TEA021045 / CTD-F and TEA021045 / CTD-R amplify the C-terminal 221-293 amino acids of TEA021045, and the EcoRI restriction site and start codon are added to the 5' end of the upstream primer, and the KpnI restriction site is added to the 5' end of the downstream primer. Using the above two pairs of primers, the plasmid pJET1.2-TEA021045 constructed and sequenced in Example 1 was used as a template to PCR amplify the truncated gene sequences TEA021045 / NTD and TEA021045 / CTD of TEA021045; the PCR product was gel-recovered and double-digested (EcoRI and KpnI) and then connected to the pJET1.2 vector that was also double-digested, and transformed into the Escherichia coli strain DH5α, and positive colonies were selected by ampicillin resistance. After overnight incubation at 37°C, an appropriate number of monoclonal colonies were picked for DNA sequencing. The subsequent construction of the expression vector and yeast eukaryotic expression were as shown in Example 1.

[0052] The expressed protein was used to treat tea tree cuttings, which were then used for cuttings. The specific operation was as shown in Example 1. The results showed that ( Fig.10 EH), the rooting rate of tea cuttings treated with the purified protein at the N-terminus of the XET gene increased significantly, while the treatment with the purified protein at the C-terminus of the XET gene had no significant promoting effect. This indicates that the N-terminal sequence of XETs plays a role in promoting the rooting of tea cuttings.

[0053] Table 2: Truncated primer sequence list

[0054]

[0055] Note: Bold letters represent additional start codons or stop codons; underlined letters represent additional restriction enzyme cutting sites.

Claims

1. Application of Novel.313 truncated protein or biological material containing Novel.313 truncated protein in promoting rooting of tea tree cuttings, characterized in that: The truncated protein sequence of Novel.313 is shown in SEQ ID NO.

4.

2. The use according to claim 1, characterized in that: The biological material containing the Novel.313 truncated protein is any one of the following A1)-A3): A1) a nucleic acid sequence encoding the truncated protein of Novel.313 as claimed in claim 1; A2) an expression cassette containing the nucleic acid sequence described in A1); A3) A recombinant vector or recombinant microorganism containing the nucleic acid sequence described in A1).

3. The use according to claim 1, characterized in that: Used in combination with indolebutyric acid.

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